Hepatocellular carcinoma (HCC) is the most common type of primary hepatic carcinoma, which is a growing public health problem worldwide. One of the main genetic alterations in HCC is the deregulated Wnt/β-catenin signaling, activation of β-catenin is associated with the progression of HCC. In the present study, we aimed to identify novel modulators in controlling β-catenin ubiquitination and stability. USP8 was overexpressed in HCC tissues and correlated with β-catenin protein level. High expression of USP8 indicated poor prognosis of HCC patients. USP8 depletion significantly decreased β-catenin protein level, β-catenin target genes expression and TOP-luciferase activity in HCC cells. Further mechanistic study revealed that the USP domain of USP8 interacted with the ARM domain of β-catenin. USP8 stabilized β-catenin protein via inhibiting K48-specific poly-ubiquitination process on β-catenin protein. In addition, USP8 depletion inhibited the proliferation, invasion and stemness of HCC cells and conferred ferroptosis resistance, which effects could be further rescued by β-catenin overexpression. In addition, the USP8 inhibitor DUB-IN-3 inhibited the aggressive phenotype and promoted ferroptosis of HCC cells through degradation of β-catenin. Thus, our study demonstrated that USP8 activated the Wnt/beta-catenin signaling through a post-translational mechanism of β-catenin. High expression of USP8 promoted the progression and inhibited ferroptosis of HCC. Targeting the USP8 may serve as a promising strategy for patients with HCC. Liver cancer is one of the most urgent public health problems worldwide, which is the sixth most common neoplasm and is the fourth leading cause of cancer-related deaths in all tumors worldwide . HepatocWnt/β-catenin pathway is a highly conserved pathway that mainly controls cell proliferation. The activation of Wnt/β-catenin pathway is associated with haematopoietic system development, hair follicle renewal, liver metabolism and regeneration, lung tissue repair and metabolism, and the maturation of osteoblast –9. This At the off state of Wnt/β-catenin signalling pathway, β-catenin binds to the cytoplasmic sides of cadherin and a degradation complex (DC) comprising APC, AXIN, CK1 and GSK3 protein is formed. β-catenin is captured and phosphorylated by DC, thus activating the ubiquitin proteasome-mediated degradation of β-catenin and keep the low level of free β-catenin in the cytoplasm . AbnormaIn the present study, we report that a deubiquitinating enzyme, USP8, regulates HCC cells proliferation, invasion, stemness and ferroptosis resistance via the Wnt/β-catenin pathway. Mechanistically, USP8 deubiquitinates and stabilizes β-catenin in a catalytic activity-dependent manner. Furthermore, USP8 is overexpressed HCC samples, suggesting that USP8 may play a role in the pathogenesis of HCC.C786A) could not increase β-catenin protein levels, indicating that USP8 increased β-catenin protein level depending on its DUB activity . Briefly, we transfected four nonoverlapping siRNA mixtures specific for each of the DUBs into LM3 cells and found that silencing USP8 significantly decreased β-catenin protein level in LM3 cells Fig. . We testity Fig. . We thenity Fig. . To furtity Fig. . IHC anaity Fig. . Besidesity Fig. . Survivaity Fig. , Table 1We then performed immunofluorescence assay to assess cellular localization of USP8 and β-catenin. Immunostaining results indicated that β-catenin and USP8 colocalized both in the cytosol of HCC cells Fig. . Co-immuSince USP8 is a deubiquitylase, we went on to examine whether β-catenin is a substrate of USP8. As illustrated in Fig. We further assessed the biological functions of USP8 in LM3 and HepG2 cells using two non-overlapping siRNAs separately. The proliferation rate of HCC cells treated with USP8 siRNAs was significantly suppressed compared with that in control cells Fig. . USP8 deHCC is an aggressive and virulent solid tumor. The underlying molecule mechanisms involved in the initiation and progression of HCC remain largely unclear. It is an urgent issue to find out more novel candidate targets and improve the treatment decisions. Ubiquitination is one of the most important posttranslational modifications, it is essential for cellular homeostasis and plays a central role in the cellular protein-degradation machinery . Ubiquitβ-catenin activity plays an important role in regulating tumorigenesis. Accurate regulation of β-catenin is important for maintaining the functions of normal cells. Growing studies have reported that the protein stability of β-catenin can be regulated through the Ub-mediated proteasomal degradation. For instance, β-catenin can be phosphorylated and subsequently recognized by the E3 ubiquitin ligase β-TrCP, which promotes the formation of K48-linked polyubiquitin chains on β-catenin, resulting in the proteasomal degradation of β-catenin . The linTo further systematically evaluate the impact of the individual members of the DUB family in β-catenin deubiquitination and tumor progression, we have screened a DUB siRNA library and conducted unbiased siRNA screening by monitoring the levels of β-catenin and identified several candidate DUBs. In addition to USP8, we also noticed a few other DUBs with potential effect on β-catenin signaling. However, siUSP47, siUSP2a, siUSP20, or siOTULIN imposed minimal effect on β-catenin in our screening systems. The seemingly contradicting results may arise from the variation of cellular context in different cell lines.USP8 is a DUB that belongs to the ubiquitin-specific processing (USP) protease family. The expression of USP8 is frequently overexpressed in multiple cancer types, including lung cancer, breast cancer, cholangiocarcinoma, gastric cancer and melanoma –38. USP8C786A. Under the treatment of the proteasome inhibitor MG132, USP8 deletion could not further affect β-catenin protein level. USP8 depletion shortened the half-life time of β-catenin protein. Ectopic expression of USP8-WT, but not USP8C786A, significantly decreased the ubiquitylation of β-catenin. In vivo deubiquitylation assays demonstrated that the ubiquitin chain on β-catenin could be directly removed by USP8 in a dose-dependent manner. To further find out which type of ubiquitin chain on β-catenin was removed by USP8, we performed ubiquitination assay using a series of ubiquitin mutants, including K6, K11, K27, K29, K33, K48, and K63. We observed that USP8 significantly decreased K48-linked polyubiquitination from β-catenin. As polyubiquitination through K48 of Ub generally results in proteasomal degradation [In the present study, we identified USP8 as a potent DUB which could deubiquitinate and stabilize β-catenin in HCC. First, USP8 and β-catenin interacted with each other. Co-IP analysis identified the interaction between USP8 and β-catenin. Second, USP8 decreased β-catenin polyubiquitination and promotes β-catenin protein stabilization in a manner that depending on its DUB activity. Deletion of USP8 markedly reduced the protein abundance of β-catenin, and the reduced β-catenin protein abundance could be restored by ectopic expression of USP8-WT, but not its catalytically inactive mutant USP8radation , 44, USPFerroptosis is a newly defined form of programmed cell death, which is caused by the accumulation of reactive oxygen species (ROS), particularly lipid ROS –47. ROS In conclusion, we demonstrated that USP8 is a β-catenin DUB that stabilizes β-catenin and promotes tumor growth, invasion, tumor stem-like properties and ferroptosis resistance though its deubiquitylation activity . LM3, HepG2, and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . All cells were cultured at 37 °C, in 5% COThe Ubi and the mutant plasmids were described in our previous study . Wild tyHipure total RNA mini kit  was used to isolate the total RNA from the cancer cells. Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit  according to the instructions. The SYBR green mix  and 7500 Fast Real-Time PCR System  were used for qRT-PCR analysis. Primers were listed as follows: GAPDH ; β-catenin ; CCND1 ; c-MYC ; LGR5 .The TOP luciferase reporter plasmid and Renilla-TK-luciferase vector were obtained from Hanbio Biotechnology Co., Ltd. . Luciferase assay was performed by using Promega luciferase assay kit  according to the manufacturer’s instructions. HCC cells cultured in 24-well plates for 24 h were co-transfected with TOP luciferase reporter (0.2 µg), Renilla reporter (0.05 µg), and siControl or siUSP8 (50 nM). At 48 h post-transfection, luciferase activities were determined using a microplate reader . The ratio of reporter plasmids was determined, each normalized to the luciferase activities of the Renilla-TK-luciferase vector.Cells were washed with pre-chilled phosphate-buffered saline (PBS) and lysed with RIPA extraction reagent  supplemented with protease inhibitors . Cell lysates were pre-cleared and incubated with indicated antibody overnight at 4 °C, The antibody associated with the protein complex were then incubated with protein A/G PLUS-Agarose beads  for additional 2 h. The beads were washed with PBS three times and boiled at 100 °C for 10 min to reverse crosslinking before SDS-PAGE immunoblotting analysis.−1protein synthesis inhibitor cycloheximide  for indicated times. Western blot was performed to measure protein levels.To measure the half-life of β-catenin, cells were treated with 10 µg mlC786A plasmid were transfected into HEK293T cells for 48 h. Cells were then treated with 10 μM MG132  for 6 h. Then cells were washed with pre-chilled phosphate-buffered saline (PBS) and lysed with RIPA extraction reagent. HA-ubiquitinated β-catenin was isolated using an anti-Myc antibody. The ubiquitination level of β-catenin was detected by Western blotting with an anti-HA antibody. In HCC cells, HA-Ub plasmid and USP8 siRNAs were co-transfected into LM3 cells. HA-ubiquitinated β-catenin was isolated using an anti-β-catenin antibody. The ubiquitination level of β-catenin was detected by Western blotting with an anti-HA antibody.For in vivo deubiquitination assay, HA-Ub, Myc-β-catenin, Flag-USP8 or Flag-USP8 NP-40 lysis buffer supplemented with protease inhibitors  was used to extract total protein. BCA Reagent  was used to measure the protein concentration. We used an SDS-polyacrylamide gel was to separate the total proteins and transferred proteins to 0.45 μm PVDF membrane . The primary antibodies used for western blot analysis are listed as follows: β-catenin , USP8 , GAPDH , Myc , HA , Flag  antibodies. Signals were detected and visualized using ECL  and ChemiDocMP imager (Bio-Rad).For cell proliferation assay, LM3 and HepG2 cells were seeded in 96-well culture plates (2000 cells per well). The proliferation rate of HCC cells was detected using Cell Counting Kit-8  assay at 0 h, 24 h, 48 h, 72 h, and 96 h according to the manufacturer’s instructions. The absorbance of each well was detected at a wavelength of 450 nm . For clone formation assay, LM3 and HepG2 cells were seeded into 6-well plates (1000 cells per well) and incubated for 14 days, cells were washed with PBS and stained with 0.5% crystal violet. EdU  incorporation assay was performed as we previously reported . Briefly5 cells were suspended without serum and were cultured into the upper compartment of a transwell insert precoated with Matrigel.. The bottom chambers were filled with 600 μl complete medium. After 24 h, the invasion cells were fixed and visualized by crystal violet staining.We performed transwell invasion assay using 8 μm pore polycarbonate membrane transwell plates . Briefly, 5 × 103 single cells were seeded into 6-well ultra-low attachment culture plates  in serum-free DMEM/F12  supplemented with 20 ng/ml EGF , B27 , and 20 ng/ml bFGF . Two weeks later, the spheres were photographed and counted.2 × 106 LM3 cells were resuspended in 100 μl DMEM and injected subcutaneously into the flanks of BALB/c nude mice aged 6 weeks (n = 6). Mice were treated with vehicle or erastin  when the tumor volume reached 50 mm3. We used a vernier caliper to measure the tumor sizes and recorded every 5 days until the end of the experiment.Animal experiments were conducted according to the protocols approved by ethnic committee of Xiangya Hospital. 1 × 10Reproducibility checklistsupplementary Figure


Lung adenocarcinoma (LUAD) therapies are plagued by insufficient immune infiltration and suboptimal immune responses in patients, which are closely associated with the hyperactive Wnt/β-catenin pathway. Suppressing this signaling holds considerable promise as a potential tumor therapy for LUAD, but Wnt suppressor development is hindered by concerns regarding toxicity and adverse effects due to insufficient targeting of tumors.in vivo efficacy, we utilized the LLC Lewis homograft model, and further validated its mechanism of action through immunohistochemistry staining and transcriptome sequencing analyses.We have synthesized a tumor-specific biomimetic Wnt pathway suppressor, namely CM-CA, by encapsulating carnosic acid within Lewis lung carcinoma (LLC) cell membranes. It possesses nano-size, allowing for a straightforward preparation process, and exhibits the ability to selectively target the Wnt/β-catenin pathway in lung adenocarcinoma cells. To evaluate its The findings from the animal experiments demonstrated that CM-CA effectively suppressed the Wnt/β-catenin signaling pathway and impeded cellular proliferation, leading to notable tumor growth inhibition in a biologically benign manner. Transcriptome sequencing analyses revealed that CM-CA promoted T cell infiltration and bolstered the immune response within tumor tissues.The utilization of CM-CA presents a novel and auspicious approach to achieve tumor suppression and augment the therapeutic response rate in LUAD, while also offering a strategy for the development of Wnt/β-catenin inhibitors with biosafety profile. Amongin vitro results to in vivo efficacy. As a result of these significant constraints, there are currently no approved drugs specifically targeting the Wnt/β-catenin pathway for safe clinical utilization.Encouraging progress has been made in utilizing Wnt/β-catenin pathway inhibition as a potential therapeutic approach against tumors: inhibition targets encompass both upstream components and downstream target genes; various types of agents, including small molecules, peptides, and antibodies, have been investigated; and numerous studies have been conducted, ranging from preclinical animal models to clinical trials –22. Nevein vivo utilization and the presence of additional toxic side effects  expression on the survival of patients with lung adenocarcinoma, an analysis was conducted using data from The Cancer Genome Atlas Program (TCGA). A cohort of 293 lung adenocarcinoma (LUAD) patients was categorized into two groups, the high-CTNNB1 and low-CTNNB1 groups, based on the expression level. According to the data presented in + T cell infiltration level.  into C57BL/6 mice. The established model mice were randomly divided into three groups (n=5/group), treated with different substances: PBS (Control), CA, and CM-CA. Specifically, when the tumor volume reached approximately 50 mm3, a 14-day administration was initiated by drug injection via tail vein at a dosage of 2 mg/kg. The frequency of administration was once every two days. The animal protocol received approval from the medical ethics committee of Xi’an Jiaotong University . The transmission electron microscopy (TEM) image, measurement of the encapsulation efficiency, particle size distribution and zeta potential data proved that the CM-CA used in the experiments possess normal particle morphology and stability with relatively satisfactory CA loading.  on differently treated tumors. As anticipated, findings confirmed expression alterations of the Wnt/β-catenin pathway in response to CA and CM-CA treatments. –44, furtGiven the considerable heterogeneity observed in LUAD tumors, it is not uncommon for certain patients to exhibit inadequate intratumoral immune infiltration, which could potentially contribute to their resistance to conventional therapeutic approaches , 45. Mot2.4in vivo , promotes the degradation of β-catenin and inhibits the activation of downstream target genes by perturbing β-catenin secondary structures near the binding site, inducing a conformational change in β-catenin and blocking the interaction with BCL9 , 46. Howin vivo , 32, 41.3in vivo retention time remains unsatisfactory  to encapsulate inhibitors, thereby rendering them inconspicuous to the immune system and enabling their targeted recognition and utilization by tumor cells  and the Gene Expression Omnibus  database. The RNA sequencing datasets in this study are available in online repository: PRJNA1000099 [NCBI Sequence Read Archive]. All the methods and materials can be found in the Supplementary Material.Clinical data and mRNA expression matrix of LUAD samples analyzed in this work are obtained from TCGA database (The animal study was approved by Medical Ethics Committee of Xi’an Jiaotong University. The study was conducted in accordance with the local legislation and institutional requirements.RG: Data curation, Formal Analysis, Investigation, Writing – original draft. XZ: Conceptualization, Investigation, Software, Writing – original draft. AJ: Investigation, Methodology, Validation, Writing – original draft. WH: Conceptualization, Project administration, Supervision, Writing – review & editing. TL: Conceptualization, Funding acquisition, Resources, Supervision, Validation, Visualization, Writing – original draft.


Currently, the epoxy resin market is expressing concerns about epoxy resins’ non-recyclability, which can hinder their widespread use. Moreover, epoxy monomers are synthesized via petroleum-based raw materials, which also limits their use. So, it is crucial to find more environmentally friendly alternative solution for their formulation. Within this context, the aim of this paper is to exploit a Cradle-to-Cradle approach, which consists of remodeling and reshaping the productive cycle of consumer products to make sure that they can be infinitely reused rather than just being recycled with a downgrading of their properties or uses, according to the principle of the complete circular economy. Indeed, after starting with a fully-recyclable bio-based epoxy formulation and assessing its recyclability as having a process yield of 99%, we obtained a recycled polymer that could be reused, mixing with the same bio-based epoxy formulation with percentages varying from 15 wt% to 27 wt%. The formulation obtained was thoroughly characterized by a dynamic-mechanical analysis, differential scanning calorimetry, and flexural tests. This approach had two advantages: (1) it represented a sustainable disposal route for the epoxy resin, with nearly all the epoxy resin recovered, and (2) the obtained recycled polymer could be used as a green component of the primary bio-based epoxy matrix. In the end, by using replicated general factorial designs  combined with a proper optimization process, after carrying out a complete thermo-mechanical characterization of the developed epoxy formulations, the right percentage of recycled polymer content was selected with the aim of identifying the most performing epoxy matrix formulation in terms of its thermo-mechanical properties. Epoxy resin is among the most used and commercialized thermosets due to its outstanding properties, such as its easy and cost-effective processing, chemical resistance, high durability, insulation, adhesiveness, low thermal expansion, and good thermal and mechanical properties ,2. These®Technologies in 2012 filed a patent (US Patent 2013/0245204 A1) [®Technologies has recently been optimized, becoming more environmentally friendly [Due to their interesting properties, epoxy resins have different applications in several industrial fields; they are used in paints and coatings, adhesives and sealants, matrices for fiber-reinforced composites, electrical and electronic components, marine sector, wind turbines, and so on. The epoxy market was estimated at USD 22.9 billion in 2021, and it is expected to expand at a compound annual growth rate (CAGR) of 7.3% from 2022 to 2030 . Even thfriendly . For theIn this work, we propose an alternative reuse strategy that starts from the produced recycled polymer (rTP), encompassing a truly circular economy (CE) approach and relying on the cradle-to-cradle (C2C) concept. The latter approach is gaining ground in the polymer field with the aim of developing innovative recycling technologies that can help establish a circular economy and sustainable, benign environment . The C2CFrom this point of view, many studies already present in the literature have proposed the synthesis of bio-based epoxy monomers from vegetable oils, lignin, eugenol, and natural acids ,32,33,34In the present paper, bio-based epoxy prepolymers and different formulations of rTPs with contents varying from 15 wt% to 27 wt% were processed with different cure cycles. All the systems were thoroughly characterized by thermo-mechanical testing. All the collected data by the test runs were statistically analyzed via a replicated general factorial design. In the end, the best formulation was identified using an optimization process relying on the desirability function approach.The epoxy resin used for this study is a bicomponent system purchased from R*Concept . Part A is a bio-based epoxy prepolymer (EEW = 180–190 g/eq), named Polar Bear, characterized by a biocarbon content of 28% deriving from pine oils . The latter was determined according to the ISO/IEC 17025:2017 PJLA #59423 standard. Part B is an amine hardener named RecyclamineTM R*101 (AHEW = 52.5 g/eq). A cleavable acetal group is embedded in the epoxy resin system’s chemical structure, which can be selectively cleaved under mild acidic conditions, thus achieving the epoxy resin’s network breakage and, in turn, its recyclability.Both part A and part B of the formulation were liquid at room temperature, hence making the mechanical mixing process easy.3 basis) from Sigma-Aldrich  were used.For the chemical recycling process, pure acetic acid from VWR International S.r.l., Milan, Italy and ammonium hydroxide  in a 1:1 ratio. During this phase, the rTP started to precipitate, and it was recovered, washed with distilled water, and dried at 50 °C for 24 h under vacuum conditions. Finally, the rTP was pulverized by using a mortar and pestle.The cured specimens were chemically recycled using an optimized chemical recycling procedure as proposed elsewhere ,35. BrieThe yield for the chemical recycling process was equal to 99%, and it was evaluated using the formula reported below:According to previous results obtained by the authors of , the obtRecycled thermoplastic (rTP) has been extensively characterized in our previous work . Its cheDue to their low toxicity and availability, carboxylic acids , alcoholTo investigate the influence of the rTP on the thermo-mechanical properties of the epoxy blends, three formulations were prepared by curing the epoxy monomer named Polar Bear with a mixture of rTP and RecyclamineTM R*101. The molar ratio between the epoxy prepolymer and the hardening mixture (RecyclamineTM R*101 + rTP) was kept fixed to 1:0.46. Moreover, the percentage of rTP varied from 15%wt up to 27%wt. For all the blends, the following procedure was used: (i.) the selected amount of rTP was mixed with Recyclamine at 70 °C to ensure full dissolution; (ii.) the epoxy prepolymer was then added and mixed under constant degassing in a vacuum using a centrifugal mixer ; and (iii.) the obtained formulation was poured into a silicone mould. Once the epoxy/thermoplastic blends were ready, they looked homogeneous, with the rTP totally dissolved in the epoxy resin.Two cure cycles were analyzed: curing at 25 °C for 24 h (C1) and curing at 25 °C for 24 h followed by an additional post-curing step at 100 °C for 3 h (C2). The samples were tested after each curing cycle to evaluate the effect on the resin properties.The cured samples, obtained by mixing different amounts of rTPs, were characterized in terms of their thermo-mechanical properties to evaluate the range of applications for their reuse from the cradle-to-cradle perspective.g), (ii.) flexural strength, (iii.) flexural modulus, and (iv.) deformation at break. The response parameters were determined using dynamic mechanical analysis (DMA), differential scanning calorimetry (DSC), and flexural test (ASTM D790), which are described below. For each experimental study, the number of replications was set equal to n = 5. Therefore, N = a · b · n = 30 runs were carried out for each considered experimental plan. In the end, once the response parameters were collected, an analysis of variance (ANOVA) was performed to investigate the statistical significance of each considered factor and their interaction. The four different experimental plans performed in the study are reported in The rTP content added (named factor A) and the curing cycle (named factor B) were selected as independent variables for the study, and their combined effect was investigated using a replicated general factorial design. In detail, factor A was varied among three different levels (a = 3) of {15, 21, 27} %wt. Conversely, factor B was varied among two different levels (b = 2) of {C1, C2}. Four different responses (dependent variables) were investigated: (i.) glass transition temperature  and B (curing cycle) were varied within the considered experimental range, i.e., among all the considered levels. The set constraints for the optimization process performed are summarized in Finally, to identify the best-performing formulation in terms of the investigated responses, an optimization was carried out by using the approach of desirability functions. This general approach first converts each response rability . To carrg for each formulation with different rTP contents and different curing cycles. These analyses were run using a TRITEC 2000 dynamic mechanical thermal analyzer  on samples with size of (10 × 6 × 4) mm3. Each analysis was performed in single cantilever deformation mode, since this is a well-known mode for characterization through glass transitions [g using the following approach: tanδ versus temperature curves were plotted for each investigated formulation, and the Tg was measured as the tanδ peak temperature.A DMA was used to measure the Tnsitions . Each anDSC analysis was carried out to confirm the obtained results from DMA analysis and to understand the effect on the cross-linking process of the addition of rTP. The calorimetric measurements were run using a Shimadzu DSC-60  instrument. For each scan, about 6 mg of sample was inserted into 40 μL sealed aluminum crucibles. Each sample was heated from 25 °C up to 250 °C at a rate of 20 °C/min in air.3. They were mechanically tested using an Instron 5985 universal testing machine  equipped with a load cell of 10 kN, in strain control mode, with a span length of 60 mm and a speed of 2.0 mm min−1. Five samples were tested for each investigated scenario measuring the flexural strength, the flexural modulus, and the deformation at break; next, the average and the standard deviation were evaluated for each of them.The flexural properties of each thermoset formulation containing varying contents of rTP were measured according to the ASTM D790 standard. The characterized samples had a size of (80 × 10 × 4) mm−1 with 32 scans and a resolution of 4 cm−1.The evolution of the chemical groups by increasing the rTP amount within the formulation was examined via Fourier transform infrared spectroscopy. The spectra, studied with a Perkin Elmer Spectrum 100 UATR  in attenuated total reflectance (ATR) mode, were recorded in the range between 4000 and 650 cmSEM (scanning electron microscopy) micrographs were obtained using an SEM EVO 15 . The post-cured samples were fractured in liquid nitrogen, and then the cryofractured surfaces were etched with stabilized Tetrahydrofuran ACS reagent  for 20 min before performing the sputter-coating process. The etching process was exploited to etch the rTP phase, thus increasing the contrast between the two phases  when phase separation occurred. The specimens were gold-sputtered with an Agar Sputter Coater AGB7340 sputter coater machine  after the etching treatment. The SEM analysis was run at different magnifications: 100×, 300×, and 500×. The electron source used was an LaB6 (Lanthanum Hexaboride) emitter, while the electron high tension (EHT) value was set at 25 kV.g evaluation of each investigated scenario are reported in The obtained results from the DMA test for the Tg of the rTP content added (factor A) and the curing condition used (factor B) was rationalized using an ANOVA analysis, whose results are summarized in p-value* < 0.0001) for the Tg value, as can be inferred from the effects diagram for the considered parameter . No anomaly was detected in the model adequacy check on the residuals, as shown in 2 parameter, equal to 0.9980 . This trend has also been reported in the literature when adding plasticizer within epoxy systems [g by increasing the rTP content in the thermoset formulation can be explained by the lower reactivity of the recycled thermoplastic compared to that of the commercial recyclable amine and by the dilution effect observed for thermoplastic/thermoset blends [g. In previous findings, the main molecular effect related to the interaction of a plasticizer with an epoxy network has been shown to be a broadening of its viscoelastic response [According to the effects diagram for the Tg , it is py matrix ,35. On t systems ,51. The t blends ,53. Bothresponse . Howeverresponse ; and secresponse . This fiThe exothermic peak of the DSC thermogram, which represents the area corresponding to the polymerization process, showed only a slightly lower variation in the peak temperatures for the increasing rTP content . TherefoThe representative flexural stress versus flexural strain curves obtained for each investigated epoxy resin formulation with a variable content of rTPs, ranging between 15%wt and 27%wt, and exploiting two different cross-linking curing cycles (C1 and C2), are shown in The data gained from the mechanical characterization, i.e., the flexural properties found for all the investigated scenarios, are summarized in p-value* < 0.0001). Moreover, even their interaction AB was influential . According to the normal probability plot reported in 2 parameter had a high value, 0.9746 , the combined effect of factor A and factor B on the flexural modulus can be determined. The ANOVA study results are reported in p-value* < 0.0001). Furthermore, as R2 = 0.9270, it can be determined that most of the variability found for the collected data for the flexural modulus parameter is related to the variation in the influential factors found inthe ANOVA. Following the same approach described for the first two responses  and focusing on the investigated scenario {A: 15%wt; B: C2}, the prevalent influence of factor A can be highlighted, since the additional post-curing step at 100 °C for 3 h allowed us to achieve an increase of about 1.7% in the flexural modulus. Compared to the results obtained for the virgin thermoset , in whicFor the results discussed so far, a common feature can be highlighted: the addition of rTP to the virgin thermoset allowed us to improve the flexural modulus, even though this varied depending on the rTP quantity added. A similar trend was found by M.I. Giannotti et al. , who perp-value∗ > 0.0001). Conversely, their interaction AB was influential . Next, considering the high value found for the R2 parameter (R2 = 0.9566)  , most ofLastly, from the normal probability plot related to the response elongation at break shown in The diagram of effects related to the elongation at break is reported in −1, 1800–1400 cm−1, and 1200–900 cm−1. By increasing the amount of the thermoplastic, an increase in the intensity of the band at 3396 cm−1 [−1 and 1506 cm−1 are linked to the stretching and bending of the C–H groups in aliphatic ethers, respectively [−1, which was associated with the stretching vibration of the aromatic alkyl C–O ether derived from the epoxydic ring opening of Polar Bear [To validate the above-described reaction mechanism, the chemical structures of the cured formulations were analyzed by infrared spectroscopy, and the related spectra are reported in 396 cm−1  was obseectively . From thlar Bear  as well.The findings related to both the mechanical and chemical characterizations were further confirmed by microscopic investigations. Indeed, according to the acquired micrographs, when a low amount of rTP was added, the epoxy/rTP blend did not present a clear or well-distributed phase separation. This behavior can be explained by the mean of two different mechanisms: (1) the catalytic behavior of the hydroxyl groups towards the cross-linking reaction between part A and part B of the formulation; and (2) the cross-linking reaction between part A of the formulation, i.e., the epoxy prepolymer, and the rTP itself, which works as a curing agent. For rTP contents below or equal to 21%wt, the two aforementioned mechanisms are well balanced, thus achieving an optimized chemical reaction which ensures a high solubility of the rTP within the blend. Conversely, beyond the identified threshold value, the rTP starts to demix from the blend, causing the appearance of a clear and homogeneously distributed particulate morphology.The phase separation within the thermoplastic/epoxy blend containing 27%wt of rTP, which led to the worst thermo-mechanical properties found, is optically visible . Here, aThe obtained results for the optimization process carried out by using the approach of desirability functions are shown in A CE/C2C approach was used as a strategy to reuse recycled thermoplastic (rTP) derived from the epoxy matrix to be recycled into the virgin epoxy matrix itself. This approach had two advantage. First, we could devise a clear plan to define how to recycle and reuse the cured resin at its EoL, thus ensuring that once it enters the production cycle, it can be infinitely reused rather than just being recycled. Second, the chosen strategy permitted us to reduce the use of petroleum-based raw materials for the synthesis of the epoxy matrix, since variable contents (ranging between 15%wt and 27%wt) of recycled material were added to the virgin epoxy formulation.g) and mechanical property as determined by flexural tests . From these results, the following conclusions were found:homogeneous epoxy/thermoplastic blends with good thermo-mechanical properties were obtained for an rTP content not exceeding a threshold value of 27%wt;by implementing an additional post-curing process at 100 °C for 3 h, it was possible to enhance the thermo-mechanical properties;the best-performing epoxy system in terms of thermo-mechanical properties, as determined by an optimization process relying on the approach of desirability functions, was the one containing 21%wt rTP which was cured exploiting the C2 curing cycles.Each of the investigated epoxy formulations containing a variable rTP content was thermo-mechanically tested to explore how the rTP’s incorporation affected the thermo-mechanical properties of the initial epoxy resin formulation. By analyzing each replicated general factorial design, it was possible to define how the rTP content and the implemented curing cycle affected each investigated thermal  for many applications when the additional post-curing step was used. For example, the commercial HexPly® M79/M79-LT thermosetting system, which requires short cure cycles at a low temperature (about 100 °C) and is characterized by a Tg equal to 95 °C, was designed for prepreg applications and to manufacture large industrial components. However, it is neither a bio-based nor a recyclable epoxy system. Conversely, the epoxy system identified as the best-performing one in terms of its thermo-mechanical properties corresponded to the configuration {A: 21%wt; B: C2}. As proposed in this experimental work, this could represent a novel thermoset system able to overcome the environmental issues related to thermosets’ typical non-recyclability and the use of petroleum-based raw materials for their synthesis.Furthermore, it was highlighted that an increase in the rTP content within the virgin epoxy system caused a reduction in the TOur recommendation for future work is an evaluation of rTP’s durability in recycling cycles, in terms of its chemical and thermal properties , using the optimized epoxy/thermoplastic blends developed in this experimental work.


TOH-containing emulgel did not cause skin irritation in mouse model of irritation and in healthy human volunteers. Moreover, an increase in skin hydration and a decrease in trans-epidermal water loss (TEWL) were observed after TOH-containing emulgel treatment on human skin. Our findings indicated that TOH-containing emulgel can be utilize as an antifungal agent for topical treatment against fungal infections on the skin.Tryptophol (TOH), a fungal quorum-sensing molecule, that possesses anti-fungal activities for controlling the growth of human pathogenic fungi. In the present study, we developed TOH-containing emulgel formulations and examined the antifungal activities and potential use as topical treatments on the skin. The results showed that TOH-containing emulgel at 1000 μM has excellent physical characteristics as homogenous, stability, and inhibits the growth of 30 species of human pathogenic fungi  Fungal QSMs have been shown to have a variety of biological activities such as cell-to-cell communication, morphogenesis, biofilm formation, and virulence, which ultimately controls the growth of another fungi2. To date, they are 4 main groups of QSMs including farnesol (FOH), tyrosol (TYR), phenylethanol (PE), and tryptophol (TOH)3.Fungi can communicate to their extracellular environments by producing and releasing their small diffusible chemical signaling molecules known as fungal quorum-sensing molecules (QSM)10H11NO, TOH; molecular weight, 161.20) is a QSM that can be isolated from yeasts such as Candida albicans and Saccharomyces cerevisiae4. Our previous studies have demonstrated that TOH can inhibit the growth of Candida albicans5 and Scedosporium apiospermum4. Thus, TOH has a high potential to be developed as an innovative product containing active antifungal agent for the treatment of fungal infections.Tryptophol  was also produced. The parameters of emulgel base, TOH- and VOZ-containing emulgel were presented in Table With the aim to develop the TOH-containing emulgel from emulgel base recipe, the emulgel prototype products containing 100 μM and 1000 μM of TOH were successfully developed. The physical and chemical properties of TOH-containing emulgel were evaluated previouslyIn-Vitro antifungal activities of manufactured TOH-containing emulgel formulations were evaluated in 30 strains of fungal pathogens using the agar spot assay. Firstly, the colonies diameters of azole-resistant/or azole-sensitive strains including Scedosporium apiospermum (CBS 117410), S. boydii (CBS 120157), Lomentospora prolificans (CM324)9, Aspergillus fumigatus (CL-262)10, A. fumigatus (AF293), Candida albicans (ATCC 96901), and C. albicans (ATCC 90028) were determined after 14 days of incubation with emulgel base, 100 μM and 1000 μM of TOH- and 1000 μM of VOZ-containing emulgel (Table Trichophyton mentagrophytes (ATCC MYA-4439), T. rubrum (ATCC MYA-4438), T. verrucosum (ATCC 42898), T. tonsurans11, Microsporum canis11, M. gypseum12, and Epidermophyton floccosum (ATCC 52066) than 100 μM of TOH- and 1000 μM of VOZ-containing emulgel. In the group of dematiaceous molds, 1000 μM of TOH-containing emulgel remarkedly lessened the growth rate of A. recifei (ATCC 36328), Fonsecaea pedrosoi, E. dermatitidis (ATCC 76204), and Bipolaris maydis than 100 μM of TOH- and 1000 μM of VOZ-containing emulgel. Moreover, 1000 μM of TOH-containing emulgel remarkedly lessened the growth rate of dimorphic fungi (Sporothrix schenckii (ATCC 58251) and Talaromyces marneffei13) than 100 μM of TOH- and 1000 μM of VOZ-containing emulgel. In pathogenic yeasts, 1000 μM of TOH-containing emulgel remarkedly lessened the growth rate of Malassezia furfur (ATCC 12078), T. asahii (ATCC 90039), C. tropicalis, C. dubliniensis (ATCC MYA-646), Cryptococcus neoformans (ATCC 32045), C. gattii (ATCC 56992), Lodderomyces elongisporus (ATCC 11503), Mucor spp.13, Rhizopus spp., and Beauveria bassiana (ATCC 74040) than 100 μM of TOH- and 1000 μM of VOZ-containing emulgel. Thus, the effective concentration of TOH-containing emulgel at 1000 μM was chosen due to its board antifungal activities.dii CBS 10157, Lomin vitro.To further determine the potential use as topical TOH-containing emulgel, human foreskin fibroblasts HFF-1) cell viability and cytotoxicity were investigated after incubating for 24 h with 100 μM and 1000 μM of TOH and 1000 μM of VOZ using MTT -2,5-diphenyl tetrazolium bromide), MitoGreen, lactate dehydrogenase (LDH), and dual Acridine Orange/Ethidium Bromide AO/EB staining assays. The results showed that HFF-1 cell viability did not show a significant change after treating with 100 and 1000 μM of TOH as determined by MTT and MitoGreen assays  of 1000 μM TOH-containing emulgel. After 24 h post-topical application, croton oil and 5% Sodium Lauryl Sulfate (SLS) were able to induce mild to moderate edema with loss of skin creases, whereas, emulgel cream base, commercial shielding cream, 1000 μM VOZ-containing emulgel and 1000 μM TOH-containing emulgel did not induce any skin changes as determined by visual assessment. The notable histological features of acute irritant contact dermatitis were spongiosis, accumulation of fluid in the epidermis, and inflammatory cells infiltrations in epidermis, dermis, and subcutaneous tissues in the group treated with croton oil  of 1000 μM TOH-containing emulgel. Severe epidermal hyperplasia, spongiosis, inflammatory cells infiltrations, and dilatation of dermal vessels were found in the group treated with croton oil after 7 days Fig. d and 14 Skin irritation test was determined in thirty healthy volunteers after topical treatment of 1000 μM TOH-containing emulgel. After 24 h post-topical application, no visual characteristics of skin irritation or dermatitis observed in the group treated with commercial shielding cream Fig. a, 1000 μHydration values measured on the skin of volunteers treated with 1000 μM TOH-containing emulgel are significantly higher than emulgel base and distilled water treated areas Fig. a. Skin h14. Several species of human pathogenic fungi can infect deeper than the superficial layer of the skin causing cutaneous mycosis15. These skin mycoses are common worldwide, which in some cases, causing cosmetically poor appearance with severe inflammation16. Antifungal drug such as voriconazole (VOZ), a triazole compound, is the drug-of-choice antifungal agent against fungal infections17. However, fungal infections that resistant to triazoles such as VOZ has become a concern with increases cases over the past decade18. Thus, the present study was conducted and developed to evaluate the effectiveness of alternative topical treatment to treat various pathogenic fungi as a tryptophol (TOH)-containing emulgel.Superficial mycosis is caused by pathogenic fungi that affects superficial layer of the skin19. TOH affects fungi morphogenesis, which provides a potential use as an antifungal agent various fungal pathogen species20. We have previously demonstrated that in vitro treatment of TOH suppresses the pathogenicity of C. albicans. In addition, treatment of TOH was found to induce C. albicans apoptosis via the transcriptional upregulation of caspase recruitment domain-containing protein (CARD)-9 and Noxa and the downregulation of Bcl-25. We also previously demonstrated that treatment of TOH at 100 μM reduces the germ tube length and biofilm formation of S. apiospermum in vitro. Moreover, TOH-containing emulgel was developed at 100 μM, which reduced S. apiospermum-induced eumycetoma formation in vivo9. Also the antifungal activity of TOH both in vitro and in vivo have been extensively studied, whether antifungal activity and safety of TOH-containing emulgel for clinical use remains to be elucidated.In general, TOH can be naturally found as a quorum sensing molecule (QSM) in seed plants, bacteria, and fungi9. Moreover, TOH-containing emulgel in the present study exhibited excellent in cosmeceutical levels including homogeneity, consistency, and stability with pH range of 5.5, which compromised with the pH of the skin21. Interestingly, we further demonstrated that 1000 μM TOH-containing emulgel exhibited excellent in antifungal activity against 30 pathogenic fungi including azole-resistant strains, hyaline and dematiaceous molds, dimorphic fungi, and yeasts than 1000 μM VOZ-containing emulgel in vitro. Thus, our study suggested that the manufactured TOH-containing emulgel at 1000 μM was optimal for topical use in vivo with antifungal activity against wide ranges of human cutaneous pathogenic fungi.In the present study, we demonstrated that TOH at 100 μM and 1000 μM was successfully incorporated into emulgel base containing SEPIGEL 305in vitro suggesting that TOH is safe to use as topical treatment in vivo. We further determined the safety use of TOH-containing emulgel in human volunteers by determining both acute irritant contact dermatitis (AICD) and chronic irritant contact dermatitis (CICD) caused by 1000 μM TOH-containing emulgel prior to commercialize the product. From the present study, our results suggested that either single or repeated exposure of 1000 μM TOH-containing emulgel does not cause skin irritation or any histological changes, which suitable for long term use22.In this report, we demonstrated that TOH caused less cytotoxic to human foreskin fibroblast (HFF-1) cells than VOZ No skin irritation was observed in human volunteers after 1000 μM TOH-containing emulgel treatments. Moreover, skin hydration and less water loss were gained after 1000 μM TOH-containing emulgel treatments. Therefore, our present study showed that 1000 μM TOH-containing emulgel is suitable to be used as an alternative antifungal product for topical treatment. Hence, it is possible to infer that the 1000 μM TOH-containing emulgel was safe for topical application on human skin without skin adverse effects. Nevertheless, the antifungal activity of 1000 μM TOH-containing emulgel need to be evaluated further in patients with cutaneous fungal infections, which we plan to investigate in the near future.The study confirmed positive effects of 1000 μM TOH-containing emulgel that exhibits antifungal activities and cosmeceutical benefits without causing skin irritation to human. The potential use of 1000 μM TOH-containing emulgel in patients with skin mycoses may be investigated in the future. Further, this study showed an innovative approach to utilize TOH-containing emulgel as a topical application for treating fungal infections as being in accordance with the growing use of emulgels in cosmeceutical and pharmacological products.™ 20, glycerin, propylene glycol, allantoin, phenoxyethanol, Acridine Orange/Ethidium Bromide (AO/EB), Sodium Lauryl Sulfate (SLS), and croton oil were purchased from Sigma-Aldrich . SEPIGEL 305™ was purchased from Seppic Inc. . Triethanolamine was purchased from Dow Chemical (Thailand). Deionized (DI) water used in all experiments was produced from in-house Milli-Q® IQ 7000 Ultrapure Water System . Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA) were purchased from Oxoid Ltd. . Roswell Park Memorial Institute 1640 medium (RPMI-1640), Fetal Bovine Serum (FBS), Penicillin-streptomycin (Pen-strep), Phosphate-Buffered Saline (PBS), and Trypsin-EDTA were purchased from Gibco™ . MTT -2,5-diphenyl tetrazolium bromide) was purchased from Bio Basic Inc. (Canada). CytoPainter MitoGreen (#ab176830) was purchased from Abcam. CytoTox 96® Non-Radioactive Cytotoxicity assay for measuring the release of lactate dehydrogenase (LDH) was purchased from Promega . All the reagents used in this study were of high purity or HPLC grade.Tryptophol , voriconazole , absolute ethanol (EtOH), Tweenet al.9. Optimization of all the emulgel formulations were performed by evaluating the effect of different concentrations of excipients based on visual changes in appearance, homogeneity, consistency, viscosity, stability, and any change in the physical characteristics prior to manufacturing the final formulations. The compositions of all the optimized emulgel formulae are provided in Table The emulgel formulations were prepared with either tryptophol or voriconazole according to Kitisin ™. TOH at 100 µM and 1000 µM or VOZ at 1000 µM were accurately prepared and added to the emulgel base and vigorously mixed until a smooth emulgel was formed9.The emulgel base was manufactured with a constant mechanical stirring of the gel-based SEPIGEL 30523.The visual appearance of manufactured emulgel formulations were examined for their physical appearances including color, homogeneity, consistency, and phase separation9. The formulated emulgel samples were left for equilibrium (30 min) at 25 °C. Measurements with the spindle were determined at 100 rpm for 10 min. The viscosity reading of each sample was recorded. Moreover, the pH of all manufactured emulgel formulations was measured by a digital pH meter 23. Prior measurement, pH meter was calibrated using standard pH buffers at 4, 7, and 10. Then, the pH for all the emulgel formulations was recorded. The pH meter probe was washed after each measurement with DI water and 70% (v/v) EtOH. Data were analyzed in tripiclate.The viscosity of manufactured emulgel formulations was evaluated at room temperature using a Brookfield viscometer  with a volume sample of 0.5 g24. To measure the spreadability of emulgel samples, 1 g of each formulated emulgel was placed onto the ground fixed glass plate and covered with another glass plate and placed on a wooden block. A definite load of about 20 g was then placed on the top of the slides for 1 min to provid the basis for the slip and drag characteristics of the emulgel. The time in seconds required to separate the two slides was recorded and analyzed in triplicate. The spreadability was calculated using the following equation: S = m * l/t; where S stands for spreadability, m stands for weight tied on upper slide; l is the length of glass slides; t, the time (s) requires to separate the slides completely25.The spreadability of manufactured emulgel formulations was investigated to determine the competence of each emulgel formulation, which is able to spread evenly after smeared on the affected skin9. For centrifugation test, approximately 10 g of sample was centrifuged at 9000 rpm for 30 min. In addition, 2 g of each prepared emulgel samples was subject to a temperature cycle test by storing in 24 h/a cycle at − 4 °C (8 h) and 40 °C (16 h) for 10 cycles. After cooling-heating and freeze-thaw, the emulgel samples were evaluated for stability changes. All measurements were performed in triplicate. Physical stability of these emulgels formulations was evaluated at the end of these tests.Physical stability studies were investigated for all the formulations using using centrifugation and temperature cycle testsScedosporium apiospermum (CBS 117410), S. boydii (CBS 120157), and Lomentospora prolificans (CM324) used in this study were kindly provided by Dr. Ana Alastruey-Izquierdo 9. Aspergillus fumigatus  was kindly provided by Asst. Prof. Dr. Marut Tangwattanachuleeporn 10. Bipolaris maydis was kindly provided by Dr. Songbai Zhang, 26. Microsporum canis11, M. gypseum12, Trichophyton tonsurans11, Talaromyces marneffei13, Mucor spp.13, Candida tropicalis (unpublished data), Fonsecaea pedrosoi (unpublished data), and Rhizopus spp. (unpublished data) used in this study were clinical or environmental isolates that previously collected and identified from our laboratory.A. fumigatus , Candida albicans , C. albicans , C. dubliniensis , T. mentagrophytes (ATCC MYA-4439), T. rubrum (ATCC MYA-4438), T. verrucosum (ATCC 42898), Epidermophyton floccosum (ATCC 52066), Acremonium recifei (ATCC 36328), Exophiala dermatitidis (ATCC 76204), Sporothrix schenckii (ATCC 58251), Malassezia furfur (ATCC 12078), T. asahii (ATCC 90039), Cryptococcus neoformans (ATCC 32045), C. gattii (ATCC 56992), Lodderomyces elongisporus (ATCC 11503), and Beauveria bassiana (ATCC 74040) were provided by the American Type Culture Collection (ATCC) . Fungal strains were cultured in SDA for yeasts at 37 ± 1 °C and PDA for molds at 25 ± 1 °C.In-vitro antifungal activity studies of manufactured emulgel formulations were evaluated using the agar spot assay according to Horváth et al.27 for yeasts and according to Muangkaew et al.28 for filamentous fungi with some modifications. Briftly, ten µl of each fungal conidia at concentration about about 5 × 106 conidia/ml were mixed with 10 µl of each manufactured emulgel formulations (TOH at 100 or 1000μM or VOZ at 1000 μM) and spotted on the surface of SDA plates. Then, the plates were incubated at 37 ± 1 °C for 14 days. The colony diameter (mm) were measured in millimeters using a vernier caliper9. Uninoculated SDA plate was used as a negative control. The experiment was performed in triplicate.2 at 37 °C. Measurements of cell viability and cytoxicity after TOH treatments were carried using MTT9, CytoPainter MitoGreen, LDH9, and dual AO/EB staining assays. As previously described, HFF-1 were seeded at a concentration of 1 × 105 cells/ml in a 96 well plate and after 24 h were treated with 100 μl of TOH at 100 or 1000μM or VOZ at 1000 μM or with absolute EtOH and RPMI 1640 at 1% (v/v) as diluent controls. Untreated HFF-1 cells were used as negative controls (100% viable). After incubation for 24 h, 10 μl of 0.5 μg/μl MTT solution or 50 μl of CytoTox 96® reagent were added and measured spectrophotometrically at 540 nm or 490 nm, respectively . To label cell viability and toxicity, treated HFF-1 cells were stained with 100 μl of MitoGreen indicator or 25 μl of 100 μg/μl AO/EB solution, respectively. Cellular morphology and apoptotic cell numbers were examined by a Zeiss Axio Imager fluorescence microscope . All of the experiments were performed in triplicate.Human foreskin fibroblasts (HFF-1) cell line (ATCC #SCRC-1041™) was used to investigate cell viability and cytotoxicity after TOH treatments. HFF-1 cells were cultured in RPMI-1640 supplemented with 10% FBS and 100 U/ml of Pen-strep and incubated under humidified conditions with 5% CO22. Briefly, all mice were topically treated with various concentrations of manufactured emulgel formulations  that do not cause necrosis of the epidermis. Five percentage of SLS and croton oil were used as irritants. Commercial shielding cream was applied as controls. For single irritation, 15 μl of the irritants were put on the 8 mm Finn Chambers on Scanpor tape , which were then applied on the dorsal skin of the animals. Polyurethane film  was used to fix them and Finn Chambers were then removed after 24 h post-application. For cumulative irritation, the procedure was repeated once a day for 1 and 2 weeks. After removing the Finn Chambers, mice were sacrificed, and skin samples were collected. The specimens were fixed in 10% buffered formalin and processed for haematoxylin and eosin (H&E) stain. All skin sections were examined with a light microscope . All animal experiments were conducted in accordance with the Animals for Scientific Purposes Act, B.E. 2558 (A.D. 2015), Thailand. Experiments on mice were approved by Institutional Animal Care and Use Committee (IACUC) of the University of Phayao, Thailand .Female BALB/c mice were purchased from Nomura Siam International Co, Ltd. (Thailand) and used to evaluate skin irritation of manufactured emulgel formulations. Each group included 5 animals  The skin irritation mouse model was produced and validated as described previously with some modification29. A single irritation test of the manufactured emulgel formulations  were put on the 8 mm Finn Chambers on the back of arm skin of the volunteers and fixed with polyurethane film. Commercial shielding cream was applied as controls. Finn Chambers were then removed after 24 h post-application. Treated areas of volunteer skin were observed immediately and 72 h after removing Finn Chambers. All in vivo studies with healthy volunteers are complied with the 2013 Declaration of Helsinki and was approved Ethics Committee (EC) at the Faculty of Tropical Medicine, Mahidol University, Thailand . Written informed consent was obtained from all the participants.To evaluate the sensitization potential of manufactured emulgel formulations toward human skin, a skin irritation test was performed by 30 healthy volunteers® with a hydration probe and a TEWL probe 30. After manufactured emulgel treatments, measurements of the skin hydration and TEWL were performed in 2 × 2 cm squares on the middle forearms, next to each other, with an interval of 5 s between each measurement. Three measurements were taken in each square.To evaluate the effects of manufactured emulgel formulations on human skin physiological functions including skin hydration and trans-epidermal water loss (TEWL), treated skins of 30 healthy volunteers with manufactured emulgel formulations were investigated using DermaLabP-value of < 0.05 was considered as statistically significant using an independent t-test or analyses of variance (ANOVA) followed by Tukey’s multiple comparison test.All the data was statistically analyzed using GraphPad Prism, version 6.05, . All experiments were performed in triplicate. Data were expressed by mean ± SD. A All animal experiments were conducted in accordance with the Animals for Scientific Purposes Act, B.E. 2558 (A.D. 2015), Thailand. Experiments on mice were approved by Institutional Animal Care and Use Committee (IACUC) of the University of Phayao, Thailand . Human studies were approved by Ethics Committee (EC) at the Faculty of Tropical Medicine, Mahidol University, Thailand .Informed consent was obtained from all human subjects involved in the study.


Advancements in neurosurgical visualization have been made possible by the introduction of the operating microscope (OM) and the emergence of exoscopic technology (EX). Both OMs and EXs provide enhanced magnification and illumination, but they come with their own set of advantages and disadvantages. OMs provide high-quality magnification and illumination and have been used successfully in a variety of surgical procedures. They can be customized to fit the specific needs of the surgeon and are a well-established technology. However, they can be bulky, expensive, and cause discomfort during extended procedures. EXs provide high-definition magnification and illumination, improved depth perception and ergonomics, and can be cost-effective. They can be customized to fit the specific needs of the surgeon and can be made using locally available materials, reducing the need for expensive imports. However, they may require adjustment and have a learning curve for surgeons who are used to operating with OMs. Additionally, they may have limited availability in some healthcare settings. The choice between OMs and EXs will depend on the specific needs of the surgeon and the healthcare setting. The integration of 3D EX systems has revolutionized neurosurgery, offering improved depth perception and ergonomics. EX's cost-effectiveness addresses accessibility concerns, making it an attractive alternative, particularly for low and middle-income healthcare settings. The exoscope seems to be a safe alternative compared to an operative microscope for the most common brain and spinal procedures. The exoscope may help expand access to neurosurgical care and training worldwide. In conclusion, both technologies have their own set of advantages and disadvantages, and the choice between them will depend on the specific needs of the surgeon and the healthcare setting. Advancements in visual technology have significantly impacted the field of neurosurgery, particularly with the introduction of the operating microscope (OM) in the late 1950s. This innovation gained momentum in microsurgery by 1962 and extended to spinal surgery in 1977, offering high-quality magnification, illumination, and depth perception [In response to these challenges and the quest for enhanced surgical visualization, a novel solution emerged in the form of exoscopic technology (EX). This revolutionary system involves positioning a high-definition scope externally, projecting an enlarged view of the surgical field onto a 2D  or 3D  high-resolution monitor. Recent advancements have led to the development of 3D EX systems, which significantly enhance depth perception for surgeons [As ongoing research delves into the capabilities and outcomes of 3D EX technology across various neurosurgical procedures , it becomes increasingly crucial to critically evaluate its effectiveness and safety compared to the traditional OM. This evaluation is paramount in paving the way for the potential integration of the 3D EX as a viable alternative in the field of spinal surgery. The utilization of EXs in the realm of neurosurgery introduces a range of models, each carrying distinct advantages. Particularly notable are models like ViTOM® and ORBEYE®, which offer features such as robotic arms and advanced optics. Diverse modes, including HD 3D, HD 2D, and 3D 4K, provide neurosurgeons with adaptable visualization options catering to specific needs. Comparative assessments of video quality between EXs and traditional OMs yield varying results, with certain studies favoring EXs for superior visual quality and enhanced surgeon comfort. One area where EXs excel is in ergonomic design, contributing to reduced surgeon fatigue and optimized efficiency . The avaFurthermore, EXs contribute significantly to the educational aspect of neurosurgery by offering detailed anatomical visualization . This isFuture research in the domain of EX neurosurgery holds the potential to significantly shape the trajectory of surgical innovation. Comparative studies are essential to comprehensively assess the extended outcomes of EX procedures, shedding light on factors such as long-term patient recovery and recurrence rates. Expanding the scope of investigation beyond spinal surgeries to encompass a diverse array of neurosurgical procedures is crucial to fully understand the versatility and efficacy of EX techniques. Additionally, an emphasis on patient-centric parameters, such as the duration of the surgical procedure, intraoperative blood loss, incidence of complications, management of postoperative pain, overall quality of life, and levels of patient satisfaction, will yield valuable insights into the comprehensive ramifications of EX interventions -5. InvesIn conclusion, the evolution of EX techniques represents a significant stride in the realm of spinal surgery, offering a potential solution to the limitations posed by traditional operating microscopes. The integration of 3D EX systems enhances surgical visualization and holds promise for various neurosurgical procedures, including spinal surgeries. Comparative assessments highlight the favorable outcomes of EX in terms of ergonomics, surgical efficiency, and educational benefits. Moreover, the cost-effectiveness and adaptability of EX address accessibility challenges, particularly in low- and middle-income settings. As ongoing research advances our understanding of EX technology, the feasibility of EX spinal surgery emerges as a necessity in resource-constrained environments. This transformation not only improves surgical techniques but also paves the way for a more inclusive and accessible future in spinal surgery.


This inactivation stops the destruction complex from phosphorylating β-catenin. As a result, β-catenin first builds up in the cytoplasm and then migrates into the nucleus, where it binds to the Lef/Tcf transcription factor to activate the transcription of more than 50 Wnt target genes, including those involved in cell growth, survival, differentiation, neurogenesis, and inflammation. The treatments that are currently available for neurodegenerative illnesses are most commonly not curative in nature but are only symptomatic. According to all available research, restoring Wnt/β-catenin signaling in the brains of patients with neurodegenerative disorders, particularly Alzheimer's and Parkinson's disease, would improve the condition of several patients with neurological disorders. The importance of Wnt activators and modulators in patients with such illnesses is to mainly restore rather than overstimulate the Wnt/β-catenin signaling, thereby reestablishing the equilibrium between Wnt-OFF and Wnt-ON states. In this review, we have tried to summarize the significance of the Wnt canonical pathway in the pathophysiology of certain neurodegenerative diseases, such as Alzheimer's disease, cerebral ischemia, Parkinson's disease, Huntington's disease, multiple sclerosis, and other similar diseases, and as to how can it be restored in these patients.Defective Wnt signaling is found to be associated with various neurodegenerative diseases. In the canonical pathway, the Frizzled receptor (Fzd) and the lipoprotein receptor-related proteins 5/6 (LRP5/LRP6) create a seven-pass transmembrane receptor complex to which the Wnt ligands bind. This interaction causes the tumor suppressor adenomatous polyposis coli gene product (APC), casein kinase 1 (CK1), and GSK-3 It is a key pathway that regulates cellular functions such as proliferation, translocation, differentiation, genetic stability, apoptosis, and stem cell renewal. This evolutionarily conserved pathway is also involved in the determination of cell polarity and cell fate during normal embryogenesis, in addition to maintaining tissue homeostasis in adults ..β [β enzβ of the destruction complex phosphorylate the N-terminal of cytoplasmic β-catenin. The phosphorylated β-catenin is ubiquitinated by the E3 ligase β-transducin repeat-containing protein (β-TrCP), thereby targeting it for ubiquitin-mediated proteasomal degradation. Hence, the nuclear translocation of β-catenin is blocked, and there is no expression of target genes (In the absence of Wnt (Wnt-OFF state), CK1 and GSK-3et genes .β-catenin and TCF/LEF transcription factors and hence is known as the β-catenin-independent pathway. It is subdivided into two pathways: the planar cell polarity pathway (PCP) and the Wnt/Ca2+ pathway. The noncanonical pathway mainly determines cell polarity [The noncanonical Wnt pathway is independent of polarity . A subclThe PCP pathway is commonly known as the Wnt/PCP pathway or the Wnt/JNK pathway. In this pathway, activated Dsh transduces the signal to small G-protein molecules (GTPases) like Rho/Rac and activates them. Subsequently, c-Jun-N-terminal kinase (JNK) also gets activated. Because of this, reorganization of microtubules and actin filaments occurs .2+ pathway, interaction occurring between the Wnt ligand and Fz receptor stimulates the intracellular release of Ca2+ from the endoplasmic reticulum (ER) via phospholipase-c (PLC), which increases the intracellular level of Ca2+ and leads to decreasing the level of cyclic guanosine monophosphate (cGMP) [2+/calmodulin-dependent protein kinase II (CamKII) as well as protein kinase C (PKC) which are Ca2+-sensitive kinases. This leads to the nuclear translocation of the nuclear factor of activated T cells (NFACT) and cAMP response element-binding protein (CREB), which later binds to its target gene and regulates gene expression (In the Wnt/Cae (cGMP) . This fupression .β-catenin, and transducers of the “noncanonical” Wnt pathway, i.e., Wnt, RhoA, and RhoA kinase (RhoAK), can cooperate in order to control the expression of P-glycoprotein (Pgp) in the cells lying in the blood-brain barrier (BBB) [Caenorhabditis elegans, a switch from noncanonical to canonical signaling leads to the termination of long-range migration of QR neuroblast descendants. The noncanonical pathway acts via PIX-1/RhoGEF, while the canonical pathway directly activates the Slt–Robo component EVA-1/EVA1C and the Rho GTPase-activating protein RGA-9b/ARHGAP required for inhibiting migration. These studies show that the cross-talk between canonical and noncanonical Wnt signaling occurs via the antagonistic regulation of Rho GTPases which are responsible for cell migration.Lately, it has been found that the transducers of the “canonical” Wnt pathway, i.e., GSK3 and er (BBB) . Any diser (BBB) . StudiesWnt signaling plays a very important role in axon pathfinding, dendritic development, and synapse assembly by recruiting presynaptic and postsynaptic elements in both the central nervous system (CNS) and the peripheral nervous system (PNS) . Wnt sigIn a study, Wnt7a knockout mice were shown to possess fewer neural progenitor cells (NPCs), leading to an increase in the length of cell cycles, a decrease in reentry into the cell cycle, and impairment of neuronal differentiation . This stβ and restoring MAP-1B, which leads to the destabilization of axonal microtubules and shields them from nocodazole depolymerization [β-catenin promote dendritic arborization, whereas sequestered endogenous β-catenin leads to a reduction in dendritic complexity.Dsh enhances the differentiation of neuroblastoma 2A cell (N2A cell) and enables neuronal outgrowth. One of the three domains of Dsh is the N-terminal DVL domain (DIX) which is required for neuronal remodeling in N2A cells. Hence, DIX is crucial for N2A cells to differentiate . Studiesrization . Also, iβ-catenin signaling enhances neurogenesis in progenitor cells [In a recent study, the effect of the Wnt signaling pathway on neural progenitor cells isolated from the cerebral cortices of newborn mice was observed. It was found that the activation of Wnt canonical signaling reduced the expression of Hes1 and Hes5 genes in differentiating cell cultures, thereby interfering with the Notch pathway. This suggests that during the period of postnatal neural development, Wnt/or cells .An inflammatory response in the brain or spinal cord is referred to as neuroinflammation. It is associated with the production of cytokines, chemokines, reactive oxygen species (ROS), and disruption of the permeability of the blood-brain barrier (BBB). Endothelial cells, peripherally derived immune cells, and glial cells like microglia and astrocytes all contribute to the production of cytokines, chemokines, and ROS which are important mediators of neuroinflammation . Wnt sigκB) pathway [β-catenin pathway and the NF-κB pathway which becomes a crucial factor in both acute and chronic inflammation. The activity of the NF-κB pathway is reduced by the activation of the Wnt/β-catenin pathway [β protein, which stimulates β-catenin breakdown and positively modulates the NF-κB pathway [Neuroinflammation is partially caused by the activation of the nuclear factor-kappa B , and cytosolic β-catenin is stabilized. The expression of target genes is induced as a result of stabilized β-catenin translocating into the nucleus and interacting with TCF/LEF. But when LRP5/6 and Fzd bind to Dickkopf (DKK) and soluble Frizzled-related protein (sFRP), respectively, they block the formation of the LRP-Wnt-Fz complex in response to Wnts [Two-thirds of dementia cases are associated with Alzheimer's disease (AD), which is characterized by gradual memory loss and cognitive impairment that makes performing daily tasks challenging. Wnt proteins are secreted glycoproteins that interact with the low-density lipoprotein receptor-related protein 5 (LRP5) or LRP6 and the extracellular cysteine-rich domain of the Frizzled (Fzd) receptor family to activate the canonical Wnt/ pathway . When Wn to Wnts . Synapseβ activity and decreased cytoplasmic β-catenin levels as a result of this suppression of Wnt signaling by DKK1 [According to a number of studies, the canonical Wnt antagonist DKK1 is upregulated in the brains of AD patients and AD mouse models. By interacting with LRP5/6 Wnt coreceptors, DKK1 prevents Frizzled and LRP5/6 from recognising Wnt proteins, hence inhibiting canonical Wnt signaling. The brains of AD patients show increased GSK-3 by DKK1 .β-catenin signaling system regulates the establishment of the BBB and cerebrovascular development [β activity and obstructing the canonical Wnt pathway. The overexpression of DKK1 was observed in every case of Alzheimer's disease, Parkinson's disease, and cerebral ischemia [During embryonic development, the Wnt/elopment . Clinicaischemia . In an eischemia .β activity and low Wnt/β-catenin signaling are correlated with high DKK1 expression [β-catenin dysregulation has been linked to LRRK2 and Parkin mutations. Wnt signaling controls a variety of cellular processes, including the growth and maintenance of midbrain dopaminergic (mDA) neurons. Due to the fact that PD is brought on by the degradation of these neurons, they are of great interest in regenerative medicine [β-catenin signaling along with glial cells regulate nigrostriatal DAergic neuronal health, protection, and regeneration. Therefore, it can be inferenced that Wnt/β-catenin signaling is capable of mediating a full neurorestorative programme in PD. Parkinson's disease (PD) has no known cure.Numerous genetic flaws that produce familial and idiopathic types of Parkinson's disease (PD) have been discovered over the past 20 years. Parallel to this, it has become clear that Wnt signaling pathways are essential for the adult brain's normal functioning and that these pathways are dysregulated in neurodegenerative diseases. Wnt signaling pathways are partially responsible for the disruption of cell biological processes in PD, and proteins encoded by PARK genes have been found to alter Wnt signaling. This raises the possibility of targeting Wnt signaling pathways in order to treat PD . In Parkpression . Additiomedicine . In one β-catenin. Godin et al. specifically discovered an abnormal cytoplasmatic β-catenin build-up in the phosphorylated form that is unable to move in the nucleus and activate the transcription of target genes by using several in vitro and in vivo HD models [β-catenin accumulation. The results showed that wild-type Htt functions as a scaffold protein, promoting β-catenin phosphorylation by the β-catenin destruction complex [Huntington's disease (HD) is a neurodegenerative disorder brought on by an increase in the number of CAG triplets in the gene that codes for the Huntington protein (Htt). It is characterized by selective neuronal death in the brain. According to recent data, the downregulation of the Wnt pathway in HD is correlated with lower transcription of Wnt prosurvival genes and consequently with higher levels of apoptosis. The Wnt canonical pathway contributes to HD progression by interfering with the turnover of D models . In one  complex .β-catenin pathway [Schizophrenia is an extremely complex syndrome that involves dysregulation in cognition, behaviour, and emotion. According to recent research, schizophrenia's pathophysiology may be influenced by immunological dysfunction and central nervous system (CNS) inflammation. Neuroinflammation in schizophrenia is correlated with disruption of the Wnt/ pathway .β-catenin coactivator, functions as one of the major transcriptional mediators of the canonical Wnt signaling [Despite the fact that no Wnt ligands have been discovered to be genes of interest in genome-wide association studies (GWAS) or other linkage studies, Wnt1 has been reported to be upregulated in the schizophrenic brain, and it has been found that some genes are linked to disease susceptibility and are essential elements of the Wnt signaling pathway . The disignaling . TCF4 haignaling . Additioβ-catenin signaling. In genome investigations of mutations linked to schizophrenia, DKK1, DKK3, and DKK4 have all been identified as genes of relevance, along with the DKK1 coreceptor KREMEN1 [Genetic tests have also linked DKK proteins to schizophrenia. Extracellular DKK proteins compete with Wnts for binding to LRP5/6 receptors and therefore damage the Fzd-LRP5/6 complex that starts Wnt/ KREMEN1 . In one  KREMEN1 .β-catenin/Tcf signaling pathway has been recognised as a crucial factor in the control of oligodendrocyte development and remyelination [Among other signaling pathways, the Wnt/lination . Numeroulination .β-Catenin is a component of the cadherin complex and has the ability to control cell-cell adhesion and cell migration. Any imbalance in the level of β-catenin may lead to serious problems that can further trigger the onset of multiple sclerosis [β-Catenin-overexpressing mice showed a considerably lower g-ratio when compared to wild-type mice [clerosis . In one ype mice .Wnt signaling has also been demonstrated to contribute to the emergence of persistent pain in multiple sclerosis. One study found that Wnt signaling pathways are activated abnormally in the SCDH of EAE mouse models, which may contribute to the development of chronic pain associated with the disease . Thus, oβ-catenin signaling pathway. The degeneration of motor neurons in ALS is caused by the significant buildup of β-catenin in those cells [β-catenin signaling activation. The conversion of microglia to a proinflammatory phenotype is also encouraged by the activation of Wnt/β-catenin signaling. Wnt/β-catenin signaling has also been found to be associated with delayed maturation of regenerated oligodendrocytes. It has been found that Wnt/β-catenin signaling encourages oligodendrogliogenesis and the restoration of degenerative myelin structure in preexisting oligodendrocytes [Studies related to amyotrophic lateral sclerosis (ALS) have demonstrated a correlation between glial cell growth and aberrant activation of the Wnt/se cells . In ALS,drocytes .β-catenin signaling activation causes glial cell growth and neuronal degeneration. The Wnt/PCP routes  are interdependent, which makes this signal network more complex. Secondly, the MuSK (muscle-specific kinase) receptor in SOD1-G93A mice has been the subject of a growing body of research, and this research suggests that gene therapy is crucial for treating ALS. Moreover, the diverse LRP4 antibody patterns across various ethnic groups point to considerable individual variations in the ALS disease processes [The malfunctioning of the complete motor unit, from axon terminal denervation to motor neuron death, appears to be mediated by Wnts, Wnt receptors, and other essential elements of the Wnt pathways . Anotherrocesses . We willβ-catenin pathway so as to treat various neurodegenerative diseases are summarized in There are currently a few methods for halting or slowing the progression of PD, AD, and ALS . AlthougThe communication between glial cells and neurons is mediated by Wnt1 and Wnt1 agonists and is responsible for providing a critical neuroprotective mechanism against oxidative stress, growth factor deprivation, and inflammation. Microglial and astrocytic cells in the brain are responsible for the primary secretion of Wnt1 and Wnt1 agonists . These tβ-catenin signaling. It is interesting to note that natriuretic hormone acts as a Wnt agonist in DA neuron-like cultures, enhancing signaling where the system is essentially not activated [β-catenin pathway in colorectal cancer cells (where this pathway is otherwise constitutively active). ANP essentially acts as a Wnt modulator that can restore the equilibrium between the Wnt-OFF and Wnt-ON states to successfully reverse aberrant Wnt signaling in a number of clinical situations. Endogenous ANP appears to be crucial for brain development, synaptic transmission, and neuroprotection in the CNS, where it is abundantly generated and mostly secreted by glial cells.A few years ago, it was discovered that the hormone atrial natriuretic peptide (ANP), which is produced by the heart and is essential for maintaining cardiovascular homeostasis, has antiproliferative effects on colorectal cancer cells by influencing Wnt/ctivated . On the β-catenin pathway. The most likely method by which DKK1 inhibits Wnt signaling seems to be the breakdown of the Fzd/LRP6 complex. As a negative regulator of the Wnt/β-catenin pathway, DKK1 is one of the most studied inhibitors to modulate Wnt in neurodegenerative diseases, including AD. DKK1-neutralizing antibodies can counteract the effects of acute exposure to Aβ oligomers. Aβ oligomers have been demonstrated to trigger DKK1 expression and synaptic site loss in mouse brain slices from AD patients [β-mediated synapse loss in corticohippocampal cells taken from transgenic AD mice [Dickkopf (DKK) proteins are LRP5/6 antagonists and act as inhibitors of the Wnt/patients . Recent  AD mice .β (GSK-3β), one of the five enzymes able to phosphorylate glycogen synthase, was initially isolated from skeletal muscle in 1980. By regulating a number of signaling pathways, including the Wnt/β-catenin pathway, GSK-3β governs several important cellular processes in the brain [β is a component of the destruction complex in the Wnt-OFF state, where it quickly phosphorylates extra β-catenin before the ubiquitin-proteasome pathway degrades it.Glycogen synthase kinase-3he brain . It is cβ results in the stability of β-catenin and activates signaling (Wnt-ON state). The GSK inhibitors SB216763 and LiCl (lithium chloride) have been shown to activate Wnt/β-catenin signaling in a cellular model of Parkinson's disease [β-catenin pathway and has recently been found to improve the process of neurogenesis in the adult hippocampus of the corresponding mouse model of Alzheimer's. Lithium and VPA have been evaluated for their efficacy in treating neurological diseases in clinical trials. However, their efficacy was not related directly to Wnt/β-catenin [β. In one of the studies, MK-801 has been shown to increase Wnt3a levels in hemiparkinsonian rats to inhibit GSK-3β, which in turn activates Wnt/β-catenin signaling and boosts hippocampus neurogenesis [On the other hand, suppression of GSK-3 disease . It has -catenin . Lithiumogenesis .(1) Triazine Derivatives (Cholinesterase Inhibitors). Triazines have received great attention as cholinesterase inhibitors. The formation of Aβ fibrils is promoted by a type-B carboxylesterase enzyme known as acetylcholinesterase (AChE). AChE is responsible for the hydrolysis of acetylcholine (Ach) along with some other choline esters and is present in the synaptic cleft. This interaction between Aβ fibrils and AChE is responsible for the production of the AChE-Aβ complex which increases Aβ-dependent neurotoxicity [β neurotoxicity which leads to the assumption that cholinesterase inhibitors should have a positive impact on the Wnt/β-catenin pathway. Cholinesterase inhibitors have already been established as one of the symptomatic methods for the treatment of Alzheimer's disease [toxicity . Subsequ disease . Owing t(2) Sodium Selenate. Selenium is a crucial trace element for the brain, playing a role in many CNS processes, such as memory and cognition [β-catenin [β plaque development [ognition . There iognition . It perf-catenin . Sodium -catenin . Selenatelopment .(3) Statins. Recent studies have reported that statins, a class of drugs used to treat hyperlipidemia in clinical settings, exhibit neuroprotective characteristics and are therapeutically beneficial for neurological illnesses like Parkinson's disease (PD), Alzheimer's disease (AD), and all forms of dementia [in vitro and in vivo conditions, simvastatin can also encourage neurogenesis [β-catenin signaling [dementia . Simvastogenesis . Althougignaling .β-catenin signaling.Several natural compounds have been identified as modulators of Wnt signaling . Numerouβ-catenin pathway has recently been explained further by Palomera-Avalos et al. [β-catenin signaling pathway was activated, leading to metabolic stress in the SAMP8 mouse model [A polyphenolic phytoalexin called resveratrol is present in a variety of foods, including blueberries, grapes, peanuts, and wines. Resveratrol has been shown to have antioxidant, cardioprotective, neuroprotective, and anticancer properties . Resveras et al. , which sse model .Curcuma longa. Traditional treatments like Ayurvedic and Chinese traditional medicine frequently employ Curcuma longa extract. Curcumin has intriguing biological properties, like anti-inflammatory, antioxidant, and neuroprotective [β and induce the expression of β-catenin to activate Wnt/β-catenin signaling has also been established. Activation of the Wnt/β-catenin pathway using curcumin can be made possible by using modern delivery strategies that enhance neuroavailability, as in the case of curcumin nanoparticles [Curcumin, a polyphenol, is obtained from the rhizome of the ginger family flowering plant otective . It can articles .Panax ginseng, ginsenoside Rg1, may exert neuroprotective effects in PD models both in vitro and in vivo through the Wnt/β-catenin pathway and has been recommended as a potential treatment strategy. Another ginsenoside, Rb1, was shown to be a robust inhibitor of synuclein fibrillation and toxicity. In addition, it has been reported to exhibit a high capacity to disintegrate fibrils and prevent the polymerization of α-synuclein in vitro [According to one of the studies, the principal bioactive component of in vitro .Ginkgo biloba extract, one of the ingredients in Chinese herbal medicines, has been shown to enhance memory along with spatial learning ability in a transgenic mouse model of Alzheimer's [Ginkgo biloba extract and ginkgolide B, one of its primary components, have been reported to activate the Wnt/β-catenin pathway, thereby promoting the development of neurons in neural stem cells [heimer's . It has em cells . InteresRhodiola rosea and has been proposed as a potent neuroprotective agent for the treatment of neurodegenerative diseases, especially PD. It is enriched with several pharmacological properties like being an antioxidative, anticancerous, antifatigue, and anti-inflammatory agent [β-catenin pathway activation. A labdane diterpenoid, andrographolide, is known to activate the Wnt/catenin pathway and drive transcription of Wnt target genes through direct inhibition of GSK-3β by employing a substrate-competitive mechanism. It is an annual herb which has been used as a natural anti-inflammatory compound for thousands of years in China and India with no reported side effects [Salidroside, a phytobioactive compound, is a phenol glycoside obtained from the antifatigue herb ry agent . A recen effects .β-catenin signaling, restoring Wnt/β-catenin signaling can be one of the potential strategies with respect to the development of innovative neurological therapeutics. Wnt signaling controls a variety of biological functions, including cell survival. In addition to being essential for neuronal survival and neurogenesis, Wnt signaling in the brain also controls synaptic plasticity, BBB integrity, and function. Many more such activities are performed by Wnt signaling. For instance, in the case of AD, in the adult hippocampus, synaptic integrity depends on Wnt signaling. Loss of synapses, long-term memory, and deficiencies in synaptic plasticity are brought on by DKK1. So, therefore, reactivating the Wnt pathway can reverse memory deficits and synapse loss.Since various pathogenic pathways in neurological illnesses significantly reduce Wnt/β, β-catenin, APC, TCF7L2, and FZD3, are connected to schizophrenia as well [Most of the neuronal processes that are damaged in neurological disorders would be improved if Wnt signaling was restored in the brains of individuals with brain diseases, especially PD and AD . The tre as well . Accordi as well . Therefoβ-catenin pathway. The evidence from the literature points to the consensus that restoring the Wnt canonical pathway to normal levels has a neuroprotective impact. Therefore, a viable therapeutic approach to prevent the onset of neurodegenerative illnesses might involve the stimulation of Wnt signaling through the exogenous injection of Wnt agonists or its suppression through the use of Wnt antagonists. With the exception of some naturally occurring chemicals like ginkgolide B, there are not many specific blood-brain barrier- (BBB-) permeant Wnt activators which could serve as a potential treatment for PD, AD, or other neurological disorders. The limited brain bioavailability of these compounds may reduce their usefulness. However, the advent of innovative delivery methods, such as drug-loaded nanoparticles or mucoadhesive formulations, established with the intention of enhancing medication neuroavailability, may be the solution to this issue. Additionally, nose-to-brain delivery is a noninvasive technique that enables the transfer of complicated medications to the CNS directly or via nanosized carriers, evading the BBB. To further circumvent the BBB and treat neurodegenerative diseases, exploring hydrogel-based biomaterials or nanoengineered drug-releasing implants could also be a good option. Finally, there are certain issues that need to be addressed regarding the safety of targeting a signal transduction system that is essential for tissue homeostasis and repair. Overstimulating Wnt/β-catenin signaling to accomplish neuroprotection and neurorepair might be tumorigenic because incorrect stimulation of this signaling has been linked to specific forms of cancer. Therefore, the reestablishment of balance between the Wnt-OFF and ON states in patients with neurodegenerative disorders can be achieved by therapeutic administration of Wnt activators or modulators which function in such a manner as to just restore the normal functioning of the Wnt/β-catenin pathway instead of overstimulating it by any means.The development of novel treatment approaches for treating neurodegenerative illnesses may result from a better understanding of the molecular processes underlying their pathophysiology. There is mounting evidence that the onset and progression of neurodegenerative disorders are significantly influenced by changes in Wnt canonical signaling. Overall, we think that the beginning and progression of the majority of neurodegenerative illnesses may be influenced by the loss or gain of function of the Wnt/


Triple-negative breast cancer (TNBC) is a subtype of breast cancer with the highest degree of malignancy and is easily resistant to drugs due to the lack of hormone receptors. Research on the resistance mechanisms in TNBC is particularly important. Keratin 17 (KRT17) is highly expressed in TNBC. Anthracycline doxorubicin (Dox) is a commonly used chemotherapeutic drug for early stage triple-negative breast cancer.This study investigated the role of KRT17 in TNBC-Dox resistance.Immuno-histochemical staining, qPCR, western blotting (WB), and immunofluorescence were used to detect the expression of KRT17 in TNBC-Dox-resistant patients and in TNBC-Dox-resistant MDA-MB-468 and MDA-MB-231. the effect of KRT17 on the proliferation and migration in KRT17 knockdown of TNBC-Dox-resistant cells was determined by the CCK8, clone formation, transwell invasion and wound healing assays were used to determine.KRT17 was highly expressed in the TNBC-Dox-resistant cells. Knockdown of KRT17 significantly reduced the IC50s of TNBC-Dox-resistant and parental strains and also reduced the proliferation and invasion abilities of TNBC-Dox-resistant cell lines. KRT17 regulated the Wnt/β-catenin signaling pathway. The inhibitory effect of KRT17 knockdown on the proliferation and migration of TNBC-Dox-resistant cells was reversed by an activator of the Wnt signaling pathway.KRT17 can inhibit the Wnt/β-catenin signaling pathway, thereby reducing the proliferation and invasion ability of TNBC-Dox-resistant cells. Triple-negative breast cancer (TNBC) is a specific subtype of breast cancer that lacks the estrogen receptor (ER)  is a type I keratin that is involved in multiple biological processes such as tumor growth and metastasis, inflammation, and signal transduction  and LRP5/6 membrane receptors, recruiting scaffold proteins such as Axis Inhibition Protein (AXIN) and Dishevelled Segment Polarity Protein (DVL). The Wnt/β-catenin signaling pathway is also regulated by the Wnt pathway regulator, Adenomatous Polyposis Coli protein (APC). β-catenin is released, enters the nucleus and is activated by binding to the Transcription Factor (TF). Transcription factors initiate epithelial-mesenchymal transition (EMT), growth, and transduction-related gene expression  and MDA-MB-231  were purchased from the ATCC . The 293T cell lines were maintained in our laboratory. The MDA-MB-468, MDA-MB-231, and 293T cell lines were preserved in MEM medium supplemented with 10% FBS. Once the cells reached 60–75% confluency, 50 nM small interfering RNA (siRNA)  of KRT17 was transfected into TNBC cells using Lipofectamine 2000 reagent  according to the manufacturer’s instructions. The cells were harvested after 48 h incubation. The siRNA sequences were as follows: 5’-GCCAACAUCCUGCUACAGAUU-3’, 5’-GCGUGACCAGUAUGAGAAGAU-3’, and 5’-CACCUGACUCAGUACAAGAAA-3’. Stable transfected cell lines were constructed by sub-cloning the most effective siRNAs, siRNA2 and si-NC, into a lentivirus vector (pLKO.1)  resulting in the construction of sh-KRT17 and sh-NC vectors. The shKRT17 and sh-NC vectors were transfected into 293T cells for lentivirus preparation. The viruses were harvested to infect MDA-MB-468 and MDA-MB-231 cells, followed by selection with puromycin.−ΔΔCq method was used to calculate the KRT17 expression levels.According to the manufacturer’s instructions, the total RNA of MDA-MB-468 and MDA-MB-231 cells was extracted using Trizol reagent and reverse-transcribed into cDNA using the PrimeScript RT kit . The SYBR® Premix Ex Taq™ II kit  was used for RT-qPCR. The RT-qPCR cycling conditions were: 95℃ for 10 min, 55℃ for 2 min, 72℃ for 2 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 1 min. The primers used for RT-qPCR analysis were as follows: KRT17 forward primer, 5′-ATCCTGCTGGATGTGAAGACGC-3′ and KRT17 reverse primer, 5′-TCCACAATGGTACGCACCTGAC-3′; GAPDH forward primer, 5′-GTCTCCTCTGACTTCAACAGCG-3’; and GAPDH reverse primer, 5′-ACCACCCTGTTGCTGTAGCCAA-3′. The 2The MDA-MB-468 and MDA-MB-231 cells were lysed according to the manufacturer’s instructions. The protein levels were measured using the BCA protein assay kit. Equal amounts of denatured proteins were transferred to a polyvinylidene fluoride membrane  and incubated  with primary antibodies KRT17 , β-catenin , APC , AXIN1 , GAPDH . The membrane was then incubated with goat anti-rabbit secondary antibody . The blots were visualized using an enhanced chemiluminescence reagent .KRT17  was used to observe the localization of KRT17 in MDA-MB-468 and MDA-MB-231 cells. The Fluorescent In Situ Hybridization Kit  was used to perform the FISH assay, according to the manufacturer’s instructions. The cell nuclei were stained with 4,6-diamidino-2-phenylindole . Images were obtained using a fluorescence microscope .3 cells/well of MDA-MB-468 and MDA-MB-231 were added to a 96-well plate and incubated for 60 min. Ten microliters of CCK8 reagent was added at 0, 24, 48, and 72 h. Optical density was measured at 450 nm using an enzyme-labeled instrument .A total of 2.5 × 105) were placed in the top chamber of the 24-well Transwell plate coated with Matrigel containing 200 µL serum-free medium. The bottom chamber contained complete medium supplemented with 10% FBS. After 48 h, cells were fixed with anhydrous ethanol for 20 min and stained with crystal violet for 15 min. Images were obtained under an inverted microscope .MDA-MB-468 and MDA-MB-231 cells . One-way analysis of variance (ANOVA) and Dunnett’s post-hoc test were performed on more than two groups of data for statistical analysis, and the Student’s t-test was used to analyze the means of the two groups. Statistical significance was set at TNBC patients are resistant to endocrine and hormone therapies due to the lack of endocrine and hormone receptors, however tumor growth and metastasis are mainly controlled by chemotherapy. Dox is a common chemotherapeutic drug used to treat breast cancer. However, after multiple chemotherapy treatments, patients develop resistance to Dox. Previous studies have shown that KRT17 is highly expressed in TNBC tissues  addition in normal and KRT17-knockdown TNBC-Dox-resistant strains. The CCK8 and clone formation demonstrated that KRT17 knockdown significantly reduced the proliferative ability of TNBC-resistant strains in the presence and absence of Dox (Fig. Aberrant Wnt signaling is characteristic of triple negative breast cancer, compared to other subtypes of breast cancer. Numerous studies have shown that TNBC is associated with the activation of the Wnt/β-catenin signaling pathway, the activation of which increases the metastasis and poor prognosis of breast cancer (Geyer et al. Since interfering with KRT17 expression can activate the Wnt/β-catenin signaling pathway, the 20 µM Wnt/β-catenin activator SKL2001 was added to KRT17 knockdown TNBC-Dox-resistant cells. SKL2001 disrupts the interaction of Axin/β-catenin by activating the Wnt/β-catenin signaling pathway. SKL2001 was added to determine whether the Wnt/β-catenin activator can reverse the inhibitory effect of interfering KRT17 on the proliferation and invasion of TNBC-Dox-resistant strains. The results of CCK8 and clone formation showed that the Wnt/β-catenin activator could reverse the inhibition of proliferation of TNBC-Dox-resistant strains Fig. A and B. Owing to the lack of endocrine and anti-HER2 targets for TNBC, most patients are mainly treated with chemotherapy. Dox is a chemotherapeutic drug commonly used in the treatment of early breast cancer. Treatment efficacy is reasonable in the early stages of cancer; however, it is prone to drug resistance in the later stages, and drug resistance is a key factor for tumor metastasis. Merkin et al. found that KRT17 is highly expressed in TNBC and HER2 receptor-negative breast cancer patients (Merkin et al. The Wnt/β-catenin signaling pathway is a well-established classical signaling pathway that controls embryonic and cancer development. Recently, the Wnt/β-catenin signaling pathway has been shown to be involved in stemness maintenance (Gangrade et al. The relationship between KRT17 and Wnt/β-catenin signaling pathway was predicted using the STRING database. The expression of APC and AXIN1 proteins was increased which is important as APC and AXIN1 proteins are repressors of the Wnt/β-catenin signaling pathway. Ran et al. also found that KRT17 could regulate the Wnt/β-catenin signaling pathway in colon cancer cells by regulating APC (Ji et al. In conclusion, KRT17 knockdown inhibited drug resistance, proliferation, and migration of TNBC-Dox cells by inhibiting the Wnt/β-catenin signaling pathway.