The thermal behavior of the prepared samples was investigated under heating in oxidative environment using thermogravimetric analysis. The hardness was measured using the Shore D hardness test. To evaluate the flammability of the samples, the ignition temperature and time-to-ignition were determined. It was concluded that sodium bicarbonate in the studied concentrations (10 and 15 wt%) is not appropriate for use as a filler capable of improving the thermooxidative stability and reducing the flammability of epoxy polymers. The improvement in the thermal properties can be achieved by using the combination of boric acid and multi-walled carbon nanotubes as fillers. The thermooxidative destruction of the samples filled with boric acid passes more slowly and more evenly via the formation of B Epoxy resins are widely used in industry as construction materials, adhesives, coatings, and matrices for advanced composites because of their superior mechanical and electrical properties, chemical resistance, and resistance to moisture ,2,3,4. TTo reinforce and modify the epoxy matrices, various nanofillers of organic and inorganic nature are used ,15,16,173) is non-toxic and inexpensive material; when heated to a temperature of 60 °C it begins to decompose into sodium carbonate, carbon dioxide, and water:Sodium bicarbonate  is another inorganic compound known as a flame retardant additive for wood, paper, and cotton [Boric acid  forms a glassy film on the burning surface of the polymer, thereby inhibiting diffusion of flammable gases into the flame. Boric acid can provide a greater effect on the improvement of the thermal stability of epoxy composites when it is used in combination with other materials. In our earlier study , it was The aim of this work is to study the effects of multi-walled carbon nanotubes, boric acid, and sodium bicarbonate separately, and their combination on the thermooxidative degradation and flammability of the epoxy composites.−1. The hardener used was Polyethylenepolyamine (PEPA), supplied by ZAO Uralkhimplast . The particles of milled sodium bicarbonate  and boric acid  with a size less of than 64 μm were used as fillers.The epoxy composites were manufactured by using the epoxy resin DER-331 supplied by Dow Chemical , which had the epoxy group content 5200–5500 mmol/kg and average molecular weight of 340 g·molThe MWCNTs (4060 grade) obtained by Shenzen Nano-Tech Port Co., China, have the diameter of 15–40 nm and the length of 3–5 µm .The epoxy resin was heated to 55 °C, and then the required amount of the fillers was added into epoxy resin using mechanical stirring for 10 min; afterwards, PEPA was added into mixture. The ratio of epoxy resin and PEPA was 6:1 by mass. The curing of the samples was conducted at room temperature (25 ± 2 °C) for 24 h. The formulations of the studied epoxy composites are given in −1 in argon–oxygen mixture with an argon flow rate of 180 mL/min and an oxygen flow rate of 20 mL/min. The samples of approximately 5 mg were placed in an Al2O3 crucible. The studied samples were grinded to powder in mortar for all the experiments.The thermal properties of the prepared samples were studied by thermal analyzer STA 449C Jupiter . The thermogravimetry (TG) and differential scanning calorimetry (DSC) was conducted from 55 to 700 °C at the heating rate of 10 °C minThe Shore D hardness test according to ASTM D2240 at 25 °C was performed on the epoxy composites to measure the hardness. The test was made by placing the studied sample under the indenter of Shore durometer. Then, a load was applied to the sample through the indenter. The tests were repeated five times and mean value is reported.To evaluate the flammability of the samples, the standard method of experimental determination of the ignition temperature of solid substances and materials was used in accordance with Russian standard GOST 12.1.044-89 “Occupational safety standards system. Fire and explosion hazard of substances and materials. Nomenclature of indices and methods of their determination”. The ignition temperature of the epoxy resin and composites was determined using the installation, which is a vertical electric furnace with two coaxially located cylinders made of quartz glass. The spiral electric heaters with a total power of at least 2 kW are wound on the cylinders, allowing the creation of a working zone temperature of 600 °C during 40 min. The principle of the installation is based on setting the temperature regime in the reaction chamber and the action of burner flame on the samples after introducing them into the reaction chamber; afterwards, the temperature characteristics of the material were monitored. The samples prepared were of cylindrical shape with a diameter of 31.4 mm, and the weight of each sample was 6 ± 0.1 g. At least six samples for each formulation were tested for the good reproducibility.The thermal behavior of the epoxy composites filled with MWCNTs, boric acid, sodium bicarbonate, and their combination at the heating was investigated by means of TG analysis. The TG curves of the studied epoxy composites are shown in T5). The midpoint temperature T50 corresponds to a 50% mass loss. The temperature T90 corresponds to a 90% mass loss. The thermooxidative degradation of the neat epoxy E0 finished nearly at 690 °C, and the residue is 0.2%. Therefore, we considered the residue at 690 °C to evaluate the thermal stability of the composites.The data obtained from the TG curves of the prepared epoxy composites are given in T5 decreased by 23.8 °C. The temperature T50 slightly increased by 1.4 °C. However, the temperature T90 for the sample E/0.5MWCNT decreased by 19.1 °C in comparison with that of the sample E0, and is linked with oxidation of filler at these temperatures. The effect of increased mass loss of MWCNT composites at 250–350 °C compared with pure epoxy was also observed in [Incorporation of 0.5 wt% MWCNTs into epoxy matrix was not found to lead to the improvement in thermal stability of the sample E/0.5MWCNT. After the addition of MWCNTs to epoxy resin, the temperature erved in ; the autBoth samples E0 and E/0.5MWCNT practically completely decomposed at 690 °C. This result can be probably explained by the poor distribution of MWCNTs in the epoxy matrix. Optical micrographs of composites are presented in T5 of the boric acid and sodium bicarbonate is much lower than that of the epoxy resin; as a result, the presence of these fillers in the epoxy composite leads to a decrease in the T5 of the composites. During combustion, these fillers can dilute the flammable gases by carbon dioxide (sodium bicarbonate) and reduce the temperature in the flame zone via water (boric acid and sodium bicarbonate), thereby inhibiting the combustion process. The thermal behavior of epoxy resin ED-20 filled with boric acid at the heating in air and in argon was investigated in [The initial temperature of degradation gated in ,43. The T5 of the samples via the early decomposition of these substances. When the samples filled with boric acid E/10B and E/15B are heated above ~360 °C, they become more stable compared to the unfilled sample E0. The temperature T50 of the samples E/10B and E/15B is higher than that of the sample E0 by 15.5 and 21.7 °C, respectively. The residue at 690 °C of the samples E/10B and E/15B is higher in comparison with that of the sample E0 by 18.3 and 22.9%, respectively. The combination of boric acid and MWCNT in the samples E/10B/0.5MWCNT and E/15B/0.5MWCNT resulted in their greater resistance to heating above the temperature 350 °C than the neat epoxy sample E0 and E/0.5MWCNT as well. The temperatures at which the samples E/10B/0.5MWCNT and E/15B/0.5MWCNT lose 50% of the mass upon heating are the highest; are 19 and 35.7 °C higher than that for the unfilled sample E0, respectively; and are 3.5 and 14 °C higher than that for the samples E/10B and E/15B, respectively. However, the residue at 690 °C for the E/10B/0.5MWCNT and E/15B/0.5MWCNT is lower compared to that of samples E/10B and E/15B. The endothermic DSC peak with the maximum around 460 °C is attributed to boric acid decomposition  is determined according to Tmax ranged from 355.0 to 366.9 °C, and the highest value was shown by E/10S sample. As seen from DTG curves, addition of boric acid and MWCNTs makes the DTG peak complex, with a broad temperature range of weight loss. Instead of metallic particles used for enhancement of epoxy resin degradation behavior [The value of behavior , the addTg shown in Tg to 160–175 °C  and 170–175 °C . Generally, the glass transition temperature is higher for composites containing sodium bicarbonate and MWCNTs  compared to composites containing boric acid.Glass transition temperature Hardness is defined as the property of material to resist penetration, abrasion, and indentation. The measured Shore D hardness values of the studied samples are given in From these results, the sample E/0.5MWCNT shows the highest hardness among all the samples. The hardness of the sample E/0.5MWCNT is 10.5% higher than that of the neat epoxy E0. Incorporation of MWCNTs into the epoxy matrix increases the cross-linking density of epoxy that restricts the motion of polymer chains and resulted in increased hardness ,46. The The digital images of the samples after flammability test are given in The ignition temperature is the minimum temperature at which a combustible substance when heated takes fire in air and continues to burn. The time-to-ignition is defined as the minimum exposure time required for the sample to ignite and sustain flaming combustion. It should be noted that ignition tests data are closer to practical application of epoxy composites under the influence of high temperatures in fire conditions than that of TG analysis data.The neat epoxy resin E0 melts and flows upon heating and burning, as shown in The maximum ignition temperature was observed in sample E/10S. The incorporation of 15 wt% of sodium bicarbonate led to a decrease in the ignition temperature by 2 °C. Sodium bicarbonate releases carbon dioxide and water upon burning the composites and causes formation of the dense char residue with a large number of small bubbles, uniformly distributed in the sample volume. This structure is typical for all the samples filled with sodium bicarbonate d,f. The In this research, the thermal properties  of the epoxy composites filled with MWCNTs (0.5 wt%), boric acid and sodium bicarbonate (10 and 15 wt%) separately, as well as in combination with MWCNTs, were compared to those of the control unfilled sample. The addition of 0.5 wt% MWCNTs in the epoxy matrix did not improve the thermal stability of the epoxy composite. The improvement in the thermal properties can be achieved by the combined use of MWCNTs and boric acid. The thermooxidative degradation of the samples filled with boric acid passes more slowly and more evenly. The ignition temperature and time-to-ignition of the neat epoxy resin and composites was determined. Based on the results of thermal analysis, it was concluded that sodium bicarbonate in the studied concentration ranges (10 and 15 wt%), despite the high ignition temperature value, is not recommended to be used as filler capable of increasing the thermal stability and reducing the flammability of epoxy polymers. In addition, the incorporation of boric acid and sodium bicarbonate to the epoxy matrix resulted in a decrease in Shore D hardness. Thus, the effect of additives deteriorating the mechanical properties of composites must be taken into account when developing recommendations for the practical use of epoxy composites. The obtained composites with enhanced thermal stability can be used as adhesives, coatings, housings, details of some machines which can be subjected to impact of heating. The fillers used in the preparation of epoxy composites make it possible to delay the degradation process upon heating.


Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck tumor. It is a high incidence malignant tumor associated with a low survival rate and limited treatment options. Accumulating conclusions indicate that the Wnt signaling pathway plays a vital role in the pathobiological process of HNSCC. The canonical Wnt/β-catenin signaling pathway affects a variety of cellular progression, enabling tumor cells to maintain and further promote the immature stem-like phenotype, proliferate, prolong survival, and gain invasiveness. Genomic studies of head and neck tumors have shown that although β-catenin is not frequently mutated in HNSCC, its activity is not inhibited by mutations in upstream gene encoding β-catenin, NOTCH1, FAT1, and AJUBA. Genetic defects affect the components of the Wnt pathway in oral squamous cell carcinoma (OSCC) and the epigenetic mechanisms that regulate inhibitors of the Wnt pathway. This paper aims to summarize the groundbreaking discoveries and recent advances involving the Wnt signaling pathway and highlight the relevance of this pathway in head and neck squamous cell cancer, which will help provide new insights into improving the treatment of human HNSCC by interfering with the transcriptional signaling of Wnt. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignant tumor in the world . HNSCC cIn 1982, Nusse found an oncogenic gene in mouse models of mammary cancer, named int1, and which has homology to the wingless gene of drosophila reported later by Sharma, and the two were collectively called Wnt . The Wnt2+ and Wnt/PCP pathways  receptors and several coreceptors, such as Lrp-5/6, Ryk, or Ror2 . Intracepathways .Xenopus laevis and the segmental polarity and wing development of Drosophila, researchers first clarified the role of this canonical pathway in embryonic development . These Wnt ligands bind to Fzd receptors. In addition, receptor tyrosine kinase-like orphan receptor 2 (Ror2), and receptor tyrosine kinase (Ryk) have been suggested as non-canonical signaling co-receptors, which are required for downstream activation. These signal transductions jointly activate the calcium-dependent signaling cascade by activating Dvl . This results in inositol 1,4,5-triphosphate-3 (IP3) production and subsequent Ca2+ release -positive and HPV-negative tumors, each of which has its unique clinical, pathological, and epidemiological significance . Increasin vitro . Accordiin vitro . More rein vitro . Due to in vitro .Components of the Wnt signaling pathway, such as Wnt ligand proteins, Wnt antagonists, membrane receptors, and intracellular conduction medium, are often disrupted by genetic or epigenetic inheritance in human tumors . It is rDNA methylation and histone modification also play important parts in the occurrence of HNSCC. Epigenetic regulation may contribute to the silencing of Wnt related genes. Because there is no changes of methylation levels in the CpG island of APC, Axin, and β-catenin genes in OSCC , downregStem cells (SCs) have the ability of self-renewal and differentiation. The maintenance and repair of tissue homeostasis depends on the activity of tissue-specific SCs. Cancer SCs (CSCs) are a subset of cells that are resistant to chemotherapy and radiotherapy and often promote relapse by stopping or evading clinical treatment . Like otin vivo and found that Wnt3a activated Wnt signals in cancer cells and CAF. The activation of Wnt increases the characteristics of CSC, such as sphere formation and invasiveness , and plays a key role in guarding CSCs . Le et asiveness . Non-cansiveness . The anasiveness . In addisiveness . The Wntsiveness  and cancsiveness .A study showed that the expression of CD44 + was essential for maintaining tumor heterogeneity in HNSCC . The CSCDue to the presence of drug-resistance CSCs, disease recurrence is the main marker of HNSCC. A large body of evidence suggests that Wnt confers chemotherapeutic resistance by upregulating CSC activity in HNSCC. The use of the Fzd/Wnt antagonist SFRP4 was found to increase the drug sensitivity of HNSCC by 25%. SFRP4 was shown to compete directly with Wnt, significantly enhancing cisplatin-induced apoptosis and reducing the activity of tumor cells . FurtherIn vitro, C59 inhibited the activity of PORCN, and then inhibited the Wnt palmitoylation, Wnt interaction with carrier protein Wntless/WLS, Wnt secretion, and Wnt activation of β-catenin reporter protein. The chick chorioallantoic membrane (CAM) experiment proved that LGK974 can inhibit the growth and metastasis of HNSCC (in vivo and in vitro. Additionally, inhibitors of tankyrase stabilize axin and antagonize Wnt signaling including XAV939, IWR, G007-LK, and G244-LM, though they have not yet entered clinical trials (Wnt signaling plays an important role in tumorigenesis and acts as a regulator of CSCs renewal in the process of cell homeostasis; thus, it is an attractive therapeutic target. To date, several approaches have been developed, and a few have moved on to clinical trials. One of them is to block the activity of Wnt with specific inhibitor. PORCN, also known as porcupine, is an enzyme which can limit the activation of Wnt signals in serine residues and promote the palmitoylation of Wnt. Using small inhibitors of PORCN, such as IWP, C59, and LGK974 caused rapid decreases in the expression of Wnt signaling . In vitrof HNSCC . Studiesl trials . Moreovel trials . ICG-001l trials . Examplel trials . To dateAs outlined above, aberrant activation of the Wnt signaling pathway may impact on HNSCC. In addition to gene mutations in the Wnt component, abnormal changes downstream of EGFR are involved in regulating the Wnt/β-catenin pathway, which can reshape the histone/chromatin structure of the target gene. Because the epigenetic alterations of Wnt antagonists are the cause of Wnt signal activation, it may become a potential biomarker for predicting OSCC recurrence in plasma. Appropriate methods are required to deal with CSC generated by aberrant Wnt signaling. Wnt signaling is one of the regulators of CSC generation involving HNSCC. Because of the complexity of non-canonical signal pathway, most of the research on Wnt in HNSCC is focused on canonical WNT signal pathway, but there are few related studies on non-canonical signal pathway. More attention needs to be paid to non-canonical signaling pathways in the future. The evaluation of various aspects of signal transduction can expand our understanding of both this key pathway and the crosstalk between signaling pathways in cells. Such advancement will enable the development of a broad range of therapeutic interventions to eradicate and respond to HNSCC recurrence.JX and LH contributed equally in conceiving the review focus, conducting the literature review, summarizing the manuscript, writing the first draft, and finalizing the manuscript. D-LZ and Y-GL designed and directed the review. JX, LH, D-LZ, Y-GL revised and made corrections to the manuscript. All authors have read and agreed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Whisker HFs were isolated from Rex rabbits and cultured in vitro to measure hair shaft growth. Meanwhile, dermal papilla cells (DPCs) were isolated and cultured in vitro. Treatment with AdWnt10b or the Wnt/β-Catenin Pathway inhibitor, XAV939, assessed the DPCs proliferation by CCK-8 assay. And the cell cycle was also analyzed by flow cytometry. We found that Wnt10b could promote elongation of the hair shaft, whereas XAV-939 treatment could eliminated this phenomenon. AdWnt10b treatment promoted the proliferation and induced G1/S transition of DPCs. AdWnt10b stimulation up-regulated β-Catenin protein in DPCs. Inhibition of Wnt/β-Catenin signaling by XAV-939 could decreased the basal and Wnt10b-enhanced proliferation of DPCs. And could also suppress the cell cycle progression in DPCs. In summary, our study demonstrates that Wnt10b could promote HFs growth and proliferation of DPCs via the Wnt/β-Catenin signaling pathway in Rex rabbits.Wnt signaling plays an important role in the growth and development of hair follicles (HFs). Among the signaling molecules, Wnt10b was shown to promote the differentiation of primary skin epithelial cells toward the hair shaft and inner root sheath of the HF cells in mice  Hair follicles (HFs) contain dermal papilla cells (DPCs) and epithelial cells, which are the two major types of cell . During Hair development and subsequent cycling follow a carefully choreographed rhythm with cycling controlled by multiple genes and pathways . SeveralIn HF development, the Wnt family appears to be the earliest and the most critical regulator for early development of the epidermis . Wnt proin vitro [In mice, Wnt10b has been shown to promote differentiation of primary skin epithelial cells toward the hair shaft and inner root sheath of the HF cells in vitro ,14, and in vitro . To undeAll experiment including animal experiments were carried out at Shandong Agricultural University. The ethics approval was also obtained from Shandong Agricultural University  and were carried out in accordance with the Guidelines for Experimental Animals of the Ministry of Science and Technology . All surgical procedures were carried out according to recommendations proposed by the European Commission (1997), and all efforts were made to minimize the suffering of animals.Four-week-old Rex rabbit was anesthetized by intravenous injection of diazepam (1.6 mg/kg) and pentobarbital sodium (30 mg/kg) followed by cervical dislocation. The anesthetic effect was assessed by the indicators include smooth breathing, muscle relaxation, no pain response, and miosis. Rabbit was confirmed dead when no breathing or heart beat was detected. And then the skin samples were taken away from the whisker or dorsal back position of the rabbit immediately for the next treatment.2 and 95% box in the general culture and cultured in one of the following basal media: Williams E medium , penicillin–streptomycin , insulin , hydrocortisone , and L-glutamine ; basal medium containing AdWnt10b  and AdGFP  at ultimate titers of 108; or basal medium containing XAV-939 (10 µmol/l) and AdWnt10b plus XAV-939. After 2 days, every culture was replaced with fresh media, and the length of the outgrowing hair shafts was determined by analyzing the digital images at 5 days of growth with stereo microscope .We used whisker HFs for the organ cultures. Whisker HFs of a 4-week-old Rex rabbit was isolated as has been previously described for mice . Early oSmall skin pieces were obtained from 4-week-old rabbits. And then the DPCs were isolated based on the previously reported method  and cultThe third generation of DPCs was plated on cover glass  in 6-well plates for culturing 3 days. Removed the basal medium and rinsed for three times using phosphate buffer solution (PBS). Added 2–3 drops of Wright-Giemsa Stain solution  for rinsing 2 min. And then washed with water, dried with the hydro-paper and observed the cell morphology by using a fluorescence microscope .The third generation of DPCs was plated on cover glass  in 6-well plates for culturing 4 days. Removed the basal medium and rinsed for three times using phosphate buffer solution (PBS). And then fixed with 4% paraformaldehyde  for 30 min in room temperature and washed with PBS for three times, permeabilized with 0.5% Trixton X-100 for 15 min in room temperature and washed with PBS for three times, blocked with goat serum  for 30 min and incubated with α-SMA antibody  /or Vimentin antibody  /or Wnt10b antibody  overnight at 4°C in wet box . For immFor overexpression of Wnt10b treatment, the DPCs were cultured with AdWnt10b at the concentration of 300 multiplicity of infection (MOI) for 2 h and then refresh the new basal medium. For inhibition of Wnt/β-Catenin pathway, XAV-939  at a concentration of 10 μM was added into the basal medium 4 h after transfection.DPCs were plated in a 96-well plate at a density of 4000 cells/well and cultured in basal medium for 24 h, and then followed by the indicated treatment. Ten microliters Cell Counting Kit-8 (CCK-8) was added to each well at 24 h after treatment, and incubated the plate for 4 h at 37°C. The optical density at 450 nm was read by an microplate reader .g for 5 min, dropped the supernate and added 0.5 ml propidium iodide (PI)  for incubating 15 min at 37°C in dark. The cells were then analyzed by flow cytometry . And the results were analyzed with the ModFit LT 5.0 software.For flow cytometry analysis, the DPCs were treatment followed by the indicated treatment for 4 days. The cells were digestion by trypsin and fixed in 70% cold ethanol for at least 18 h, centrifuged at 500 Total protein of DPCs was extracted with the cell active protein extraction kit . And the protein concentration was measured with a BCA Protein Assay Kit . For the Western blot analysis, equal protein amounts (40 µg per well) were denatured for 10 min at 100°C and separated with 12% gel electrophoresis. The protein was transferred onto a polyvinylidene fluoride (PVDF) microporous membrane at 200 mA and 4°C for 2 h. After 1 h of blocking in block solution  at room temperature, membranes were incubated at 4°C overnight in the following primary antibodies: Wnt10b antibody ; β-catenin antibody ; Tubulin antibody . The blots were then soaked with anti-mouse IgG-conjugated horseradish peroxidase  for 4 h at 4°C, and the protein bands were detected by Super Signal West Femto Maximum Sensitivity Substrate (Thermoscientific) or ECL . The results were visualized by exposing the blots to X-RAY film (Kodak). The films were scanned by a scanner (HP ScanJet 6100C) and signal intensity (Quantification) was conducted using ImageJ 1.43 software .P < 0.05 was regarded as statistically significant.All the data were analyzed with SAS software . A one-way ANOVA model was used to evaluate the means among various groups. The data were shown as means ± SEM. P < 0.0001)  D.P < 0.0001) (P < 0.0001). Cell cycle analyzed by flow cytometry revealed that AdWnt10b treatment could reduced the number of cells in G0/G1 phase (P < 0.0001) and promoted the cells into S and G2/M phase (P < 0.0001)  . DPCs we 0.0001) B.P < 0.0001). Treatment with XAV-939, which is a specific inhibitor of Wnt/β-catenin signaling that promotes the degradation of β-catenin to block the Wnt/β-catenin signaling pathway [P < 0.0001).As shown in  pathway , could rP < 0.0001)  A–D.P < 0.0001). Cell cycle analyzed by flow cytometry also revealed that the promotion of DPCs proliferation by Wnt10b was inhibited by XAV-939 (P < 0.0001)  B.HFs are known to be constantly renewed with alterations between the phases of anagen, catagen, and telogen . In HFs,In our study, we examined the effects of Wnt10b on developed HFs in Rex rabbits. For this purpose, we used organ cultures of whisker HFs because they have been shown to be cultured for a prolonged period . Like whWnt signals have been implicated in the control of HF development. And the Wnt/β-catenin signals are generally required for the initiation, development, and generation of HFs . β-Catenin vitro and enhance the proliferation of DPCs in vitro via the Wnt/β-catenin signaling pathway. We believed that Wnt10b was deeply involved in the Rex rabbit HF development through the Wnt/β-Catenin signaling pathway.In conclusion, Wnt10b can promote growth of Rex rebbits HFs


We found that in the hyperglycaemia-induced increase in the level of AGEs, pro-inflammatory changes were significantly suppressed by allithiamine. However, allithiamine could not enhance the activity of transketolase, but it exerts a potent antioxidant effect. Collectively, our data suggest that allithiamine could alleviate the hyperglycaemia-induced endothelial dysfunction due to its potent antioxidant and anti-inflammatory effect by a mechanism unrelated to the transketolase activity.Diabetes mellitus-related morbidity and mortality is a rapidly growing healthcare problem, globally. Several nutraceuticals exhibit potency to target the pathogenesis of diabetes mellitus. The antidiabetic effects of compounds of garlic have been extensively studied, however, limited data are available on the biological effects of a certain garlic component, allithiamine. In this study, allithiamine was tested using human umbilical cord vein endothelial cells (HUVECs) as a hyperglycaemic model. HUVECs were isolated by enzymatic digestion and characterized by flow cytometric analysis using antibodies against specific marker proteins including CD31, CD45, CD54, and CD106. The non-cytotoxic concentration of allithiamine was determined based on MTT, apoptosis, and necrosis assays. Subsequently, cells were divided into three groups: incubating with M199 medium as the control; or with 30 mMol/L glucose; or with 30 mMol/L glucose plus allithiamine. The effect of allithiamine on the levels of advanced glycation end-products (AGEs), activation of NF-κB, release of pro-inflammatory cytokines including IL-6, IL-8, and TNF-α, and H In recent decades, several research have focused on the pharmacologically active, plant-derived compounds . ExtensiAllium sativum L.) is among the most studied ones [1-vitamin) derivative and, similar to the molecules mentioned above, has a prop-2-en-1-yl disulfanyl moiety. According to a recent study, allithiamine is also accumulated in red sweet pepper (Capsicum annuum L.) seeds, implying that its occurrence is more frequent than as thought until now. Nevertheless, several studies revealed that numerous garlic compounds have beneficial effects on hyperglycaemia in diabetes mellitus [Among plants having significant pharmacological activity, garlic , and so consequently, AGEs play a major role in diabetic microvascular complications . HyperglTo date, there is no preclinical evidence for the antidiabetic effect of allithiamine, therefore, the main objective of our current research was to study whether this compound is able to exert a beneficial effect on diabetes. Primary cultured human umbilical cord vein endothelial cells (HUVECs) were used as a unique hyperglycaemic model, which appeared to be ideally capable to investigate the level of AGEs, antioxidant status, and pro-inflammatory cytokines.All reagents were obtained from the distributor of iBioTech Hungary Ltd.  and DIAGON Ltd. Hungary .Preparation and purification of allithiamine were carried out based on the method of our recent allithiamine-oriented study . BrieflyThe HUVECs were isolated from human umbilical cords and maintained according to the method previously described . In our HUVECs were incubated with four fluorescent dye-labeled antibodies, involving fluorescein-isothiocyanate (FITC)-labeled mouse anti-human CD31, phycoerythrin (PE)-labeled anti-human CD54, allophycocyanin (APC)-labeled mouse anti-human CD106, and PerCP-Cy5.5-labeled mouse anti-human CD45 , and then characterized by using a FACSAriaIII Cell Sorter  .v/v) isopropyl alcohol, 9% (v/v), 1 M HCl, 10% (v/v) Triton X-100) and the absorbance was measured at 465 nm by using a Clariostar microplate reader . Cell viability at different allithiamine concentrations was expressed relative to 100% of the control group.Cell viability was determined by an MTT  assay. Cells were plated into 96-well plates at a density of 20,000 cells per well in quadruplicates and treated with allithiamine of different concentrations  and without allithiamine (control group) for 24, 48, and 72 h. The medium was removed and incubated with 100 μL of MTT solution (0.5 mg/ml dissolved in Dulbecco’s modified Eagle’s medium) for 3 h. Subsequently, the formazan crystals were dissolved in 100 μL solubilizing solution (81% (1(5)) dye. The decrease in the DilC1(5) intensity indicated mitochondrial depolarization, i.e., the onset of early apoptotic processes of HUVECs [1(5) working solution (50 nM dissolved in Dulbecco’s modified Eagle’s medium). After incubation, cells were washed twice with PBS and the fluorescence of DilC1(5) was measured at 630 nm excitation and 670 nm emission wavelengths by using a Clariostar microplate reader . The results were expressed relative to 100% of the control group.The mitochondrial membrane potential of the HUVECs was determined by using 1,1′,3,3,3′,3′-hexamethylindodicarbocyanin iodide  Competitive ELISA Kit .The assay was performed according to the manufacturer’ instructions by using an OxisSelectThe assay was performed according to the manufacturer’ instructions by using a Human NF-κB p65 Sandwich ELISA Kit −1 and the released amount of IL-6, IL-8, and TNF-α was determined by using a MILLIPLEX MAP Human cytokine/chemokine Magnetic Bead Panel  based on the manufacturer’s recommendation.HUVECs were seeded into a 6-well plate , and were incubated in 5 mMol/L glucose and 30 mMol/L glucose with or without 5 μg/mL allithiamine for 6, 12, or 24 h. Supernatants were collected, centrifuged for 10 min 10,000 r·minTransketolase activity was measured by adding 100 μL cytosolic fraction to 200 μL reaction mixture containing 14.8 mMol/L ribose-5-phosphate, 253 μMol/L NADH, 185 U/mL triosephosphate isomerase, and 21.5 U/mL glycerol-3-phosphate dehydrogenase in Tris buffer (pH = 7.9). The optical density was measured at 340 nm immediately and then every 10 min for 2 h by using a Clariostar microplate reader . The activity was calculated from the difference in the optical density readings at 10 and 80 by min using the extinction coefficient of NADH. Results are expressed in nMol/min/mg protein.The protein concentrations were determined in the cell lysate by using a Pierce BCA Protein assay . Protease inhibitor cocktail  was added to the cell lysate prior to its storage or measurement.2O2; or with 100 μMol/L H2O2 plus 5 μg/mL allithiamine. Fluorescence  was assessed by using a Clariostar microplate reader .The cells were seeded in a 24-well plate and exposed to 100 μMol/L 2′,7′-dichlorofluorescin diacetate (DCFDA) for 1 h at 37 °C to label the intracellular ROS. After incubation, the cells were washed twice with PBS and divided into three groups: incubating with M199 medium as control; or with 100 μMol/L Ht-test for repeated measures according to Bonferroni’s method. Data were presented as mean ± SEM. Differences were considered statistically significant, when p < 0.05.For multiple comparisons, results were analyzed by an ANOVA followed by a modified The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the University of Debrecen (registration number RKEB/IKEB 3712-2012).2 spectrum  for 24, 48, and 72 h. We found that up to 5 μg/mL allithiamine did not decrease the viability of HUVECs in this time window .1(5) and SYTOX Green dyes). The results show that allithiamine, in line with the MTT data, did not induce a necrotic and apoptotic process and can be used without the risk of any biologically relevant cytotoxic actions in this concentration range , a routinely used oxidative agent [2O2 significantly increased the production of ROS. Allithiamine was able to eliminate a significant part of this increment, indicating the potent antioxidant effect of allithiamine  (examination of apoptosis) and SYTOX Green (investigation of necrosis) dyes. The viability tests of HUVECs after exposure to allithiamine at different concentrations (0.1–5 µg/mL) for up to 72 h indicate that cells were not significantly affected.Primarily, HUVECs were characterized by flow cytometric analysis using antibodies against specific marker proteins including CD31, CD45, CD54, and CD106. The results proved that the cells used in our experiments were endothelial cells, indeed. Subsequently, the non-cytotoxic concentration of allithiamine was determined by using an MTT assay and fluorescent labeling with DilCIn parallel with the research on the garlic component which showed a suppressed formation of AGEs , excelleIn order to investigate the effects of allithiamine on inflammatory processes, we examined NF-κB which has been proven to be upregulated under hyperglycaemic conditions. NF-κB has a pivotal role in the inflammatory process as a transcriptional factor of several pro-inflammatory cytokines . We foun2S donor [2O2 as an oxidative agent. Allithiamine was able to eliminate the significant part of ROS after the administration of H2O2. Consequently, our results clearly indicate that allithiamine exhibits a potent antioxidant effect, which may be responsible for the improvement in hyperglycaemia-induced endothelial dysfunction.Seeking possible reasons behind the positive effect of allithiamine, two plausible explanations were raised. At first, we assumed that allithiamine is able to enhance the transketolase activity, resulting in a decreased flux of hyperglycaemia-induced pathologic pathways involving the polyol, AGEs, PKC, and hexosamine pathways, similar to thiamine  and benf2S donor ,43. AlliCollectively, in this study, we observed that—without influencing the viability, necrosis, or apoptosis of HUVECs—allithiamine was able to attenuate particular negative effects of an elevated glucose level by its potent antioxidant effect and a mechanism unrelated to the transketolase activity. Further studies are needed to elucidate the mechanisms of action of allithiamine as well as its supposed beneficial role in the spectrum of plant-derived bioactive molecules. However, our results contribute to a better understanding of this relatively less-researched compound, with particular respect to its beneficial effects on hyperglycaemia.


Simultaneously, lithium (Li) and copper (Cu) can activate the canonical Wnt and HIF-1α signaling pathway, respectively. Moreover, emerging evidence has shown that the canonical Wnt and HIF signaling pathways are related to coupling osteogenesis and angiogenesis. However, it is still unclear whether the lithium- and copper-doped bioactive scaffold can induce the coupling of the osteogenesis and angiogenesis in BMSCs and the underlying mechanism. So, we fabricated a lithium- (Li+-) and copper- (Cu2+-) doped organic/inorganic (Li 2.5-Cu 1.0-HA/Col) scaffold to evaluate the coupling osteogenesis and angiogenesis effects of lithium and copper on BMSCs and further explore its mechanism. We investigated that the sustained release of lithium and copper from the Li 2.5-Cu 1.0-HA/Col scaffold could couple the osteogenesis- and angiogenesis-related factor secretion in BMSCs seeding on it. Moreover, our results showed that 500 μM Li+ could activate the canonical Wnt signaling pathway and rescue the XAV-939 inhibition on it. In addition, we demonstrated that the 25 μM Cu2+ was similar to 1% oxygen environment in terms of the effectiveness of activating the HIF-1α signaling pathway. More importantly, the combination stimuli of Li+ and Cu2+ could couple the osteogenesis and angiogenesis process and further upregulate the osteogenesis- and angiogenesis-related gene expression via crosstalk between the canonical Wnt and HIF-1α signaling pathway. In conclusion, this study revealed that lithium and copper could crosstalk between the canonical Wnt and HIF-1α signaling pathways to couple the osteogenesis and angiogenesis in BMSCs when they are sustainably released from the Li-Cu-HA/Col scaffold.The combination of osteogenesis and angiogenesis dual-delivery trace element-carrying bioactive scaffolds and stem cells is a promising method for bone regeneration and repair. Canonical Wnt and HIF-1 More than 1.6 million people receive bone grafts because of tumor resection, pathological deformation, congenital deformation, sports injury, and infection treatment each year in the USA . Currentβ, thereby inhibiting the phosphorylation and degradation of β-catenin [β-catenin is transported to the nucleus and initiates transcription of the osteogenesis gene (Wnt target genes) by binding to T cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription reporters [The osteogenesis of BMSCs involving many molecular signaling pathways and the canonical Wnt signaling pathway has been shown to be one of them. The Wnt signaling plays an important role in bone development, homeostasis, osteoblast differentiation, and bone formation . Lithium-catenin . Accumuleporters . In receeporters , 12. Addeporters , 14. Theα signaling pathway [α (HIF-1α) in mesenchymal stem cells enhances the VEGF expression and secretion to promote blood vessel formation [α by prolyl hydroxylases (PHDs) to stabilize HIF-1α expression [α) [α's interaction with the cofactors SRC-1, CBP, and p300 and then downgrade the expression of the HIF-1α target gene  [α signaling pathway [The process of angiogenesis in a physiological and pathophysiological manner is highly related to the activation of the HIF-1 pathway , 16. Incormation . Copper,pression . Simultasion [α) . The FIH1, etc.) . In addi pathway . Thus, cα signaling pathways. Kaidi and colleagues revealed that β-catenin could be used as a cotranscription factor to bind with HIF-1α and promote the expression of HIF-1α signaling pathway downstream genes [α in the Wnt signaling pathway is still controversial. In neurogenesis and bipolar disorder, Valvezan and Klein had reported that the HIF-1α promotes Wnt target gene expression in stem cells [α inhibits β-catenin-T cell factor-4 (TCF-4) complex formation and transcriptional activity in hypoxia [α signaling pathway activated by lithium and copper can couple and enhance the osteogenesis-angiogenesis in the BMSCs is still unclear.Interestingly, there are inextricable and extensive connections between canonical Wnt and HIF-1am genes . While tem cells . However hypoxia , while w+-) and copper- (Cu2+-) doped organic/inorganic (Li-Cu-HA/Col) scaffold to explore their effects on coupling osteogenesis-angiogenesis. And then, we screened the optimal concentration of lithium and copper to evaluate their ability as an agonist of canonical Wnt and HIF-1α signaling pathways. Finally, we investigated the osteogenesis-angiogenesis coupling ability in BMSCs under the costimulation of optimal lithium and copper concentration and explore the underlying mechanism.In this study, we fabricated a lithium-  and collected by centrifugation at 250 g for 10 min. Resuspend the cells with 2 ml Ammonium-Chloride-Potassium Lysis Buffer , and add 6 ml DMEM to stop the reaction after 5 minutes. Then, centrifuge at 250 g for 5 minutes to obtain BMSCs, and the cells were cultured in DMEM, supplemented with 10% fetal bovine serum  and 1% (v/v) antibiotic/antimycotic . When cells grew to 80% confluence, 0.25% trypsin  was used to passage the cells.The BMSCs were isolated from 6-to-8-week old Balc/c mice. Femurs and tibias from mice were dissected, and the bone marrow was flushed with Dulbecco's Modified Eagle's Medium . Bone marrow-derived cells were filtered through a 70 BMSCs were seeded into 6-well plates and cultured in the complete medium. According to the manufacturer's instructions, osteogenic and adipogenic induction was stimulated by using commercial kits . Osteogenesis effects were detected by Alizarin Red staining  after 3-week induction, and the Oil Red O was used for adipogenic detection after 3-week induction. Chondrogenic differentiation was induced using the micromass culture technique. BMSCs were collected and washed three times with chondrogenic induction medium  under centrifugation at 350 g for 5 min. The pellet was cultured in 0.5 ml chondrogenic induction medium in the 15 ml tube. After 2 days, the pellet was suspended by slightly knocking the bottom of the tube. The medium was replaced every two days, and the pellet was cultured for 28 days. Chondrogenesis effects were detected by Alcian blue staining.For Alizarin Red staining of cultured cells, BMSCs were washed with 1× Phosphate-Buffered Saline (PBS) and fixed with 10% formalin for 20 min. Then, cells were washed in tap water three times before staining with Alizarin Red solution  for 15 min. Finally, cells were rewashed with tap water three times and viewed under an inverted microscope .For Oil Red O staining of cultured cells, BMSCs were washed with 1× PBS and fixed with 10% formalin for 20 min. Then, cells were washed in tap water three times before staining with Oil Red O solution  for 15 min. The excess stain was removed by washing with 70% ethanol three times. Finally, cells were rewashed with diluted water three times and viewed under an inverted microscope .For Alcian blue staining, tissues generated from the pellet cultures were fixed in 10% formalin for 2 h and then washed twice with 1× PBS before the addition of 0.1% stock solutions of Alcian blue. After 30 min incubation at room temperature, the dye solution was removed and the constructs were washed with distilled water. Moreover, the staining results were recorded under an inverted microscope .3)2·4H2O and NH4H2PO4  were dissolved in deionized water separately to form 0.1 M and 0.06 M solution. And then, add LiNO3 and Cu (NO3)2 solid with designed Li/(Li+Cu+Ca) and Cu/(Li+Cu+Ca) molar ratios of 0.025 and 0.01, separately, to form the mix solution. Subsequently, the pH of the mix solution and NH4H2PO4 solution was buffered close to 12 by NH3·H2O, and then, pour the adjusted NH4H2PO4 solution into Ca (NO3)2·4H2O solution slowly. Then, the above resolution was transferred into an autoclave (60 ml) and heated in a microwave oven  for 120 min at 130°C. After cooling, the products were collected by centrifugation, washed three times with deionized water and ethanol solution, and then dried at 60°C. The obtained products were labeled as Li 2.5-Cu 1.0-HA.We firstly synthesized lithium- and copper-doped hydroxyapatite (Li 2.5-Cu 1.0-HA) powder through a microwave-assisted hydrothermal method . The Li 2O2 foaming method [2O2, 7.5 ml of polyvinyl alcohol (PVA), and 60 ml of deionized water were mixed to form a ceramic slurry. This slurry was heated for 2 min in a microwave to generate gas and then molded to obtain the porous ceramic scaffold. Afterward, the scaffold was dried at 80°C for 12 h, following sintering at a heating rate of 5°C·min−1 for 6 h until 1200°C, and then, the ceramics in the furnace were cooled until room temperature. All the samples were cut into Φ10 mm × 10 mm cylinders, and the Archimedes principle method was used to test the sintered Li 2.5-Cu 1.0-HA scaffolds' porosity.The porous ceramic structures of the Li 2.5-Cu 1.0-HA were produced and designed using the Hg method . Approxi−1 concentration to 20 g·ml−1, and it was adjusted using 5% w·v−1 acetic acid to a pH ranging from 4.0 to 6.5. After that, the scaffold was fully immersed into the type I collage solution and sealed in a high-pressure vessel. And then, the vacuum infusion with a pressure of 10 Pa was used to fill the collage I solution in the porous Li 2.5-Cu 1.0-HA scaffolds, which sustained for 2 h to allow the full saturation of the ceramic materials. Ultrasonic vibration and a repeated process were conducted for vacuum infusion. Finally, the Li 2.5-Cu 1.0-HA/Col scaffold was lyophilized at -20 C° and sterilized by γ-rays (60Co) at a dose of 25 kGy for 1 h for use.And then, we used the vacuum infusion method, described by Zhou et al., to establish the Li 2.5-Cu 1.0-HA/Col scaffold according to the “brick-and-mortar” reinforcement theory . We were−1 to 500 cm−1. The uniaxial compression testing was conducted to investigate the mechanical properties of HA, Li 2.5-Cu 1.0-HA, and Li 2.5-Cu 1.0-HA/Col scaffolds using a universal mechanical testing machine . Briefly, Φ10 mm × 10 mm scaffolds were fixed on the testing platen. And then, we used the load displacement measurements to obtain the stress-strain curve via a 10 kN load cell at a crosshead speed of 0.5 mm·min−1. After that, we got the maximum compressive strength (maximum load) through the stress-strain curve. In addition, the scaffolds' microstructural characterization was observed using scanning electron microscopy .The chemical compositions of the scaffolds were analyzed by Fourier Transform Infrared Spectroscopy  with scanning from 4000 cmw1, w2, and w3) under different conditions. w1 is the mass of the dried sample (30 min vacuum treatment), w2 is the mass of the saturated sample weighed in the air, and w3 is the apparent mass of the saturated sample weighed in liquid. And then, we use Equation  were placed in clean vial bottles and completely immersed in 5 ml of SBF. Then, the bottles were sealed and put into a 37°C water bathing constant temperature vibrator, and the SBF was refreshed every 3 days. The scaffolds were taken out at designated time intervals of 2, 4, 7, 10, 14, 17, 21, 28, and 30 days. An obsolete release medium was gathered for measuring ionized lithium/copper/phosphate concentrations.The solution-mediated degradation properties of scaffolds were tested in the simulated body fluid (SBF). HA/Col and Li 2.5-Cu 1.0-HA/Col scaffolds  on day 1, 3, 5, and 7 time points. The BMSC coculture scaffolds were washed twice with PBS and incubated with 200 μl MTS solution plus 800 μl culture medium for 3 h in a humidified incubator. After that, the absorbance of the culture medium was read by using Synergy™ Mx Monochromator-Based Multi-Mode Microplate Reader  at 490 nm.The cell proliferation rate of BMSCs on the scaffold was measured using CellTiter 96® AQAfter coculture of the scaffold with P3 BMSCs for 3 days, a Live-Dead Cell Staining Kit  was used for detecting the dead cells. The BMSC coculture scaffolds were washed twice with PBS and stained with Live-Dye  and propidium iodide . After incubation for 15 min at 37°C, live and dead cells were visualized by fluorescence microscopy .Take the sample culture medium into a centrifuge tube and centrifuge at 3000 rpm for 10 minutes at 4°C. Collect the upper layer of medium and freeze it at -80° for use. The alkaline phosphatase (ALP), osteocalcin (OCN), and VEGF protein concentration in the culture medium were detected using an enzyme-linked immunosorbent assay kit . The test was performed on five samples per treatment, according to the manufacturer's protocol.ueous One Solution  was added to each well in a 1 : 10 dilution in the culture medium. After incubation for 4 h at 37°C and 5% CO2 in the humidified atmosphere, absorbance was measured in a microplate reader at 490 nm . Simultaneously, the BMSC number was counted by using Cellometer Mini Automated Cell Counter  on day 7.BMSCs at P3 were cultured under different interventions for 1, 3, 5, and 7 days. And then, CellTiter 96R AQΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as a housekeeping control. The primers used for the RT-qPCR are listed in Supplementary Table Total RNA was subsequently extracted using the RNeasy Mini Kit following the manufacturer's instructions  and reverse-transcribed into cDNAs using SuperScript IV Reverse Transcriptase . RT-qPCR was performed in an ABI StepOnePlus™ instrument  using the SYBR® Premix Ex Taq™ . Each RT-qPCR analysis was performed using six independent biological samples. Relative mRNA expression levels were calculated using the 2For western blotting, BMSCs were harvested with radioimmunoprecipitation assay lysis buffer  supplemented with protease inhibitor cocktail . Protein concentrations were determined using a protein assay kit  according to the manufacturer's instruction. The Invitrogen NuPage® Novex® Gel System was used for protein separation in 4–12% Bis-Tris Protein Gels at 200 V for 50 min. Proteins were then transferred to a Polyvinylidene Fluoride (PVDF) membrane by using iBlot™ Transfer Stack. Membranes were blocked in 5% nonfat milk in the TBST  and 0.1% polysorbate 20 ) for 1 h, followed by incubation with a primary antibody (diluted in 1% nonfat milk in 0.1% TBST) overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies  for 1.5 h at room temperature, washed with 0.1% TBST, and imaged with the ChemiDoc™ Touch Imaging System . Antibody information is provided in Supplementary Table β-catenin, anti-HIF-1α, and anti-IgG (negative control) using salmon sperm DNA/protein A agarose beads overnight at 4°C with gentle rotation. Crosslinking was reversed for 4 h at 65°C and was followed by proteinase K digestion. DNA was purified by a standard phenol/chloroform method, reverse transcriptased, and subjected to real-time PCR. Primer sequences are available in Supplementary Table The chromatin immunoprecipitation (ChIP) assay was performed as described previously . Brieflyβ-catenin and VEGF and the secondary antibody against rabbit or mouse IgG. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a fluorescence microscope .For IF analysis of cultured cells, BMSCs were fixed with 10% formalin for 15 minutes. Permeabilize cells with 0.1%. And then, they were blocked in PBS with 5% bovine serum albumin for 30 minutes at room temperature. Immunostaining was performed using the primary antibody against P value < 0.05, ∗∗ indicates a P value < 0.01, and ∗∗∗ indicates a P value < 0.001.The GraphPad Prism 7.0 software  was used for the statistical analyses. Data are presented as the mean ± standard error (SE). One-way ANOVA was used for the comparison between multigroups' results, followed by the comparison between each two groups using the Tukey analysis. ∗ indicates a To characterize cells isolated by the adherence method, we detected the osteogenesis, adipogenesis, and chondrogenesis potential and surface markers of mouse bone marrow mesenchymal stem cells (BMSCs). The Alizarin Red, Oil Red O, and Alcian blue staining results revealed that the BMSCs had osteogenesis, adipogenesis, and chondrogenesis differentiation potential Figures . The celμm to 400 μm  had absorption peaks of hydroxyapatite, and the Li 2.5-Cu 1.0-HA/Col powders also presented the absorption peaks of collagen . After tr 7 days , while, Based on the excellent physical and chemical properties of Li 2.5-Cu 1.0-HA/Col, we further explored the biocompatibility and osteogenic and angiogenic properties of the scaffold. As expected, the Li 2.5-Cu 1.0-HA/Col scaffold promotes the proliferation of BMSCs and benefits the vitality of BMSCs Figures . Moreove+ (125 μM-1 mM) was proven to be beneficial to BMSCs' proliferation, and 500 μM was the optimal stimulation concentration, which was confirmed by the MTS assay  can rescue the inhibitory effect of 10 μM XAV-939 on the canonical Wnt signaling pathway in BMSCs. On the 500 μM lithium and 10 μM XAV-939 costimulation group, the expression of β-catenin, osterix, and p-GSK3β was similar to that of the Wnt signaling pathway-activated group  can promote the VEGF expression, and 25 μM copper ions have the same efficacy with 1% oxygen microenvironment on stimulating the secretion of VEGF  released from the bioactive scaffold can promote osteogenesis or angiogenesis in bone regeneration , 13, 26.The normal bone repair involves multiple overlapping processes such as inflammation, osteogenesis, and vascularization et al. The coupling of osteogenesis and angiogenesis is an indispensable part of bone regeneration and repair. More and more studies have shown that the dual-delivery scaffold coupling osteogenesis and angiogenesis is a feasible method for bone regeneration , 28. In −1) gavage-fed to the rat for 2 weeks could promote the bone formation in the area of periapical bone lesions, while continuous lithium chloride treatment for 4 weeks impaired periapical bone healing [μM lithium rescued the inhibition effect of 10 μM XAV-939 on the canonical Wnt signaling pathway and osteogenesis differentiation in BMSCs. XAV-939 has been widely proven to be an inhibitor of the Wnt signaling pathway [μM.Lithium has been used as a medicine to treat psychiatric patients for more than half a century, and it can activate the canonical Wnt signaling pathway to promote the MSC proliferation, migration, osteogenesis, etc. –32. Acco healing . Consist pathway . Recentl pathway , 38. Yanα signaling pathway to enhance the VEGF secretion and blood vessel formation [μM copper has the similar effect with 1% oxygen condition on the activation of the HIF-1α signaling pathway. One percentage of oxygen microenvironment is a lower oxygen tension than the physoxic state of most tissue and organs [α would sustain expression and promote the VEGF expression in cells under the 1% oxygen stimulation [α was mainly in the cytoplasm under 5% hypoxia condition and transferred to the nucleus when stimulated under 1% hypoxia condition [μM copper is an appropriate concentration to activate the HIF-1α signaling pathway in BMSCs.Copper is well known to be an essential trace element needed by the human body. A significant amount of information has been gained from understanding its role in angiogenic processes associated with tumor development . In BMSCormation , 42. In d organs , 44. Andmulation , 46. Simondition . The ectα and promote angiogenesis [α signaling pathway can promote the differentiation of stem cells to the direction of osteogenesis and promote the expression of osteogenesis-related genes by combining with transcription promoters such as HRE in the gene promoter region [β-catenin has a direct-acting effect on the HIF-1α target gene. Kaidi et al. reported that there is an overlap between β-catenin- and HIF-1α-regulated genes. As cotranscription factors, β-catenin and HIF-1α bind to the promoter region of angiogenesis-related factors to promote the expression of VEGF, Glut 1, etc. [β-catenin could bind to the TCF to upregulate the VEGF expression [β-catenin could combine in the promoter region of VEGF to enhance its expression in BMSCs. In addition, the osteogenesis of BMSCs was also enhanced under costimulation. And we investigated that the osterix promoter was enriched with HIF-1α and the expression of osterix significantly upregulated under the costimulation. Osterix is a zinc finger-containing transcription factor encoded by the Sp7 gene, which can regulate the osteoblast differentiation of BMSCs [α could activate the osterix expression [β-catenin and HIF-1α could activate the VEGF (HIF-1α target gene) and osterix (Wnt target gene) to crosstalk the canonical Wnt and HIF-1α signaling pathway and then enhance the coupling of osteogenesis and angiogenesis under the costimulation of lithium and copper in BMSCs. However, the mechanism we reported may be just one of the crosstalks between canonical Wnt and HIF-1α signaling pathways in BMSCs. And more researches are needed to explore the interaction between the canonical Wnt, HIF-1α, and other signaling pathways in coupling osteogenesis and angiogenesis.It is common knowledge that there are extensive connections and communications between intracellular signals. Previous studies had shown that the expression of proteins such as c-Myc and AKT downstream of the Wnt signaling pathway could upregulate the expression of HIF-1ogenesis , while tr region , 50. Mor 1, etc. . In addipression . Consistof BMSCs . As a maof BMSCs . The ostof BMSCs . This ispression . Hence, α signaling pathway, respectively. Finally, we indicated that lithium- and copper-mediated osteogenesis and angiogenesis coupling was enhanced through the crosstalk between canonical Wnt and HIF-1α signaling pathways. The results of our study highlight that collaboration of lithium- and copper-doped porous organic-inorganic scaffolds with BMSCs is a possible strategy for the treatment of bone defect.Our study identified that lithium and copper ions' release from the HA/Col scaffold could couple the osteogenesis and angiogenesis in vitro and the Li-Cu-HA/Col scaffold had good mechanical properties and biocompatibility. In addition, we found that the optimal concentration of lithium and copper could effectively activate the canonical Wnt and HIF-1


In vitro experiments validated the pH-responsiveness of MnS@BSA at pH 6.8 and both H2S gas and •OH radicals were detected during its degradation. In vivo experiments showed efficiently tumor turn-on T1-weighted MRI, significantly suppressed tumor growth and greatly prolonged survival of tumor-bearing mice following intravenous administration of MnS@BSA. Our findings indicated that MnS@BSA nanotheranostics hold great potential for traceable H2S gas therapy primed CDT of cancer.Manganese-based nanomaterials have piqued great interest in cancer nanotheranostics, owing to their excellent physicochemical properties. Here we report a facile wet-chemical synthesis of size-controllable, biodegradable, and metastable γ-phase manganese sulfide nanotheranostics, which is employed for tumor pH-responsive traceable gas therapy primed chemodynamic therapy (CDT), using bovine serum albumin (BSA) as a biological template . The as-prepared MnS@BSA can be degraded in response to the mildly acidic tumor microenvironment, releasing hydrogen sulfide (H T1) and transverse (T2) MR contrast agents, respectively 2+). Therefore, the development of γ-MnS-based nanotheranostics is promising for tumor pH-responsive T1-weighted MRI of cancer.Cancer nanotheranostics provide a new solution for cancer management using nanotechnology to integrate medical imaging and therapy of cancer 2O2) into the toxic hydroxyl radical (•OH) at tumor tissues, thus killing tumor cells 2 shell was decomposed in tumor microenvironment to release Mn2+, which can catalyze H2O2 to produce •OH at the presence of HCO3- in physiological environments by a Fenton-like reaction manner as that catalyzed by iron ions 2+ from tumor pH-responsive γ-MnS nanoparticles could be also used for CDT of cancer.For cancer therapy, chemodynamic therapy (CDT) can convert hydrogen peroxide (H2S) and hydrogen (H2) 2S gas is an important endogenous bio-signaling molecule, similar to NO and CO 2S gas shows a substantial anti-proliferative effect on MCF-7 breast cancer cells 2S gas has also been reported in living organisms 2S donors, such as Na2S and NaHS, the H2S gas release is too fast to maintain a long-term effect, while organic compounds, such as GYY4137, are too complicated to obtain 2S donors is highly desirable for cancer gas therapy.Additionally, gas therapy is an emerging therapeutic strategy based on the bioeffects of several kinds of gases, such as nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide  as a template for tumor pH-responsive T1-weighted MRI guided the integration of CDT and gas therapy (Scheme 1). BSA is used to regulate the size of MnS nanoparticles by tuning the ratio of BSA and Mn2+. The as-prepared MnS@BSA is tumor pH-responsive and will be dissociated in acidic tumor microenvironment, producing H2S gas and releasing Mn2+ ions simultaneously. The former can be used for gas therapy while the latter can provide CDT and serve as a contrast agent for T1-weighted MRI.The combination of CDT and H3)2 to a Na2S solution. Typically, different amounts of BSA  was added into 400 µL of Na2S (0.5 M). The volume was later set to 40 mL using deionized water and pH was adjusted to 7.4-7.8 using 0.5 M H2SO4. After 5 min of vacuuming to remove O2, the system was protected by N2. Then 2 mL of Mn(NO3)2 (0.05 M) was added to the mixed solution by a micro-injection pump in 30 min. A flesh pink product was produced, washed and concentrated by centrifugation . The obtained nanoparticles were named as MnS@BSA-2.5, MnS@BSA-5 and MnS@BSA-10. The MnS@BSA-10 with uniform size was used for further studies.MnS@BSA were synthesized by gradually adding Mn was added into a 3500 Da dialysis bag with PBS buffer (50 mL 10 mM) at pH 6.8 and 7.4, respectively. The solutions were kept stirring at 37 °C and 2 mL was removed for analysis at 0, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h. Mn element quantification was performed by using an ICP-AES. For H2S, 400 μL of MnS@BSA (5.5 mM) was added into a 3500 Da dialysis bag with HEPES buffer (50 mL 10 mM) at pH 6.8 and 7.4, respectively. 1 mL of the solution was removed as a working solution for H2S analysis at 0, 1, 2, 5, 10, 20, 30, 60 min. H2S concentration was analyzed using a standard MB method2, 20 μL of DMPD (20 mM) and 20 μL of FeCl3 (30 mM) were mixed, kept at room temperature for 15-20 minutes, and the optical density (OD) at 663 nm was measured.For Mn2 was added into 10 μg/mL MB solution which containing 25 mM NaHCO3 and 10 mM H2O2. The OD@665 nm of the above mixture solution was monitored by UV-Vis spectrophotometer at different time points from 0 to 60 min. For ESR detection, 25 mM of NaHCO3 were used as the solvent for H2O2 (10 mM) and DMPO (100 μM). Five groups were tested: H2O2, MnS@BSA, MnCl2, H2O2 + MnS@BSA, and H2O2 + MnCl2. The concentrations of MnCl2 and MnS@BSAwere set to 5 μM and the total volume was 0.5 mL. All reagents were mixed and kept for 30 min before hydroxyl radicals were measured.MnS@BSA or MnCl2, Na2S ([Mn]/[S] = 0-200 μM) for 24 h. To assess the cytotoxicity of MnS@BSA, various concentrations of antioxidant lascorbic acid  in 10 µM with different concentration of MnS@BSA ([Mn]=0-200 μM) were tested. The standard MTT assay was carried out to evaluate the cell viability.MCF10A and 4T1 cells were seeded into 96-well plates at a density of 5000 cells per well. After 12 h in the incubator, the cells were incubated with MnS@BSA, MnCl5 of 4T1 cancer cells were digested and resuspended into 2 mL of DMEM and subcultured into φ15 confocal laser scanning microscopy (CLSM)-exclusive culture disks for another 12 h. Subsequently, the medium was removed and the disks were rinsed by PBS twice before adding 1 mL of DMEM containing 50- 200 μM of MnS@BSA, 200 μM of MnCl2. Finally, the medium was removed and the fluorescence probe addition: 25 μM of non-fluorescent DCFH-DA was added to reduce hydroxyl radical for 30 min, then the fluorescence of DCF was observed on CLSM. For H2S, 2.5×105 4T1 cancer cells were subcultured into 24-well plates for 12 h. After incubated with 100 μM of the WPS-5 probe for 30 min, then treated with 0, 50, 100, 200 μM of MnS@BSA and 200 μM of Na2S. Then added HEPES containing 100 μM of CTAB . After 10 min, fluorescence images were acquired.5×102). T1-weighted images were acquired by FSE sequence at 0, 1,2, 4, 8, and 24 h and the following parameters were applied: TR=700 ms, TE=14.3 ms, Flip Angle=145 º, matrix size, 160 x 160, slice thickness, 1.5 mm. Signal intensities were measured in defined regions of interest (ROIs) with software named Image J. MRI were performed at 0, 1, 2, 4, 8, 24 h post-injection.Tumor-bearing mice were scanned with a 3T clinical United Imaging 790 MRI scanner  before and after intravenous administration of 2.5 mg/kg of Mn (MnS@BSA or MnClin vivo bio-distribution of MnS@BSA.Six tumor-bearing mice were divided into two groups (n=3). After administration of 2.5 mg/kg of MnS@BSA through their tail veins. One group was euthanized after 4 hours, and the other was euthanized after 24 hours. Tumors and major organs  were obtained and washed with PBS. Each organ was immersed in 3 mL of nitric acid overnight, and then heated to 150 ℃. The final volume was set to 1.5 mL. The concentration of Mn was measured by ICP-AES to estimate the 2, Na2S, MnS@BSA -2.5, MnS@BSA -5, MnS@BSA -10 and MnS@BSA -10+AA. Their body weights and tumor volumes were measured every two days to evaluate the therapeutic performance. FL imaging of mice were recorded on day 2, 4, 6, 8 and 14 post-treatments. Before FL imaging, 15 mg/mL of D-luciferase potassium salt was intraperitoneally injected at a dose of 10 μL/g, and bioluminescence imaging was performed at 10-15 min post-injection for tumor growth evaluation.The therapeutic effects of MnS@BSA were examined on 4T1-Luciferase mammary tumor xenograft on BALB/c mice. Tumor-bearing mice were randomly divided into seven groups: 5 mice per group, administered dose was 2.5 mg/kg MnS@BSA. The seven groups were: PBS, MnClFigure ). Briefly, manganese source (MnCl2) was slowly added to a mixture solution of Na2S and BSA, which was vacuumed and protected with N2 gas to obtain a pink solution , the typical color of γ-phase MnS. XRD pattern (JCPDS Card No.40-1289) further confirmed that MnS@BSA is γ-phase MnS . As shown in Figure  MnS@BSA exhibited well-defined sphere. The size of MnS@BSA can be controlled from 300 to 150 nm by adjusting the amount of BSA from 2.5 to 10 mg. More BSA were added, the size of MnS@BSA were smaller . The MnS@BSA (10 mg of BSA) with the smallest size (~ 150 nm) was selected for the following experiments. As shown in the element mapping of MnS@BSA , C, N and O elements were assigned to BSA, S and Mn elements were assigned to MnS. These results indicated that γ-phase MnS was successfully synthesized using BSA as template, which can improve its solubility and biocompatibility.The α-MnS was commonly synthesized by the high temperature decomposition of manganese oleate in oil phase Figure  and 2B, MnS@BSA was degraded much faster in the acidic solution than in the neutral solution. These results are consistent with the levels of Mn2+ released from MnS@BSA in PBS buffer at different pH values . After 8 h, 98.3% of MnS@BSA was degraded in acidic solution, while only 57.3% of Mn2+ was released at pH 7.4. Afterwards, the degradation of MnS@BSA in 4T1 cells was observed using bio-TEM imaging at 8 and 24 h post-incubation . A lot of MnS@BSA were observed inside 4T1 cells after 2 h incubation. After 24 h incubation, sphere MnS@BSA were hardly found in 4T1 cells. These results indicated that MnS@BSA could be degraded in 4T1 cells gradually. Meanwhile, the concentration of released H2S from MnS@BSA under different pH conditions was measured using the methylene blue (MB) method. The H2S gas from MnS@BSA was released much faster in the acidic solution than in the neutral solution . The concentration of H2S can reach up to 12 µM, which is able to kill cancer cells efficiently. These results suggested that pH-responsive MnS@BSA could be fully degraded in aqueous solutions at pH 6.8, followed by the release of Mn2+ and H2S gas, which promises for cancer diagnosis and treatment.The as-synthesized MnS@BSA can be easily decomposed and oxidized in aqueous solutions containing oxygen, especially under acidic conditions. As shown in 2+ and HCO3- in the blood constitute the catalysts for Fenton reactions, which can consume enriched H2O2 in tumor microenvironments to generate •OH radials. In order to evaluate the Fenton-like reactivity of MnS@BSA, MnS@BSA was added into MB solution containing HCO3- and H2O2, as shown in Figure . The degradation of MB indicated the concentration dependency of manganese concentrations. Both MnCl2 (used as a control) and MnS@BSA produced •OH that could degrade MB at a similar rate . A unique quadruple peak of •OH was observed in electron spin resonance (ESR) spectra , but was not observed in control groups. All results indicated that released Mn2+ ions from MnS@BSA can generate a lot of •OH under physiological conditions.Manganese element is a trace element in living organisms, which may catalyze Fenton-like reactions in physiological environment to produce highly toxic •OH radicals in vitro experiments of MnS@BSA were performed to evaluate its combination cancer therapeutic effect and cytotoxicity. As shown in Figure , cell viability showed a concentration-dependent therapeutic effect on 4T1 cells when incubated with Na2S, MnCl2 and MnS@BSA (10-200 µM), which is in agreement with the live/dead cell staining results . The therapeutic effect of MnS@BSA on tumor cells can be attributed to the bioeffects of both H2S gas and •OH radicals. The mortality of 4T1 cells caused by H2S gas and •OH radicals was calculated in Figure . As one can see that 65-69% of cytotoxicity was induced by •OH radicals, which suggested •OH radicals were more potent than H2S gas at all tested concentrations. Interestingly, Na2S, MnCl2 and MnS@BSA showed negligible cytotoxicity on MCF10A cells (a control cell line) as evidenced by the maintained cell viability at ~75% at all tested concentrations (10-200 µM) . The different cytotoxicity between 4T1 and MCF10A cells was attributed to the levels of H2O2 inside cells. Indeed, the H2O2 concentration of 4T1 cells was 2.2 times higher than that of MCF10A , followed by HeLa and A375 cells. This higher concentration of H2O2 allowed for the enhanced •OH generation for responsive CDT treatment of 4T1 cells but no other cell lines. Therefore, MnS@BSA elicited an improved therapeutic effect on tumor cells with high levels of H2O2. To further demonstrate the therapeutic mechanism, reductive L-ascorbic acid (AA) was used to neutralize •OH radicals at cell level. By adding 10 µM of AA to different concentrations of MnCl2  or MnS@BSA , 4T1 cell viability could increase by 10-21%. In particular, with the increase of AA, toxic effects of •OH radicals produced by MnS@BSA were gradually decreased . These results indicated that therapeutic effects of MnS@BSA were attributed to the combination effects of •OH radicals and H2S gas.Next, in vitro, 4T1 cells were incubated with different concentrations of MnS@BSA for 4 h. Then the probe of oxidative stress, 2',7'-dichlorofluorescin diacetate (DCFH-DA), was added. After deacetylates with intracellular esterase to form the non-fluorescent 2',7'-dichlorofluorescin (DCFH), DCFH would be further oxidized into bright green fluorescent dye, 2',7'-dichlorofluorescein (DCF) by reactive oxygen species (ROS) including •OH radicals Figure ). These results are consistent with the results of Fenton-like reaction mentioned above. Similarly, free MnCl2 also could induce the generation of ROS. Meanwhile, H2S gas released in 4T1 cells was observed by adding a Washington State Probe-5 (WSP-5) H2S fluorescent probe . It is based on 2-pyridyl disulfide fluorescent, which release the fluorophores and turn on the fluorescence by tandem nucleophilic subsitution-cyclization reaction. WSP-5 probe can selectively and rapidly react with H2S in cells to generate a green fluorescence signal 2S group, further indicating that MnS@BSA can release H2S gas inside cells. All these results proved that MnS@BSA can generate •OH radicals and H2S in 4T1 cells, promising for combined CDT and H2S gas therapy.In order to directly observe the generation of •OH radicals 2+ ions from MnS@BSA are not only a catalyst for the Fenton-like reaction, but also a contrast agent for tumor pH-responsive T1-weighted MRI. Since MnS@BSA is pH-responsive in acidic solutions, its longitudinal relaxivity (r1) was measured under different pH conditions, as shown in Figure S7. The T1 relaxivity of MnS@BSA, r1, was calculated to be 23.67 mM-1s-1 in a pH 6.8 buffer at 2 h. Encouraged by the high r1 value of MnS@BSA at pH 6.8, we performed in vivo tumor pH-responsive MRI of MnS@BSA on 4T1 tumor-bearing mice after intravenous (i.v.) injection with MnCl2 as a control. Tumor signal of 4T1 bearing mice treated with MnS@BSA clearly increased over time, while the tumor signal kept no change for mice treated with MnCl2 . The tumor to normal ratio (TNR) of mice treated with MnS@BSA reached to a peak number of 2.26 at 2 h post-injection . The mice treated with MnCl2 showed negligible MR contrast enhancement. These results were further confirmed by the biodistribution study of Mn element at 4 h post-injection . Since Mn2+ can be gradually excreted through metabolism, we also monitored Mn levels in excrements . At 48 h post-injection, Mn concentration in mice was reduced to 0.02%, suggesting that MnS@BSA could be fully excreted within one day. These results suggested MnS@BSA could be fully cleared from the body of mice and exempt from the risk of potential long-term toxicity.The released Mnin vivo MnS@BSA-induced therapeutic effect was studied on 4T1-luc tumor-bearing mice. Mice were randomly divided into 5 groups: (i) saline, (ii) MnCl2, (iii) Na2S, (iv) MnS@BSA, (v) MnS@BSA+AA. The tumor volumes were monitored every 2 d during 2 weeks and normalized to their initial size in Figure . The saline control group showed fast tumor growth, whereas the MnS@BSA treated group exhibited higher tumor suppression as compared with MnCl2 and Na2S treated group. The MnS@BSA+AA treated group exhibited obvious tumor regrowth after day 11, due to the neutralization of •OH radicals by AA. These results suggested the combination effect of CDT/gas therapy over any single modality treatment solely. For all groups, mice showed no noticeable body weight change, suggesting no systemic toxicity of MnS@BSA . Meanwhile, the survival of mice administered with MnS@BSA was greatly prolonged . More importantly, no obvious toxicity was induced by MnS@BSA, as evidenced by results of blood biochemistry, hematology analysis, and acute toxicity measurement of all major organs . Furthermore, all tumor growth was also monitored using bioluminescence imaging during treatment period. As shown in Figure  the tumor luminescence was located on right hind of all groups in the whole treatment process. Especially, only the tumor of MnS@BSA group showed obviously suppression effect, but tumors of other groups kept growth. After different treatments, H&E staining images of all treatment groups were taken . Tumors of saline, MnCl2 and Na2S groups show more chromatin and large nuclei, indicated silght effect of tumor cell proliferation. But for MnS@BSA treated group, the tumor slices shown that the chromatin was pyknotic or even absent in the slice. MnS@BSA-AA group can consume some ·OH to reduce the oxidative damage, which show more chromatin than MnS@BSA group. The tumors treated with CDT/gas combination therapy showed much higher damage than other groups.Finally, 2S gas therapy. The as-prepared metastable-phase MnS@BSA is able to response mildly acidic microenvironment and can release Mn2+ for Fenton-like reaction to generate •OH at the presence of endogenous H2O2 of tumor cells. The MnS@BSA can also generate H2S to benefit gas therapy of cancer. Furthermore, the size-controllable MnS NPs can be used as an MRI contrast agent for treatment monitoring owing to its high r1 relaxivity of Mn2+ ions. This work provides a new design strategy of a nanotheranostic agent for traceable H2S gas therapy ydrogen primed CDT. Our findings open new horizons for the biomedical applications of manganese-based cancer theranostics.In conclusion, we have developed a tumor pH-responsive metastable-phase MnS@BSA for combined CDT and H