Since H2O2 is rapidly degraded, we additionally exposed the cell cultures to glucose oxidase (GOx), resulting in sustained exposure to H2O2. Using these model systems, we have focused on the effects of short- and long-term oxidative stress on viability, migration, differentiation, and signaling. All cellular functions examined were affected by the applied oxidative stress. The differences that occur between pulsed and sustained oxidative stress indicated higher oxidative stress in MSC upon direct H2O2 exposure, whereas the GOx-induced prolonged exposure to H2O2 seems to allow for better cellular adaptation. The mechanisms underlying these different responses are currently unknown.Reactive oxygen species (ROS) can irreversibly damage biological molecules, a process known as oxidative stress. Elevated ROS levels are associated with immune cell activation. Sustained immune system activation can affect many different cells in the environment. One cell type that has been detected in almost all tissues of the body is mesenchymal stem/stromal cells (MSC). MSC possess proliferation and differentiation potential, thus facilitating regeneration processes. However, the regenerative capacity of MSC might be impaired by oxidative stress, and the effects of long-term oxidative stress on MSC functions are sparsely described. The examination of oxidative stress is often performed by exposure to H ROS aree (H2O2) . A decree (H2O2) . Elevated ROS levels are often connected to the activation of immune cells  releasing ROS, which is known as an oxidative burst. As part of innate immunity, this process is essential for the degradation of internalized pathogens  . HoweverMSC have a high proliferation and differentiation potential. These properties allow them to renew the fraction of mesenchymal progenitor cells or to differentiate into tissue-specific cell types ,17. Thus2O2 [2O2 is one of the most important compounds for the study of ROS signaling due to its physicochemical properties, which are relatively low reactivity and the ability to diffuse through membranes [2O2-induced oxidative stress include superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) [2O2 or ROS, are still unclear. The effects of long-term ROS exposure are of particular interest as persistent ROS production has been reported in the chronification of inflammatory processes [The effects of oxidative stress are often illustrated in vitro by exposure to the oxidative stressor H2O2 ,25,26. Hembranes . A numbese (GPx) . Howeverrocesses ,30. ROS 2O2 exposure on MSC from adipose tissue  in vitro. Not only were the adMSC treated with H2O2, but also with glucose oxidase (GOx), an enzyme that produces H2O2 by glucose oxidation [2O2 was induced in the culture medium. Thus, we induced two different characteristics of H2O2 exposure: the first rather pulsatile, the second rather persistent. Therefore, this study focused on understanding the effects of short- and long-term oxidative stress induced by Hxidation . In this2O2 and ROS present or generated after treatments with these oxidative stressors and investigated their effects on the proliferation, viability, migration, and differentiation of adMSC. In addition, the underlying signaling processes were investigated. These studied aspects are important influencing variables of the regeneration and wound healing process [In this study, we quantified the amount of H process ,33. Unde2O2 in the cell culture medium, the Amplex UltraRed assay was performed. This measurement allows us to assess the amount of H2O2 added or produced by GOx and the scavenging capacity of the cell culture medium. For this purpose, different concentrations of H2O2 and GOx  were added to the cell culture medium . The quantification of H2O2 was carried out for 48 h at different time points. Directly added H2O2 in the cell culture medium was almost completely degraded over a period of 48 h . After 24 h of exposure, a maximum of 10% of the initially applied amount was detectable in each case  and thereafter exposed to H2O2 or GOx, and the resulting fluorescence was quantified. The intracellular ROS amount increased dose- and time-dependently (significant at 0.25 mM H2O2) and was nearly saturated after 50 min in H2O2-treated adMSC  were determined after the exposure to H2O2 and GOx in different concentrations (as stated above). The H2O2 and GOx treatment took place every second day. This results in different treatment frequencies depending on the respective analysis time point. After 1 day of treatment, no significant change in adMSC number  from 0.05 mM H2O2 , thioredoxin-1 (Thio-1), paraoxonase 2 (PON2), heat shock protein-70 (HSP-70), ADAM metallopeptidase 1 (ADAMTS1), and sirtuin 2 (SIRT2) (To investigate the extent of stress exerted by Hproteins . For thiantified . The pro (SIRT2) . The mos (SIRT2) .2O2, and 2.5 mU/mL of GOx for 48 h showed an increased release of IL-8 in the cell culture supernatant. The most significant effect was observed in 0.1 mM H2O2-treated adMSC, where 322 pg IL-8/mL was detected compared to 46 pg/mL in the untreated control cultures (2O2 for 24 h (2O2 concentration (0.05 mM) and 2.5 mU/mL of GOx did not induce marked differences in nuclear translocation at the two tested time points of 8 and 24 h.The influence of oxidative stress on inflammatory reactions of adMSC was investigated by the quantification of the proinflammatory cytokine Interleukin-8 (IL-8). adMSC treated with 0.05, 0.1 mM Hcultures A. To furfor 24 h B. The lo2O2 exposure on the intracellular calcium (Ca2+) level was investigated. The Ca2+ concentration was measured by flow cytometry after the treatment of adMSC with different concentrations of H2O2 and GOx for 24 h  in response to oxidative stress using a ‘Human Adipokine Array kit’ , were differentially regulated by exposure to the oxidative stressors  for 24 h before scratch and were not further treated with the oxidative stressors after scratching. Second setting: cells were both pre-treated with the oxidative stressors and further treated with the oxidative stressors after scratching. In setting one, the fluorescent depiction of the scratch area indicated an increased migratory capacity in all treated adMSC compared to the control, as more cells covered the scratch area . This test was performed under two different settings. First setting: the cells were treated with the oxidative stressors  led to an apparent reduction in the number of lipids  were free of lipid accumulation C. Notablf lipids C. The que to GOx D.2O2-/GOx-treatments. A dose-dependent decrease in relative cell amount was observed in adMSC repeatedly treated with H2O2 and GOx compared to the osteogenically stimulated, non-oxidatively stressed control (significant at 0.05 mM H2O2 and 5 mU/mL GOx) (p < 0.001) in osteogenically stimulated adMSC with increasing concentrations of H2O2 and GOx . Where the osteogenic stimulation and simultaneous treatment of adMSC with H2O2 and GOx reduce the ALP activity in a dose-dependent manner (significantly at 0.05 mM H2O2 and 5 mU/mL GOx with p < 0.05)  activity, a bone-specific enzyme, and an early marker for osteogenic stimulation. The relative cell amount was determined after 21 days of osteogenic stimulation and repeated H/mL GOx) A. In con and GOx B. The de and GOx C. In con and GOx C. Osteog < 0.05) D.ROS serve as an essential signaling molecule in the maintenance of cellular functions ,34. ROS 2O2 on oxidative stress response in MSC [. The stability of H2O2 in biological environments is fundamentally limited, and antioxidant substances in biological environments  shorten the shelf life of H2O2 [2O2 as a model substance for oxidative stress. For this reason, we have additionally chosen exposure with GOx for our in vitro model. GOx produces H2O2 in a two-step redox reaction, and the concentration of the end product (H2O2) depends on enzyme activity and substrate (glucose) availability [2O2 [A number of in vitro studies have investigated the effects of He in MSC ,42,43,44 of H2O2 . This balability . Treatmeity [2O2 .2O2 concentration in the medium after direct exposure to H2O2 is significantly reduced over 24 h. In contrast, the H2O2 concentration after GOx exposure in the medium is relatively stable over the period of 48 h. Therefore, we refer to direct H2O2 exposure as pulsed H2O2 exposure in the following discussion, whereas we refer to GOx treatment as sustained H2O2 exposure. It is important to note that the resulting maximal amounts of H2O2 detected were almost the same for the direct H2O2 treatment or with GOx activities we investigated. However, adMSC direct exposure to H2O2 leads to a significantly higher intracellular ROS accumulation compared to exposure to GOx (H2O2 induces more than twice as high ROS quantities). We assumed that cellular response to the exposure of both oxidative stress types could differ.Our results confirm that the H2O2 and GOx for 24 h led to an elevation of stress proteins, PON2, ADAMTS1, Sirt2, and HSP-70, also antioxidant proteins, SOD2 and Thio-1. We observed a more prominent elevation of these proteins in adMSC exposed to GOx (2.5 mU/mL). The increase in these proteins was confirmed for their cytoprotective effect. For example, HSP-70 works together with antioxidant systems to inhibit the ROS effect and impair the apoptotic mechanism [2O2 exposure compared to pulsed H2O2 exposure that we have shown could be due to the higher increase in antioxidant factors in GOx. However, exposure of adMSC to oxidative stressors for 48 h led to a decrease in the expression of the mentioned antioxidant factors. This indicates that prolonged ROS generation might induce exhaustion of antioxidant enzymes. Moreover, disturbed redox equilibrium between ROS and antioxidants confers cellular dysfunction and progresses to oxidative damage [Cells can activate several signaling pathways in response to the elevation of ROS production . These sechanism . Elevatie damage ,7.α (HIF-1α) [2O2, and in turn, pro-inflammatory cytokines IL-6 and IL-8 confirmed ROS facilitated the production of these cytokines. GOx-induced H2O2 exposure in adMSC instigates lower inflammatory responses.Furthermore, ROS initiate several signaling processes, including the expression of genes responsible for survival through the activation of transcription factors, like NF-ҡB and hypoxia-inducible factor-1(HIF-1α) . ROS ini(HIF-1α) . Our res2+ regulates the generation and degradation of ROS, thereby maintaining the redox state in the cell [2+ levels. Exposure of adMSC to oxidative stressors increased the cytosolic Ca2+ levels dose-dependently. However, we observed a more pronounced increase in cytosolic Ca2+ in adMSC exposed to GOx-induced H2O2 than direct H2O2 exposure. An increase in intracellular Ca2+ has also been described upon H2O2 treatment in human endometrial stem cells [2+ is bidirectional; increased Ca2+ concentrations activate ROS-generating enzymes and free radical formation, leading to increased ROS production [Several studies have shown that intracellular Cathe cell . Therefoem cells . The intoduction . There i2O2 and GOx before induced migration  or their continuous exposure during induced migration (setting 2). We found that both oxidative stress types had a stimulatory effect on adMSC migration and that preconditioning enhanced their capacity to migrate dose-dependently.In a disease state with increased inflammation and oxidative stress, the migration of MSC is another important property required for their regenerative activity . However2O2 rather than GOx. There is evidence that pre-incubation of bone marrow or Decidua Basalis MSC with H2O2 leads to enhanced migration when the migration process is induced without sustained oxidative stress [2O2 on the migration of MSC are sparsely studied, conclusions can be drawn from the light treatment of MSC with resulting ROS formation. Light treatment (low-level laser) of MSC induced increased intracellular ROS levels and was found to accelerate MSC’s migration through the extracellular signal-regulated kinases (ERK) 1/2 and focal adhesion kinase (FAK) pathways. Gingival fibroblasts have also been shown to migrate to a greater extent when treated with a blue diode laser. adMSC treated with light of different wavelengths have been shown to stimulate their migration capacities [Furthermore, the migratory capacity of adMSC was improved by preconditioning with He stress ,25. Sincpacities . Conside2O2 or GOx, resulted in a concentration-dependent decrease in adipogenic and osteogenic differentiation in adMSC. Recent studies have described the influence of ROS on adipogenic or osteogenic differentiation of MSC through the activation or inhibition of the signaling cascade involved in the respective differentiation process [2O2 [The differentiation capacity of MSC is another important function in regeneration processes. In our study, all kinds of induced oxidative stress, even at very low concentrations of H process ,52. Rodr process . Atashi  process . Osteogeess [2O2 ,54. Oxidess [2O2 , and mayHowever, a literature review on the adipogenic differentiation capacity of MSCs under oxidative stress reveals ambiguities, as some studies have reported reduced adipogenic differentiation, which may be related to oxidative stress-induced cell aging and senescence ,57,58. I2O2 is degraded more rapidly by this compound [Ultimately, there is agreement that ROS and oxidative stress have an influence on MSC differentiation. It must be emphasized that all the results have arisen from differently designed in vitro models and the various models used to show apparent differences in their setup. For example, we deliberately omitted pyruvate from our experimental approaches, as the Hcompound . These d2O2 or sustained H2O2 exposure through the enzymatic reaction of GOx, as some differences between these two types of treatment were revealed:2O2 concentrations, direct pulsed exposure of adMSC to H2O2 resulted in significantly higher ROS development compared to sustained GOx-induced H2O2 exposure.Despite nearly equal maximum H2O2 showed higher cytotoxicity than sustained GOx-induced H2O2 exposure.Direct pulsed exposure of adMSC to H2O2 exposure.Antioxidant enzymes were more abundant during sustained GOx-induced H2O2 exposure.Release of inflammatory factors was higher during pulsed H2O2 before migration and showed high sensitivity to the degree of oxidative stress since exposure to higher but non-cytotoxic pulsed H2O2 concentrations during migration completely blocked their migratory activity.Migratory activity of adMSC was increased by exposure to H2O2 exposure conditions.Both adipogenic and osteogenic differentiation is reduced under all HThis study extends the current knowledge on the effects of long-term exposure of adMSC to pulsed H2O2 exposure induces higher oxidative stress in adMSC in vitro than in GOx-induced, sustained H2O2 exposure. This is probably triggered by a better cellular adaptation to the oxidative stress conditions when they are constant. However, patient-specific characteristics, such as age, gender, chronic diseases, and genetic variants might influence the resistance of MSC to oxidative stress [These results indicate that the direct pulsed He stress ,62. ThesTM , hence referred to as the ‘complete culture medium’ and cryopreserved in passage 2 based on the previously described procedure [2 flask  [2 in plates 4- to 96-well . DNA staining with DAPI  microscopically confirmed the absence of mycoplasma contamination. Primary human adipose tissue-derived mesenchymal stem/stromal cells (adMSC) were isolated from liposuction tissue of healthy patients with an average age of 50 ± 10 years. The aspirated tissue was transported overnight at room temperature (RT), and the stromal vascular fraction (SVF) was isolated according to a previously described protocol . CD34-porocedure . For allGermany) . All exp2O2  or GOx  with a final concentration of 0.01, 0.05, 0.1, 0.25 mM and 0.1, 0.5, 1, 2.5, and 5 mU/mL, respectively. Unless otherwise stated, these were the treatment media used for the experiments. adMSC with media without these oxidative stressors will be referred to as control hereafter. Treatments were repeated every 2–3 days throughout the experiment. After 72 h of cell cultivation in a complete culture medium without pyruvate, cells were supplemented with H2O2 in the treated media was quantified using Amplex UltraRed  according to the manufacturer’s instructions. Complete media treated with different concentrations of H2O2 and GOx were measured for 1 h to 48 h. Relative fluorescent intensity was measured using a microplate reader . The H2O2 concentration was calculated using a standard curve. The amount of H2DCFDA, Thermo Fischer Scientific, Waltham, MA, USA) ROS indicator. 72 h after seeding, CM-H2DCFDA was added to the cells as a probe at a final concentration of 10 µM in pre-warmed phosphate-buffered saline  without Ca2+ and Mg2+ and incubated at 37 °C for 30 min. After incubation, the fluorescent probe was removed, and the cells were washed with 1 × PBS without Ca2+ and Mg2+. Finally, cells were exposed to different concentrations of H2O2 and GOx, as described above, whereas cell cultures without treatment served as controls. Intracellular ROS accumulation was measured using a microplate reader . Fluorescence was quantified at 492 nm excitation and 527 nm emission every 10 min for 50 min until saturation was reached. The results were normalized to the cell count to estimate the amount of accumulated ROS per individual cell. Intracellular ROS were detected using 2′-7′-dichlorofluorescein diacetate . Cells were washed three times with PBS without Ca2+ and Mg2+ and incubated with 5 µg/mL Hoechst H33342 for 10 min in the dark, and the washing step was repeated. Images were acquired using the Hermes WiScan system  and analyzed using the Athena software . The total number of nuclei and the average and standard deviation of triplicates for each treatment were calculated for the cell number. This process was also performed on days 1, 3, and 7. Quantification of cell number was conducted with Hoechst H33342 . Cells were first washed with PBS without CaThe metabolic activity of adMSC was determined with the CellTiter 96 Aqueous One Solution Cell Proliferation Assay  on days 1, 3, and 7 after treatment, according to the manufacturer’s instructions. For this purpose, the cells were incubated with the MTS solution for 1.5 h, using the MTS solution without cells as a blank. Afterward, the supernatant and the blank were transferred into an ELISA reading plate , and the absorbance was measured in a microplate reader  at an absorbance wavelength of 490 nm (reference wavelength 620 nm). 2O2, and 2.5 mU/mL GOx for 24 and 48 h. The cell supernatants were then collected and stored at −80 °C for further cytokines analyses. Whereas cells were lysed for 30 min at 4 °C with a lysis buffer containing a cocktail of protease inhibitors . The lysates were centrifuged at 14,000× g for 5 min at 4 °C. For each analysis, 250 µg of protein was pooled from the lysates of 5 individuals. Cell stress-related proteins were detected using the kit according to the manufacturer’s instructions. For this, chemiluminescence blots were recorded with ChemiDoc XRS , and protein intensity was quantified by densitometric analysis of the blot using the Image Lab 3.0.1 software .26 cell stress-related proteins  were detFor the determination of pro-inflammatory cytokines, IL-8 ELISA and NF-ҡB translocation were performed.For the IL-8, the previously processed cell supernatants were used and analyzed with the ELISA  according to the manufacturer’s instructions. The measured IL-8 concentration in pg/mL was normalized to the concentration of the lysates for each treatment. 2O2  and GOx (2.5 mU/mL). As a positive control, adMSC incubated with 50 ng/mL TNFα  for 30 min was included. Cells were then fixed with 4% PFA, and NF-ҡB antibody staining  was performed according to the manufacturer’s instructions. The cell nuclei were stained with Hoechst H33342, as previously described. After staining, imaging was performed using the Hermes WiScan microscope . NF-ҡB translocation was quantified by the fluorescence intensity of the nuclei using Athena software . The average intensity of the nuclei was calculated from the sum of all mean intensities divided by the number of nuclei measured. NF-ҡB nuclear translocation was determined after 8 and 24 h treatment with the same concentrations of HThe previously processed cell supernatants were used for the detection of 58 different obesity-related adipokines  with a P2+ levels were determined via flow cytometry by staining the cells with 5 µM Fluo-3-acetoxymethyl ester AM (fluo-3/AM) calcium indicator . For this approach, cells were first seeded in a 24-well plate and treated with 0.01, 0.05, 0.1, 0.25 mM H2O2 and 0.1, 0.5, 1, 2.5, and 5 mU/mL GOx for 24 h. After treatment with the stressors, the cells were washed, trypsinized, and the cell suspension was recovered in the FACS tube . The intracellular basal Ca2+ level was determined according to a previously published protocol [The intracellular basal Caprotocol . CellQue2 in a 96-well plate to achieve complete cell spreading and an almost 100% confluent monolayer. 24 h after seeding, the medium was replaced with a complete culture medium, without phenol red diluted with a red cell tracking dye, DMSO free  according to the manufacturer’s instructions. The cells were divided into two settings according to their experimental arrangement. 72 h after seeding, the cells were incubated with 0.01, 0.05, and 0.1 mM H2O2 and 0.1, 0.5, 1, and 2.5 mU/mL GOx.Cell migration was investigated with a scratch assay. Cells were seeded at 29,000 cells/cm2O2 and GOx. Afterward, the confluent monolayer was scratched with an Incucyte® Woundmaker . The cells were washed three times with PBS without Ca2+ and Mg2+, and the media were replaced with supplemented low serum media (1% FCS) without pyruvate to limit the proliferation factor. Live-cell imaging was observed immediately after scratch using the Hermes WiScan system  at 10-fold magnification in a humidified atmosphere with 37 °C and 5% CO2 for 20 h. Quantification of the wound area was performed using Fiji ImageJ software version 1.8.0_72 [The first settings consisted of cells preconditioned for 24 h with H1.8.0_72 , and the2O2 and GOx described above for 2 h. Subsequently, the scratch was made, and the cells were treated again with the same concentrations of H2O2 and GOx in the supplemented media, with 10% FCS without pyruvate. Imaging was performed immediately after the scratch and continued for 15 h. The second setting was 2 h preconditioned adMSC and 15 h in treatment. The cells were incubated with the same concentrations of HThe cell number of adipogenically differentiated cells was determined 14 days after stimulation and of osteogenically differentiated cells, 14, 21, and 28 days after stimulation. Quantification of cell number was performed using crystal violet staining, as previously described .In addition, an MTS assay was conducted on the days mentioned above to check the metabolic activity of the cells and was carried out as previously described.2O2 and 0.1, 0.5, 1, 2.5, and 5 mU/mL GOx. The procedure of adipogenic differentiation was performed based on a previously published protocol [2O2 and GOx were added to the medium. In contrast, cells without treatment served as controls (US control). The second group was the adipogenically stimulated (AS) adMSC, in which the adipogenic media  was added to the cells. At the same time, they were simultaneously treated with the above-defined concentrations of H2O2 and GOx, and the control cells (AS control) without treatment. After 14 days of stimulation and treatment, adipogenic differentiation was determined by Bodipy staining, as previously described [To observe the effect of oxidative stress on adipogenic differentiation, cells were treated with 0.01, 0.05, and 0.1 mM Hprotocol . The celescribed .2O2 and GOx were added to the medium. Cells without treatment served as controls (US control). The second group was the osteogenically stimulated (OS) adMSC, in which the osteogenic media  was added to the cells. At the same time, they were simultaneously treated with the above-defined concentrations of H2O2 and GOx, and the control cells (OS control) without treatment. ALP quantification was carried out according to a previously established protocol [2O according to a protocol published [The osteogenic differentiation capacity was determined by quantifying the enzyme alkaline phosphatase (ALP) activity. Stimulation of osteogenesis was realized by replacing the medium every 2–3 days and simultaneous repeated treatment with the stressors. The cells were divided into two groups, the first group being the unstimulated (US) adMSC, in which the above-defined concentrations of Hprotocol  and was ublished , and imap-value of 0.05.All experiments were performed independently on cells from five or more different donors, each in triplicates, and the mean of each triplicate was used for one individual. Data were visualized and statistically analyzed with Microsoft Excel 2010  and GraphPad Prism, Version 6.05 . Data were presented as bars with standard deviations and boxplots. The horizontal line within the box plot indicates the median, with whiskers showing the minimum and maximum data points. The statistical significance of the data set was calculated using a two-way analysis of variance, followed by Dunnett’s multiple comparison tests, and the level of significance was set to a All tissue samples analyzed for this experiment were collected with the patient’s consent. The use of the tissue samples was approved by the local ethics committee under registration A2019-0107.


Bone marrow mesenchymal stem cells (BMSCs) can effectively alleviate liver fibrosis, which is a pathological injury caused by various chronic liver diseases. This study aimed to investigate the antifibrotic effects of BMSCs and elucidate the underlying mechanism by which BMSCs affect liver fibrosis in vitro and in vivo.4), BMSCs were administered for 4 weeks, and histopathological analysis and liver function tests were performed. T6 hepatic stellate cells (HSC-T6 cells) were stimulated by TGF-β1, and the activation and proliferation of cells were analyzed by CCK-8 assays, flow cytometry, real-time PCR, western blotting and enzyme-linked immunosorbent assay (ELISA).After the rat liver fibrosis model was induced by continuous injection of carbon tetrachloride (CClOur data demonstrated that BMSCs effectively reduced the accumulation of collagen, enhanced liver functionality and ameliorated liver fibrosis in vivo. BMSCs increased the sub-G1 population in HSC-T6 cells. In addition, coculture with BMSCs reduced the expression of α-SMA, collagen I, cyclin-D1, and c-Myc in HSC-T6 cells and activated the phosphorylation of GSK3β. The GSK3β inhibitor SB216763 reversed the effect of BMSCs. The Wnt/β-catenin signalling pathway was involved in BMSC-mediated inhibition of HSC-T6 cell activation.Our data suggested that BMSCs exerted antifibrotic effects by activating the expression of GSK3β and inhibiting the Wnt3a/β-catenin signalling pathway. Although the liver is remarkably regenerative, fibrosis due to various chronic liver diseases can cause cirrhosis, hepatocellular carcinoma and liver failure , 2. At pIn recent years, there has been hope that stem cells can ameliorate hepatic disease due to advances in stem cell research , 5. BoneAn increasing number of molecules have been shown to play important roles in the initiation and development of fibrosis, and GSK3β is one of the important targets. GSK-3β, a key kinase in the Wnt/β-catenin pathway, participates in embryonic development, apoptosis, cell differentiation, translation and transcription , 11. ActIn this study, we aimed to investigate the antifibrotic role of BMSCs and expose the underlying mechanism of BMSCs on liver fibrosis in vitro and in vivo. First, the rat liver fibrosis model was induced by the continuous injection of CCl4, and BMSCs were administered for 4 weeks. Second, histopathological analysis and liver function tests were performed in vivo. Finally, the activation and proliferation of HSC-T6 cells stimulated with TGF-β1 were analysed by CCK-8, flow cytometry, real-time PCR, western blotting and enzyme-linked immunosorbent assay (ELISA). This study was the first to validate the reduction in BMSCs in rat liver fibrosis and found that BMSC treatment exerted antifibrotic effects by activating the expression of GSK3β and inhibiting the Wnt3a/β-catenin signalling pathway. These findings may reveal strategies for the application of BMSCs as an effective to treat liver fibrosis.2.Rat BMSCs were purchased from Cyagen Biosciences(China). T6 hepatic stellate cells (HSC-T6 cells) were purchased from Procell Life Technology (China). BMSCs were cultured in OriCell Sprague Dawley Rat Bone Marrow Mesenchymal Stem Cell Basal Medium , and HSC-T6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  with 10% foetal bovine serum (FBS)  and 1% penicillin streptomycin  at 37 °C and 5% CO4)  at a dose of 3 mL/kg body weight twice per week for 8 weeks. Then, CCl4-treated rats were randomly divided into two groups (n = 10): CCl4 + PBS (model group) and CCl4 + BMSCs (treatment group). Rats that were not treated with CCl4 served as the control group (n = 10). Rats in the CCl4 + BMSC group were injected with 1 × 106/200 µL BMSCs through the tail vein, and rats in the CCl4 + phosphate buffer saline (PBS) group rats were injected with 200 µL PBS. Four weeks later, the rats were euthanized, and the serum and livers were collected to evaluate the therapeutic effects of BMSCs on liver fibrosis by western blotting and ELISA. The animal experiment protocols were approved by the Animal Experiment Ethics Committee of Southern Medical University (Ethics No.: L2017062).Sprague Dawley (SD) rats  were purchased from Guangdong Medical Experimental Animal Centre and were housed in a specific pathogen-free environment with a day and night cycle of 12 h. To induce the liver fibrosis model, rats were intraperitoneally (i.p.) injected with 30% carbon tetrachloride  were used to physically separate the two cell populations. HSC-T6 cells were seeded in the lower chamber, and BMSCs were seeded in the upper chambe in DMEM with 10% FBS. After 24 and 48 h, the samples were collected for subsequent tests.Helicobacter pylori (HP) and catalase (CAT) levels in rat serumwere measured by ELISA kits . The concentrations of AST, ALP, HP and CAT were determined quantitatively according to the corresponding standard curves.Blood samples and urine were collected from the rats, and serum was collected after centrifugation for 10 min at 3000 rpm. Aspartate aminotransferase (AST), alkaline phosphatase (ALP), Total RNA was isolated from HSC-T6 cells according to the specifications of RNA the reagent , and reverse transcription of RNAs was performed by using the PrimeScript™ RT Master Mix kit . Then, real-time PCR was conducted by using SYBR Premix Ex Tap™ II . After normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the relative amount of the PCR products was calculated using the 2 − ΔΔCt method. The primers were designed by Primer 5.0 and are shown in Table HSC-T6 cells and liver tissue were obtained and lysed using radioimmunoprecipitation assay (RIPA) buffer . The protein concentrations of the samples were measured using a BCA protein assay kit . Then, the cell extracts were separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene fluoride (PVDF) membranes . Then, the samples were visualized by incubation with primary antibodie against alph-smooth muscle actin (α-SMA) , collagen I , matrix metalloproteinase-7 (MMP7) , Wnt3a , β-catenin , p-GSK3β , cyclin-D1 , c-Myc  and GAPDH  at 4 °C overnight, followed by incubation with HRP-labelled goat anti-mouse/rabbit IgG (H + L) secondary antibodies  for 1 h. The immune complexes were immunoblotted, and the immunodetection was performed by using enhanced chemiluminescence reagents .3 cells were seeded into 96-well plates CCK-8 reagent was added, and the plates were incubated for 2 h at 37 °C according to the manufacturer’s protocol. Optical densities were measured using a multifunctional microplate reader at 450 nm (OD450).A CCK-8 kit  was used to determine the proliferation of HSC-T6 cells. First, 5 × 10HSC-T6 cells were harvested and fixed for the cell cycle assay. The samples were fixed in 70% ethanol overnight at 4 ℃. After being centrifuged, the cells were stained with propidium iodide (PI) at 37 ℃ for 30 min using a Cell Cycle Analysis Kit . The distribution of cells in the different phases of the cell cycle were analysed on a FACSCalibur flow cytometer .p < 0.05, **p < 0.01, and ***p < 0.001 were considered significant.The data are presented as the mean ± SD. All statistical analyses were performed by GraphPad software (v6.0). Statistical significance was determined using one-way analysis of variance (ANOVA) and Tukey’s test or Student’s t test. Allexperiments were repeated three times. Values of *4-induced liver fibrosis model in rats to determine whether BMSCs could exert therapeutic effects on liver fibrosis. The rats were injected with CCl4 for 8 weeks and then treated with BMSCs for 4 weeks  . In addition, the serum and urine levelsl of CAT in the BMSC-treated group were higher than those in the model group (p < 0.05 or p < 0.01)    and β-catenin (p < 0.01) was significantly inhibited in the BMSC treatment group, while the expression of p-GSK3β in the BMSC treatment group was significantly increased (p < 0.01), which suggested that BMSCs could effectively activate GSK3β and inhibit the Wnt/β-catenin signalling pathway in rat liver fibrosis. CCK-8 and cell cycle assays showed that TGF-β1 significantly increased HSC-T6 proliferation, while FH535, a specific inhibitor of Wnt/β-catenin signalling, inhibited the proliferation of HSC-T6 cells , collagen I (p < 0.01), cyclin-D1 (p < 0.05), c-Myc (p < 0.05), MMP7 (p < 0.05), Wnt3a (p < 0.01) and β-catenin (p < 0.01) in the HSC-T6 cells. FH535 also promoted GSK3β phosphorylation (p < 0.001). These results suggested that the Wnt/β-catenin signalling pathway might be involved in the activation of HSC-T6 cells and liver fibrosis.The expression levels of proteins involved in the Wnt/β-catenin signalling pathway in rat liver tissues were analysed by western blotting Fig. A. Comparp < 0.01) and β-catenin (p < 0.01) and inhibited GSK3β phosphorylation (p < 0.001). BMSC coculture attenuated the effect of TGF-β1 on HSC-T6 cells (p < 0.01)  and β-catenin (p < 0.001), and it could also inhibit the phosphorylation of GSK3β (p < 0.01)  Fig. A. To ana01) Fig. B. Cocult01) Fig. C. In add01) Fig. D. TherefAlmost all chronic liver injury causes liver fibrosis, but the current clinical treatment of liver fibrosis is not satisfactory, and there is a lack of effective drugs, resulting in the development of cirrhosis or hepatocellular carcinoma in some patients and leading to more than 1 million deaths every year , 20. Chr4-induced rat liver fibrosis model histopathological analysis and liver function tests were performed in vivo. The data revealed that BMSC treatment could reduce the protein expression of α-SMA, collagen I, and MMP7, and it could correct the CCl4-induces increases in the expression of AST, ALT and HP. Moreover, BMSCs also reduced the rat liver tissue fibrosis index, decreased fatty degeneration and significantly enhanced liver functionality. These results suggested that BMSC treatment had an effect on the rat liver fibrosis model induced by CCl4.To investigate the therapeutic effects of BMSCs on the CClHepatic stellate cells (HSCs), which are the main effector cells in liver fibrosis, play an important role in liver fibrosis , 24. HSCIn recent years, the Wnt/β-catenin signalling pathway has been confirmed to be involved in the pathogenesis of different kinds of diseases . Some stTo further identify the role of the Wnt/β-catenin signalling pathway in treatment of liver fibrosis, CT99021 was added to HSC-T6 cells that were cocultured with BMSCs, and CT99021 could abrogate the effect of BMSCs on Wnt3/β-catenin signalling in HSC-T6 cells. CT99021 reversed the inhibitory effect of BMSCs on the proliferation and activation of HSC-T6 cells and promoted the expression of α-SMA, cyclin-D1, c-Myc, collagen I, MMP7, Wnt3a and β-catenin in HSC-T6 cells. These results suggest that BMSCs can effectively inhibit liver fibrosis through Wnt3/β-catenin signalling.In summary, our study showed that BMSCs could effectively alleviate the rat liver fibrosis and inhibit the activation and proliferation of HSC-T6 cells. These effects were associated with interference with the Wnt/β-catenin signalling pathway in hepatocytes.


Small Rho GTPases are molecular switches that are involved in multiple processes including regulation of the actin cytoskeleton. These GTPases are activated (turned on) and inactivated (turned off) through various upstream effector molecules to carry out many cellular functions. One such upstream modulator of small Rho GTPase activity is delta-catenin, which is a protein in the p120-catenin subfamily that is enriched in the central nervous system. Delta-catenin affects small GTPase activity to assist in the developmental formation of dendrites and dendritic spines and to maintain them once they mature. As the dendritic arbor and spine density are crucial for synapse formation and plasticity, delta-catenin’s ability to modulate small Rho GTPases is necessary for proper learning and memory. Accordingly, the misregulation of delta-catenin and small Rho GTPases has been implicated in several neurological and non-neurological pathologies. While links between delta-catenin and small Rho GTPases have yet to be studied in many contexts, known associations include some cancers, Alzheimer’s disease (AD), Cri-du-chat syndrome, and autism spectrum disorder (ASD). Drawing from established studies and recent discoveries, this review explores how delta-catenin modulates small Rho GTPase activity. Future studies will likely elucidate how PDZ proteins that bind delta-catenin further influence small Rho GTPases, how delta-catenin may affect small GTPase activity at adherens junctions when bound to N-cadherin, mechanisms behind delta-catenin’s ability to modulate Rac1 and Cdc42, and delta-catenin’s ability to modulate small Rho GTPases in the context of diseases, such as cancer and AD. Small Rho GTPases are enzymes that bind and hydrolyze GTP. They act as molecular on-off switches by cycling between their GTP-bound active state and their GDP-bound inactive state through their interactions with a wide variety of regulatory and effector molecules. While GTPases are involved in almost all cellular processes, the Rho subfamily is best known for cytoskeletal reorganization. Small Rho GTPases have many modulators of their activity, with one being delta-catenin . Delta-cDelta-catenin—also known as NPRAP or neurojungin —is a memThe Rho subfamily of small GTPases is part of the Ras superfamily of proteins. While more than 20 Rho GTPases are known to exist, the most fully characterized are RhoA (Ras homologous member A), Rac1 (Ras-related C3 botulinum toxin substrate 1), and Cdc42 (cell division cycle 42). GTPases are small G-proteins that bind to and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). These GTPases cycle between their active GTP-bound states and their inactive GDP-bound states through the involvement of guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and GDP dissociation inhibitors (GDIs). GEFs enhance the exchange of GDP for GTP to increase GTPase signaling, GAPs increase GTP hydrolysis to decrease GTPase signaling, and GDIs prevent the exchange of GDP for GTP to inhibit GTPase signaling . CyclingDrosophila mushroom body neurons, and Xenopus retinal ganglion cells  and dominant negative (DN) mutations. As these GTPases have roles in actin reorganization, modulating their activity in various neuronal model systems has shown that small Rho GTPases play a substantial role in dendritic arbor development. RhoA tends to have an inhibitory effect on neuronal dendrites, as CA RhoA reduces dendritic arbor growth in mouse and rat hippocampal neurons, on cells . Convers neurons . Further neurons . These s neurons . While C neurons . These d neurons . OverallXenopus retinal ganglion cells and rat primary cortical neurons, while DN Rac1 results in a significant reduction in dendritic length and branching in Xenopus retinal ganglion cells, rat primary cortical neurons, Drosophila mushroom body neurons, and mouse cortical pyramidal neurons , which is a neurodevelopmental disorder that affects behavioral and social attributes . MultiplThere have additionally been several mutations identified that appear relevant to delta-catenin contributions in schizophrenia. GWAS studies have shown that both CNVs and SNPs of delta-catenin are associated with schizophrenia . Furthervia consequent effects upon small Rho GTPases (Despite a relatively diminished presence in non-neuronal tissue, delta-catenin has been implicated in several non-neurological diseases. Both delta-catenin and small Rho GTPases have been well implicated in multiple cancer types. Given that the roles of delta-catenin and small Rho GTPases in cancer have been reviewed in detail previously , and tha GTPases . Aside f GTPases . HoweverAlthough the field has advanced delta-catenin as an established modulator of small Rho GTPases in neuronal morphology and function, understanding delta-catenin’s more defined mechanisms of action will in many cases require further work. In general, delta-catenin expression inhibits RhoA and activates Rac1 and Cdc42 . HoweverWhile both delta-catenin and small Rho GTPases have been implicated in many of the same pathologies, links between the two in the context of disease have rarely been studied, particularly in neurological pathologies . As dendMD and PM wrote and finalized the manuscript. MD and YS drafted and revised the figures. PM guided the writing process. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Canonical and non-canonical Wnt signaling pathways are involved in cell differentiation and homeostasis, but also in tumorigenesis. In fact, an exaggerated activation of Wnt signaling may promote tumor growth and invasion. We summarize the most intriguing evidence about the role of Wnt signaling in cutaneous carcinogenesis, in particular in the pathogenesis of non-melanoma skin cancer (NMSC). Wnt signaling is involved in several ways in the development of skin tumors: it may modulate the inflammatory tumor microenvironment, synergize with Sonic Hedgehog pathway in the onset of basal cell carcinoma, and contribute to the progression from precancerous to malignant lesions and promote the epithelial-mesenchymal transition in squamous cell carcinoma. Targeting Wnt pathways may represent an additional efficient approach in the management of patients with NMSC. Wnt signaling is responsible for the regulation of different intracellular signal transduction pathways, which are essential for embryogenic development, cellular migration, polarization and differentiation as well as stem cell biology control and growth ,2. HowevWnt signaling activation is related to the binding of Wnt ligands to a specific cell surface receptor which belongs to the Frizzled (Fzd) family , thus inThe Fzd receptor interacts with other coreceptors, such as the receptor-like tyrosine kinase (Ryk), the receptor tyrosine kinase-like orphan receptor 2 (Ror2) or the low-density lipoprotein receptor-related protein (Lrp)-5/6, with the consequent activation of Disheveled (Dvl) and the downstream pathways . General2+) and Wnt/Planar cell polarity (PCP) pathways [2+) pathway, the activated Fzd-co-receptor-Dvl complex induces phospholipase C γ, which converts phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3), resulting in the increased release of intracellular Ca2+. The release of Ca2+ leads to the activation of calcium-dependent kinases, such as Ca2+-dependent phosphatase calcineurin (CaN), Ca2+-calmodulin dependent kinase II (CAMKII) or protein kinase C (PKC) [The non-canonical Wnt signaling pathways are divided into Wnt/Calcium (Capathways . In part C (PKC) . The act C (PKC) ; on the  C (PKC) . Cdc42 a C (PKC) . On the  C (PKC) . These p C (PKC) ,15.IL6, IL8 and TNFA, encoding tumor necrosis factor-α (TNF-α) [β-catenin and, thus, with an increased production of collagen [Chronic inflammation is a widely recognized risk factor for several skin cancers, especially cutaneous squamous cell carcinoma (cSCC) . The rol (TNF-α) ,18,19,20 (TNF-α) ,22. In p (TNF-α) . Moreove (TNF-α) . Neverth (TNF-α) . This ph (TNF-α) . On the  (TNF-α) . The imb (TNF-α) . Canonic (TNF-α) . An incr (TNF-α) . The defcollagen . It is wcollagen . Preventcollagen . In conccollagen .C-C motif chemokine ligand 4 (CCL4) in a mouse model. This results in defective infiltration and activation of dendritic cells and T-cells. Another mechanism involved in immune escape concerns the crosstalk between tumor cells and tumor-associated macrophages (TAMs). Tumor cells may stimulate IL-1β production in TAMs via Snail, a transcription factor of canonical Wnt pathway. In turn, IL-β may increase the availability of β-catenin in tumor cells. Finally, canonical Wnt pathway may influence the activity of T regulatory cells, enhancing their survival [As in cutaneous inflammatory and autoimmune diseases, the Wnt pathway may modulate inflammatory responses in cutaneous cancers and influence tumor microenvironment (TME), thus promoting tumor development . Wnt patsurvival . A combisurvival .As more broadly described in the next paragraphs, several components of Wnt signaling pathways are involved in pathogenesis of NMSCs, both basal cell carcinoma and squamous cell carcinoma. Basal cell carcinoma (BCC) is the most frequent skin tumor. Sun exposure is the most important risk factor for BCC. In particular, intermittent, intense sun exposure appears more associated to BCC than cumulative, long-term UV exposure . Other rBCCs are characterized by nuclear localization of β-catenin  and its Cutaneous squamous cell carcinoma (cSCC) is the second most common tumor after BCC and the second most frequent cause of death from skin cancer after melanoma . CSCC arc-myc, widely known for its role in tumorigenesis [β-catenin expression gradually modifies from AKs to cSCC. As mentioned above, β-catenin in normal epidermal tissues is located in the cell surface, whereas in pathological conditions, it moves to cytoplasm or nucleus. β-catenin expression on cell membrane shows a gradual down-regulation in AKs, BD and cSCC, while nuclear or cytoplasmatic expression results progressively increased. An immunohistochemical study showed that nuclear or cytoplasmatic expression rates of β-catenin were 56.25%, 91.67% and 96.00% for AKs, BD and cSCC, respectively . One of igenesis . Additioigenesis . Severaligenesis ,64,65. Iigenesis . Depletiigenesis ,69 -related cSCC would be driven by Wnt/β-catenin signaling activation, in particular in immunocompromised patients . Both WnDifferent studies found that Wnt antagonists were down-regulated in cSCC. Two classes of extracellular Wnt antagonists are recognized according to the mechanisms of action: the first class, including Cerberus, Wnt inhibitory factor-1 (WIF-1) and secreted Frizzled-related proteins (SFRPs), binds to Wnt ligands; the second class, consisting of Dickkopf (Dkk) proteins, binds to Lrp5-6, which are part of Wnt receptor complex . SFRPs mThe canonical Wnt pathway is involved in the epithelial–mesenchymal transition (EMT), which is essential for the invasion and progression of tumors . The conThe non-canonical Wnt pathway may be involved in the pathogenesis of cSCC, although its role seems to be negligible compared to that described for the canonical pathway .The non-canonical Wnt pathway may influence SCC development through EMT dysregulation as well as survival and proliferation of cancer cells. For this reason, the non-canonical Wnt pathway may also be considered a possible target to stop tumor progression. An experimental study demonstrated that TAM67 (dominant-negative c-Jun), an inhibitor of the transcription factor AP-1, reduced tumor progression not only through the modulation of the expression of genes involved in inflammation, invasion and metastasis, but also through Wnt5a reduction, whose role in the maintenance of tumor phenotype was previously observed. In fact, Wnt5a knockdown led to inhibition of Signal transducer and activator of transcription 3 (STAT3) target genes, including metalloprotease 3 or cyclin D1, thus suppressing cSCC growth . The inhWnt pathways play a significant role in the pathogenesis of NMSCs through the activation of inflammatory processes involved in carcinogenesis as well as stimulation of cancer cell proliferation and invasion. Moreover, Wnt signaling activation sustains mechanisms of resistance to SHH pathway inhibitors, which currently represent the most advanced therapy for locally advanced or metastatic BCC. Both canonical and non-canonical Wnt pathways are involved in cSCC pathogenesis, sometime synergistically. The activation of the canonical Wnt pathway may contribute to the progression from precancerous to malignant lesions, thus sustaining cutaneous cancer stem cells, and participates with the non-canonical pathway to epithelial–mesenchymal transition. Therefore, the Wnt pathway may be considered a possible therapeutic target for the management of NMSCs in order to reduce tumor burden and improve immune checkpoint inhibitors effects in patients with BCC, also slowing the progression from AKs to cSCC and reducing the recurrences and metastatic invasion of cSCC. For this reason, Wnt signaling still represents an interesting topic to study in different research areas, such as oncology, pharmacology as well as dermatology, in order to find new possible therapeutic targets for diseases that are difficult to treat, including NMSCs.


In this work, by means of MD simulations, the folding dynamics of intact t-HOPO in its neutral (t-HOPO0) and in its deprotonated state (t-HOPO4−) were studied. The results indicated that the deprotonation of t-HOPO in the aqueous phase significantly narrowed the accessible conformational space under the simulated conditions, and it was prepared in a conformation that could conveniently clamp the cations. The simulation of UIV-t-HOPO showed that the tetravalent uranium ion was deca-coordinated with eight ligating O atoms from the t-HOPO4− ligand, and two from aqua ligands. The strong electrostatic interaction between the U4+ ion and t-HOPO4− further diminished the flexibility of t-HOPO4− and confined it in a limited conformational space. The strong interaction between the U4+ ion and t-HOPO4− was also implicated in the shortened residence time of water molecules.The octadentate hydroxypyridonate ligand 3,4,3-LI ( CaL = 10.28) in 1947 - complex could not survive geometric optimization unless a distance restraint was imposed.As shown in OPO ring , which aobserved . It couloworkers , the meaoworkers  reportedt-HOPO)]0 complexes reported in previous experimental and computational studies 2−∙(H2O)2 and [UO2(κ6-t-HOPO)(H2O)2]2−, which showed the competing coordination of t-HOPO and aqua ligands with actinides.The elusive coordination interaction between actinide ion and ligating water molecules in the [U, which benefited from its more negative atomic charge than the deprotonated Oh site, built stronger interaction with U, as seen in the shorter UIV-Oc distance than that of UIV-Oh.In the [U4+, the t-HOPO4− became more compact in view of its smaller radius of gyration (Rg) . The fluctuations of θ1 and θ2 were directly relevant to the conformational stability of the t-HOPO4− ligand; thus, the smaller fluctuations of θ1 and θ2 in the UIV-t-HOPO than those in the free t-HOPO4− indicated the significantly narrowed conformational space of the ligand upon its coordination with U4+.Upon binding with Uion (Rg) a and itsion (Rg) b. This at-HOPO4− . In t-HOIV-t-HOPO complex than that for t-HOPO4−, indicating that t-HOPO4− was confined in a limited conformational space upon binding with U4+. To analyze the details in the conformational changes in the UIV-t-HOPO complex, a smaller cutoff value of 0.05 nm was used in the analysis of the UIV-t-HOPO complex to obtain a distribution of the clusters with the population of the cluster 1 .Clustering analysis. With the same cutoff value, the conformational clustering analysis gave a significantly smaller number of clusters for the Ur 1 51%, c compara complex , which fIV-t-HOPO complex at about 50 ns, as seen from the sharp change in RMSD  was calculated according to the following thermodynamic cycle:Binding free energy. The binding free energy of U3 represents the energy cost to transfer a dummy atom from water to HOPO and is equal to zero. Following the thermodynamic cycle, the binding free energy may be calculated as1 and ΔG2 were calculated by using the free energy perturbation (FEP) method.where ΔGt-HOPO4− showed strong affinity for U4+, which was calculated to be up to −81.3 kcal/mol. The strong interaction between t-HOPO4− and U4+ benefitted from the strong electrostatic interaction between them. In addition, as discussed above, the t-HOPO4− showed much lower conformational flexibility than its neutral counterpart t-HOPO0 as a consequence of the balance between the hydrophilic and hydrophobic interactions. The negatively charged t-HOPO4− adapted better in the aqueous phase than t-HOPO0 and could be populated mainly with its dominant conformation  complex.The decomposition of the interactions between U ligands  showed m4+ were taken from Merz et al. [All molecular dynamics simulations were conducted by employing the Gromacs 5.1.4 package . The bonz et al. . Long-raz et al. . The cutz et al.  to allowT = 2.0 ps [P = 1.0 ps [All simulation systems were first relaxed by 1000 steps of energy minimization (the steepest descent), equilibrated 300 ps for NVT and then for 500 ps at a constant pressure of 1 atm and temperature of 310 K . The equilibrated model systems were then sampled at 310 K and 1 atm with the NPT ensemble for 100 ns for analysis. During the simulations, the temperature was kept constant using the v-rescale weak coupling scheme with τ= 2.0 ps  and the = 1.0 ps .t-HOPO, t-HOPO4− and UIV-t-HOPO complex taken into account, and the central structures of the most populated clusters in the simulations were used as the reference to distinguish the transition between the most populated cluster and other clusters in each simulation. The time evolution of RMSD with the starting structure as the reference was also calculated and is provided in the Atomic positional root-mean-square deviation (RMSD) analysis was performed with all atoms in t-HOPO, t-HOPO4− and UIV-t-HOPO complexes, using the same cutoff value in the analysis of the three model systems would result in the too-sparse distribution of conformations of t-HOPO  or too few clusters of UIV-t-HOPO (bigger cutoff), as shown in t-HOPO, 0.18 nm for the t-HOPO4− and 0.05 nm for the UIV-t-HOPO complex, respectively. The intra-molecular weak interaction analysis of t-HOPO and t-HOPO4− was carried out by using the independent gradient model (IGM) [In the clustering analysis, the algorithm of Daura et al. was used . Owing tel (IGM)  implemenel (IGM) .mi is the mass of atom i located at distance ri from the center of mass of the complex. The calculation included U4+ and t-HOPO4− in the complex.The compactness of the complex was estimated by calculating its radius of gyration (Rg)t-HOPO in its neutral and deprotonated states, the two C atoms and one N atom on each hydroxypyridinone ring  method [4+ was perturbed from the gas phase to the aqueous phase, and each perturbed state was controlled by two variables λvdW and λcoul for the perturbation of van der Waals and Coulomb interactions between U4+ and the solvent/ligand, respectively. The increment in each perturbation factor was 0.05, and in total, there were 41 windows. Each window underwent an equilibration phase composed of two sequential 1 ns simulations under NVT and NPT ensembles, respectively, which was followed by a 100 ns production run for analysis. The Gibbs free energy change in the two states was calculated using the Bennett acceptance ratio method [The binding free energy of ) method . U4+ waso method  based ont-HOPO in its neutral and deprotonated states and the dynamics of UIV-t-HOPO were studied using MD simulations.The conformational dynamics of t-HOPO showed high flexibility, and there were frequent transition events among the conformational spaces accessible under simulation conditions. The four hydroxypyridinone rings of t-HOPO exhibited propensity to be parallel in pairs and benefitted from the weak π-π stacking and H-bond interactions between hydroxypyridinone rings, which were not sufficient to resist the perturbation from the solvent, resulting in frequent exchange between their parallel and anti-parallel conformations.In its neutral state, t-HOPO4−), its accessible conformational space was significantly smaller, and the propensity of the four hydroxypyridinone rings to stay in paralleled states in pairs was enhanced. The analysis of the trajectories indicated that, besides the π-π stacking and H-bond interactions between hydroxypyridinone rings, there were also Na+ ions to lock the pairs of hydroxypyridinone rings via electrostatic attractions between the Na+ and t-HOPO4− ligand, which could resist the disturbance of the water solvent.In contrast, in its deprotonated state (4+, t-HOPO could immobilize the cation by its eight oxygen atoms coordinating with the cation. Meanwhile, there were two water molecules in the first shell of U4+, resulting in the U4+ ion being deca-coordinated. Conformational analysis showed that the whole complex displayed less flexibility than t-HOPO in its neutral and deprotonated states owing to the strong electrostatic attraction between U4+ and t-HOPO4−.Upon binding with U


The antimicrobial activity of gA is associated with the formation of hydroxyl free radicals and the imbalance of NADH metabolism, possibly a consequence caused by the conductance of cations. The ion conductivity of gramicidin A can be blocked by Ca2+ ions. However, the effect of Ca2+ ions on the antimicrobial activity of gA is unclear. To unveil the role of Ca2+ ions, we examined the effect of Ca2+ ions on the antimicrobial activity of gramicidin A against Staphylococcus aureus (S. aureus). Results showed that the antimicrobial mechanism of gA and antimicrobial activity by Ca2+ ions are concentration-dependent. At the low gA concentration (≤1 μM), the antimicrobial mechanism of gA is mainly associated with the hydroxyl free radical formation and NADH metabolic imbalance. Under this mode, Ca2+ ions can significantly inhibit the hydroxyl free radical formation and NADH metabolic imbalance. On the other hand, at high gA concentration (≥5 μM), gramicidin A acts more likely as a detergent. Gramicidin A not only causes an increase in hydroxyl free radical levels and NAD+/NADH ratios but also induces the destruction of the lipid membrane composition. At this condition, Ca2+ ions can no longer reduce the gA antimicrobial activity but rather enhance the bacterial killing ability of gramicidin A.Gramicidin A (gA) is a linear antimicrobial peptide that can form a channel and specifically conduct monovalent cations such as H Theof helix . Gramiciof helix ,5,6,7,8.of helix ,6,8. Theof helix . Unlike of helix ,10,11,12of helix ,11,12. Tembranes ,12,13,14+/NADH synthesis [Gramicidin A has antibiotic and antimalarial activities ,16. Two ynthesis .6.3-helix mediated antimicrobial and antimalarial activities [The antimicrobial activity is dependent on the structural state of gramicidin A ,20. Two tivities . In a retivities . In thistivities . All the+, Na+, and K+ cross-membrane lipids [2+ ions [2+ ions has remained unclear. Therefore, in the present study, we investigated the effect of calcium cations on the antimicrobial activity of gramicidin A. Our results showed that the antimicrobial mechanism of gA is highly dependent on its concentration. The antimicrobial mechanism is mainly linked with increased hydroxyl free radical and NAD+/NADH ratios at low gA concentrations, while gramicidin A may act like a detergent and cause bacterial death through the disruption of membrane lipid composition. Ca2+ ions can only effectively inhibit the antimicrobial activity of gramicidin A at low gA concentrations through the inhibition of the antimicrobial activity of gramicidin A is associated with hydroxyl free radical formation and NADH metabolic imbalance. Due to the different antimicrobial mechanisms induced by the high concentration of gA, Ca2+ ions cannot inhibit but enhance the antimicrobial activity of gramicidin A. The gramicidin A channel can specifically conduct monovalent cations such as Lie lipids ,13,14. T[2+ ions ,23. HoweGramicidin A was purchased from Merck . Calcium chloride, magnesium chloride, strontium chloride, Luria–Bertani (LB) broth, and trifluoroethanol were purchased from Sigma-Aldrich . 30-(p-hydroxyphenyl) fluorescein was purchased from Invitrogen . NAD/NADH Assay kit (ab176723) was purchased from Abcam PLC . All other chemicals were reagent grade and used without further purification. Staphylococcus aureus (S. aureus) (ATCC-25923) was purchased from Bioresource Collection and Research Center (BCRC), Taiwan. Twenty-five mL of S. aureus was grown in LB broth medium in a 250 mL flask at 37 °C overnight. This overnight S. aureus culture was diluted to OD600 = 0.1 with LB medium. This S. aureus was then grown to lag phase (OD600 = 0.1–0.4), exponential phase (OD600 = 0.4–0.9), and stationary phase (OD600 > 0.9). The stock solution of gramicidin A was prepared in a final concentration of 100 μM in trifluoroethanol. At the designated phase, the S. aureus samples treated with or without gramicidin A and cations with the designed concentration were incubated at 37 °C. The optical density at a wavelength of 600 nm (OD600) was used to determine the growth curve of S. aureus using a microplate reader  every 30 min.S. aureus samples at exponential phase treated with or without gA or cations were taken every hour for 3 h. The fluorescence intensity at an emission wavelength of 545 nm with an excitation wavelength of 488 nm was used to determine the related fold of hydroxyl radical using a microplate reader .To detect hydroxyl radical formation, the fluorescent reporter dye 30-(p-hydroxyphenyl) fluorescein (5 mM) was used. In all experiments, +/NADH ratio was measured using an NAD+/NADH assay kit . The detailed experiments were performed according to the procedures provided by the kit. S. aureus treated with or without gA and cations incubated at 0, 1, 2, and 3 h were harvested and centrifugated at 10,000× g for 15 min at 4 °C. The cells were resuspended in a lysis buffer to a final concentration of 107–108 cells/mL. The resuspended cells were incubated at room temperature for 15 min and centrifugated at 2500 rpm for 5 min. The supernatant was transferred to a new tube and kept on ice. The NAD+ extraction buffer was added to neutralize NADH extraction. Similar procedures were performed for NAD extraction, except that NAD extraction buffer was used to extract NAD, and NADH extraction buffer was used to neutralize the reaction. For the NAD+/NADH ratio, seventy-five μL of NADH reaction enzyme mixture was added into each well and incubated at room temperature for 2 h. The related concentration of NAD or NADH was measured using a fluorescence microplate reader  with an excitation wavelength of 540 nm and emission wavelength of 590 nm.For NADH extraction, twenty-five μL of the sample with or without gA and cations was added to a well in a 96-well plate. Then, twenty-five μL of NADH extraction buffer was added to the well containing the sample and incubated for a further 15 min at 37 °C. After incubation, twenty-five μL of NADTM, JPK instruments, Berlin, Germany) installed on an inverted optical microscope . The AFM probes used were oxidize-sharpened silicon nitride probes  with a spring constant of 0.02 N/m. Ten microliters of samples treated with or without gramicidin A or Ca2+ ions at the designed concentration were placed on a cleaved mica disc  for 20–40 min. The mica disc was then rinsed twice with distilled water and dried for 24 h. The dried samples were imaged in an AFM operating in a contact mode. Images were acquired at a scanning rate of 1–2 Hz and a resolution of 512 × 512 pixels. Image processing and analysis were performed using SPM software v. 3.16 .All images were collected using an AFM  at 90 min, suggesting that the addition of a high concentration of Ca2+ ion can inhibit the imbalance of NAD+/NADH ratios caused by gramicidin A.Previously, we showed that hydroxyl radical formation is associated with an imbalance of the NADDH ratio . We examt 90 min A. The NA+/NADH ratio for the treatment of 5 μM gA was 4.2-fold higher than that of the control (SA only) at 90 min . At 90 min, the NAD+/NADH ratio was 3-fold in the addition of 100 mM Ca2+ ions and 5.0-fold for the 500 mM of Ca2+ compared to that of the control (SA only). Our results demonstrate that Ca2+ ions cannot inhibit the imbalance of NAD+/NADH metabolism induced by the 5 μM of gA. This result is consistent with the trends of hydroxyl free radical levels for S. aureus treated with 5 μM gramicidin A. The NADt 90 min B, indica2+ effects on the antimicrobial activity of gramicidin A were gA concentration-dependent. To further confirm the Ca2+ role, AFM microscopy was applied to observe the effect of Ca2+ ions on the morphology of S. aureus. The morphologies for S. aureus under the treatment of 1 and 5 μM gA and various concentrations of Ca2+ cations were delineated in S. aureus in the absence of gA with or without Ca2+ ions were round, smoothed, and aggregated, and the integrity of the membrane surface was maintained , the morphologies of S. aureus do not show severe membrane destruction and detergent-like shape. Gramicidin A mainly induces hydroxyl free radical formation and NADH metabolic imbalance. This is possible through the translocation of cations across the cell membranes.The antimicrobial mechanisms of gramicidin A are concentration-dependent. The AFM morphologies for S. aureus tend to be more destructive and form a detergent-like shape. The formation of detergent-like morphology may indicate that the higher concentration of gramicidin A may aggregate on the membrane surface and disrupt the membrane lipid composition more severely than the lower concentration of gramicidin A. Hence, gramicidin A mainly causes severe cell membrane permeabilization and disrupts the membrane integrity with a level-up of hydroxyl free radicals and NAD+/NADH ratios. The former corresponds to the channel function, which is more likely associated with the disruption of cell membrane composition by the aggregation of antimicrobial peptides on the membrane surface.At high concentrations (5 μM gA), the morphologies of 2+ ions on the antimicrobial activity of gramicidin A is dependent on the concentration of gramicidin A. At the concentration of gramicidin A ≤ 1 μM, Ca2+ ions can significantly inhibit the antimicrobial activity of gramicidin A. The reduction of antimicrobial activity is dependent on the Ca2+ ion concentration. The high Ca2+ concentration inhibits the antimicrobial activity more effectively than the low Ca2+ concentration. The inhibition of antimicrobial activity is well correlated with the reduction of hydroxyl free radical formation and NAD+/NADH metabolic imbalance [+, by Ca2+ ions [The effect of Cambalance . One of a2+ ions ,23. 2+ ions on 1 μM gA, the inhibitory ability of Ca2+ ions on the antimicrobial activity of 5 μM gramicidin A was almost abolished. The addition of Ca2+ ions cannot significantly rescue the growth rate of S. aureus and inhibit the hydroxyl free radical formation and the imbalance of NADH metabolism under the treatment of 5 μM gA, although a slight increase in the survival rate of S. aureus was observed for the Ca2+ ion concentration ≤ 100 mM. Two possible reasons may account for this loss of the inhibitory ability of Ca2+ ions on the antimicrobial activity of 5 μM gramicidin A. One is that a much higher concentration of Ca2+ ions is needed for complete inhibition of the antimicrobial activity of 5 μM gramicidin A. The other possibility is that 5 μM gramicidin A undergoes a different antimicrobial mechanism. Taking the antimicrobial mechanism into account, we suggest that undergoing a different antimicrobial mechanism is the most likely reason for abolishing the Ca2+ inhibitory effect on the antimicrobial activity of 5 μM gramicidin A. Similar observations are also reported by a previous study [2+ ions on the antimicrobial activity becomes ineffective under the treatment of 5 μM gramicidin A. Furthermore, it is worth notifying that when the concentration of Ca2+ ions > 100 mM, Ca2+ ions can enhance the antimicrobial activity, as the growth rate, hydroxyl free radical level, and NAD+/NADH ratios are all increased compared to those of 5 μM gA only.Unlike the inhibitory effect of Caus study . Hence, 2+ ions, we disclosed that the antimicrobial mechanism of gramicidin A is dependent on the gA concentration. At lower concentrations (≤ 1 μM), the antimicrobial mechanism of gramicidin A is mainly linked with the imbalance of NADH metabolism and the formation of free radicals caused by the translocation of cations across the bacterial membrane lipids, while at higher concentrations (≥5 μM), gramicidin A acts like a detergent and cause the disruption and destruction of bacterial membrane lipids. Hence, Ca2+ ions can only significantly inhibit the gA antimicrobial activity at the low concentration of gramicidin A (1 μM) by blocking the translocation of cations across the bacterial membrane lipids.In conclusion, using Ca


Arthritis is a general term for various types of inflammatory joint diseases. The most common clinical conditions are mainly represented by rheumatoid arthritis and osteoarthritis, which affect more than 4% of people worldwide and seriously limit their mobility. Arthritis medication generally requires long-term application, while conventional administrations by oral delivery or injections may cause gastrointestinal side effects and are inconvenient for patients during long-term application. Emerging microneedle (MN) technology in recent years has created new avenues of transdermal delivery for arthritis drugs due to its advantages of painless skin perforation and efficient local delivery. This review summarizes various types of arthritis and current therapeutic agents. The current development of MNs in the delivery of arthritis drugs is highlighted, demonstrating their capabilities in achieving different drug release profiles through different self-enhancement methods or the incorporation of nanocarriers. Furthermore, the challenges of translating MNs from laboratory studies to the clinical practice and the marketplace are discussed. This promising technology provides a new approach to the current drug delivery paradigm in treating arthritis in transdermal delivery. Arthritis is one of the important diseases affecting contemporary human health, and generally refers to inflammatory diseases that occur in human joints and surrounding tissues . The cliIn the early stage of arthritis, drug treatment is the mainstay. To inhibit the development of inflammation and reduce pain, non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids (GCs), disease-modifying antirheumatic drugs (DMARDs), immunotherapy drugs (ASOs), cytokine inhibitors , and cytokines are typically used . AlthougHowever, the skin is a powerful biological barrier against infectious viruses and harmful substances . In partMicroneedles (MNs) are a promising transdermal delivery approach that physically penetrates the skin and is more efficient in delivering drugs; they have gradually gained attention in the field of transdermal delivery . SpecifiIn this paper, we further review the application of MNs delivering drugs for the treatment of various types of arthritis including RA, OA, GA, and JIA. The advantages and disadvantages of MN-based therapies for arthritis are further discussed, which will provide a strategy for designing and developing new MNs for arthritis in the future.Arthritis is an umbrella term that encompasses more than 100 joint pain or disease conditions for which there is not yet a uniform classification standard . HoweverRA is an autoimmune illness that is chronic, symmetrical, and inflammatory . The poiOA is the most common type of arthritis in the world. It mainly occurs in the elderly and is caused by degenerative lesions with articular cartilage damage . OA affeAccording to a systematic investigation of the Global Burden of Disease Study, GA is a self-inflammatory disease that often occurs in men over 40 years old . The gloApart from above types of arthritis, which have attracted much attention in the clinical environment, other types of arthritis, such as JIA, PSA, ankylosing spondylitis (AS), and reactive arthritis (ReA), also affect the health of a group of people.JIA is a broad term for all types of chronic childhood arthritis. It is defined as arthritis that occurs in children under 16 and lasts for at least 6 weeks for unknown causes . JIA is PSA is an inflammatory joint disease associated with psoriasis, embodied by psoriatic rash accompanied by joint inflammation . It has AS, similar to RA, is an autoimmune rheumatic disease with similar clinical and genetic characteristics. It initially attacks the tiny joints of the hands and feet and primarily affects the spinal joints, sacroiliac joints, and the soft tissues around them such as tendons and ligaments . Severe ReA is inflammatory arthritis that occurs after gastrointestinal or genitourinary tract infection. Infection, HLA-B27 anomalies, and immunological diseases are related to the development of ReA . The comAlthough different types of arthritis have different causes, they all show inflammation of joints. The common goal of treatment for arthritis is to alleviate pain, reduce inflammation, and slow joint degeneration to prevent disease progression and joint deformity or impairment. Therefore, the therapeutic drugs for different types of arthritis have certain similarities. In this section, we summarized the various types of drugs and their delivery methods, as well as the potential of MNs in their application. The drugs that have been approved for the treatment of arthritis as of 20 April 2022 are summarized in In the first-line treatment of arthritis, oral NSAIDs are used to reduce pain and inflammation rapidly . NSAIDs Some conventional synthetic DMARDs are also used to treat various types of arthritis. Because of their effective anti-inflammatory and immunomodulatory ability and good tolerance, DMARDs have been regarded as the first choice for the treatment of arthritis. Methotrexate (MTX), a small-molecule immunosuppressive agent, is a folate antagonist responsible for inhibiting the synthesis of purine, pyrimidine and polyamines, and adhesion of inflammatory molecules . HoweverFor some drugs which are not suitable for oral administration such as glucocorticoids (GCs) and biological drugs, e.g., TNF-α inhibitors, injection may be a more appropriate way of administration. GCs are anti-inflammatory drugs that act faster than NSAIDs. They inhibit the development of inflammation by activating glucocorticoid receptors and inhibiting pro-inflammatory transcription factors . Since GTNF-α is a cytokine that can promote joint inflammation. TNF-α inhibitors can prevent the release of TNF-α-driven inflammatory factors and the further development of inflammation . The majDue to the gastrointestinal and cardiovascular adverse effects of NSAIDs, topical NSAIDs have become a potential alternative to oral administration. The FDA approved two topical NSAID formulations for OA in 2007: diclofenac sodium 1% gel and diclofenac sodium 1.5% in 45.5% dimethylsulfoxide . TopicalTripterygium wilfordii is commonly used to alleviate the symptoms of arthritis in China, but oral administration has been limited in clinical application because of its hepatotoxicity and reproductive toxicity [Tripterygium wilfordii Hook F gel, made from Tripterygium wilfordii extract, is for topical use in hospitals [Chinese external therapy (CET) is a topical application method using Chinese herbal medicines as the main ingredients. It has been used in Chinese medicine for thousands of years to relieve arthritis symptoms. toxicity . Tripterospitals , which cMost NSAIDs and synthetic small-molecule DMARDs require long-term administration to treat arthritis, known to cause relevant side effects. Most corticosteroids and biologic drugs require constant injections, and long-term injections not only cause irreversible damage to skin vessels and other tissues but also increase the patient’s distress. Furthermore, due to the skin barrier or articular cavity barrier, some water-soluble or macromolecular drugs cannot be delivered efficiently by transdermal administration. MNs have the advantages of both non-invasive  and invasive (injectable) drug delivery methods, which can overcome the main limitations mentioned above. MNs are considered topical transdermal, and drugs delivered by MNs are free from first-pass effects, which avoids gastrointestinal reactions. Moreover, MNs are easily handled by patients, providing efficient drug delivery. Thus, MNs have become a new generation of topical drug delivery system for arthritis drugs .MNs can be classified based on component materials, preparation methods, applications, or designs. In regard to their designs, the types of MNs widely studied today include solid MNs, hollow MNs, coated MNs, dissolving MNs, hydrogel-forming MNs, and other new types ,55. The The presence of a stratum corneum lipophilic barrier makes it suitable to deliver only drugs with logP in the range of 1.0–3.0 and small molecules (molecular weight of >500 Da) via topical transdermal administration . Solid MHollow MNs are made of metal or silicon, with a certain space inside and an opening tip of the MN . When hoCoated MNs are derived from solid MNs, in which a drug or a vaccine is coated on the surface of the solid MN . When coThe needles of dissolving MNs are made of water-soluble or biodegradable biomaterials that encapsulate drugs ,85. AfteHydrogel-forming MNs, first proposed in 2012, are formed by swellable hydrogel polymers . After iStimulus-responsive MNs are a new type of MNs that can release loaded drugs in a targeted or timed manner according to pH, pathological conditions, or external stimuli  . At presThe bionic MNs developed so far mainly include mosquito-like MNs , North AThe essential factors for designing MNs include the shape, length, tip diameter, and needle spacing  to passively penetrate into the body. Mehtab Jabla et al. used solid MNs to form hydrophilic microchannels across the skin, which provided the passive permeation of MTX . The lagAlthough the application of solid MNs is relatively limited, they can be used to topically deliver different drugs by optimizing the formulations. For example, encapsulated liposomes, ethosomes, and nanoparticles can further promote drug penetration; encapsulated nanoparticles or microsponge gels can induce long-lasting drug release combined with solid MNs ,154,155.Conventional hollow MNs deliver drugs in a manner similar to subcutaneous injection. After the insertion of hollow MNs into the skin, the drug enters the body through holes in the interior of each needle. Hollow MNs are a better choice for delivering high-molecular-weight drugs such as proteins and monoclonal antibodies  . Bushra Recently, a novel hollow MN for the delivery of biological drugs has been reported by Carcamo et al. . These hw/v) carboxymethyl cellulose was added to the coating solution to enhance the viscosity and 0.5% (w/v) Lutrol F-68 NF was added as a surfactant, resulting in a better coating of the drug. Compared to joint injections, the duration of analgesia delivered by coated MNs increased from 2 h to 2 days. In addition, intra-articular injection of tramadol significantly reduced the levels of the pro-inflammatory cytokines TNF-a and IL-1b and increased the expression of anti-inflammatory cytokine IL-10. However, the levels of TNF-a, IL-1b, and IL-10 were not significantly different from the non-treatment group after 2 h, indicating that the duration of the anti-inflammatory effects produced by intra-articular tramadol injection was short. Contrarily, the treatment of tramadol delivered by coated MNs reduced the levels of TNF-a and IL-1b and increased the level of IL-10 up to 6 days [Coated MNs have the characteristics of rapid drug release and precise dose control. Since coated MNs can only deliver the drug being loaded on the surface of the MNs, they have limited drug loading capacity, which is applied to deliver drugs with strong pharmacological efficacy in a low dosage, such as vaccines or analgesics ,157. Moso 6 days . This stDissolving MNs are known for their high drug delivery efficiency compared to various other MNs due to the large amount of biocompatible matrices that can be used to develop dissolving MNs. Therefore, dissolving MNs currently account for the majority of MN research in delivering arthritis therapeutics.The basic dissolving MNs are prepared by mixing a single polymer material with a drug solution; the preparation process is simple and convenient. Korkmaz et al. used carboxymethylcellulose (CMC) to prepare solubilized MNs encapsulated with TNF-α inhibitors by using the micromilling/spin-casting fabrication method at room temperature. Due to the continuous and simple preparation process, 500+ MN arrays could be prepared within 6 h, which is expected to be easily scaled up for automated industrial production . AlthougDifferent polymers have different properties. Thus, the properties of MNs can be optimized by using different polymers. Amodwala et al. used hydrophilic polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) as the matrix to make fast-dissolving MNs loaded with meloxicam . PVA proNanocarriers can be added to dissolving MNs to enhance drug delivery. Shende et al. prepared poly (lactic-co-glycolic acid) (PLGA) microspheres encapsulated with folic acid using a multiple-emulsion system (w/o/w type). The microspheres were loaded into methotrexate-containing dissolving MNs. Thus, the MNs can co-administrate folic acid and methotrexate into the skin and achieve synergistic effects. Moreover, PLGA microspheres have a controlled release effect. In vitro release studies showed that more than 80% of methotrexate and nearly 60% of folic acid are released from the MN array within 8 h, followed by a slow controlled release over 24 h, which results in longer-lasting effects of both drugs . Hu et aIn addition to the above innovations in MNs, the technologies for externally enhancing drug delivery, such as iontophoresis (ITP), can also be combined with MNs to improve drug penetration into the skin. ITP is an active energy-release process that uses microcurrents to promote the entry of ionic drugs into the body . Vemulap2/h. The HA dissolving MNs prepared by Du et al. showed a delivery rate of 13.8 μg/10 × 10 in their MN array for MTX. Compared to the above methods, the hydrogel-forming MNs combined with a drug reservoir developed by Chen et al. could deliver MTX at a high steady-state flux (506.8 ± 136.9 µg·h/cm2) and sustained a delivery rate of 6.8 ± 0.4 mg/cm2 of MTX for 24 h [Hydrogel-forming MNs have been widely used in recent years due to their long-lasting drug-releasing ability, high biocompatibility, and because they do not leave any residue in the skin . In a stfor 24 h .ClinicalTrials.gov using MNs as a keyword revealed 130 clinical trials worldwide, 85 of which have been completed, including two phase IV clinical trials. Most of these clinical trials are focused on anesthesia, vaccine delivery, skin diseases, and aesthetics [Because transdermal drug delivery has many irreplaceable advantages over other traditional drug delivery methods, the application of transdermal drug delivery in clinical settings is gaining overwhelming attention. It has great potential for market growth . The glosthetics ,164. Solsthetics , which sThere are still many challenges in developing approved MNs for clinical application from laboratory studies. First, the main obstacle limiting clinical translation is the relatively low drug loading capacity of MNs. The in vitro and in vivo models used in the laboratory require lower doses of drugs to show the therapeutic effects compared to the patients. Currently, some research is gradually beginning to address this issue by expanding the area of MN arrays or utilizing new manufacturing strategies to increase the maximum loading capacity of individual arrays, allowing MNs to carry sufficient doses of drug and deliver them to the human body ,167. In In general, MN array patches for drug delivery are currently under development and are still in the early stages of manufacturing for commercial applications. The biggest hurdle in scaling up MN production from the laboratory to the factory is establishing a standard manufacturing process . The firUp to now, the application of MNs in delivering arthritis drugs has mostly been in the laboratory research stage, and only one MN have been tested in clinical trials . HoweverArthritis occurs in a wide range of people of different genders and ages. It seriously affects the quality of life of a lot of patients. The current drugs for arthritis are mostly administrated by oral or injection routes, which will need long-term treatment. The gastrointestinal side effects and patients’ fear for needles have prompted the development of other arthritis treatment strategies, including transdermal drug delivery technology. As an emerging technology in the field of transdermal drug delivery, MNs have quickly attracted the interest of researchers due to their variety and potential functions. MNs have shown significant advantages in the delivery of biological drugs or drugs that require local or frequent injections, filling the gap in traditional delivery methods for arthritis drugs. Specifically, the use of biocompatible polymers allows for the stable loading of biologic DMARDs and effective delivery. Hydrogel-forming MNs can be used to obtain a slow release of drugs, which reduces the frequency of drug injections. Fast-dissolving MNs can be used for safely and rapidly loading precise doses of steroids into the joint to relieve inflammation, which also greatly reduces the systemic responses of steroids.To date, most of the studies on MN administration have focused on the treatment of RA, but since other arthritis have similar symptoms and drugs, the experimental results and development experience can also provide an important reference for the development of drug-loaded MNs in the therapy of other types of arthritis. In addition, arthritis is an inflammatory disease with typical characteristics; thus, MN studies in arthritis also provide valuable ideas for the therapy of other types of inflammatory diseases. Furthermore, most studies of MNs on delivering arthritis drugs are undergoing research in laboratories, and have not been applied in the clinic. Therefore, more clinical studies to determine the effectiveness and safety of MNs are needed. There is a lack of study on the geometry and mechanical strength of MNs that are suitable for topical use in joints. To deliver drugs into the joint cavity, MNs will need to penetrate the skin and the joint cavity, which requires a certain length and mechanical strength of MNs. However, long MNs may cause pain and increase the risk of MN breakage in patients. Therefore, it is important to balance the penetration ability with safety and reduce the pain in designing MNs for administrating the drug to local joints. In addition, the movement of the joint may cause needle breakage or dislodgement of the locally applied MNs, resulting in intradermal injury. Thus, we believe that rapid-dissolving MNs are the most suitable type of MNs for topical administration at the joint due to their rapid dissolution feature, which shortens the administration time. Moreover, even if the MN needles break within the skin, the residuals of dissolving MN needles can be dissolved on their own without causing further damage to the skin. For patients, dissolving MNs can be applied by patients without needing to visit the clinic, which has great commercial potential. For drugs, small therapeutic doses of water-soluble drugs are most suitable for the microneedle’s transdermal delivery route due to the limitation of the lower drug loading capacity of MNs and the fact that the matrix of MNs is mostly hydrophilic. Although the FDA has established some regulations for MN products, the manufacturing guidelines and standards of MNs need to be further improved to enable this new technology to be applied to patients as soon as possible.