| Inflammatory bowel disease(IBD)is a chronic gastrointestinal inflammatory disease.It is currently believed that genetic susceptibility,intestinal flora imbalance and abnormal immune response are related to the occurrence and development of IBD.The intestinal epithelial barrier is the central link in regulating the balance of the three,and maintaining its integrity is particularly important for the treatment of IBD.The destruction of the intestinal epithelial barrier leads to the occurrence of "leakage",and intestinal bacteria enter the lamina propria,causing an abnormal immune response and inducing Ulcerative colitis(UC).At the same time,inflammatory factors secreted by immune cells(such as TNF-α,IFN-γ,and IL-13)act on intestinal epithelial cells,increase intestinal permeability,and further trigger an inflammatory cascade,leading to delayed IBD prolongation.Intestinal epithelial permeability is a key indicator for measuring the epithelial barrier,mainly composed of transepithelial pathway and paracellular pathway,both of which are closely related to Tight junction(TJ)Myosin light chain kinase(MLCK)pathway is one of the key pathways regulating TJ.The activation of its pathway promotes the cytoskeletal actin filaments to slide,and the myoglobin ring around TJ shrinks.The TJ relaxes to form a cell gap,resulting in an increase in intestinal epithelial permeability.At present,there is no clear and effctive method for restoring the protective mechanism of intestinal epithelial barrier.As a main active ingredient of Huangqi Decoction,wogonoside has the highest concentration in the colon and has obvious therapeutic effects on UC mice.It has been reported in the literature that wogonoside treat UC by inhibiting NLRP3 and NF-κB pathways,but its protective effect on the barrier and its mechanism have not been reported.Based on this,and in view of the important role of barrier function in the treatment of UC,this study used Dextran sodium sulfate(DSS)to induce UC mouse model and Tumor necrosis factor-α(TNF-α)stimulated monolayer Caco-2 cell injury model,preliminary study on the protective effect of wogonoside on intestinal epithelial barrier and its mechanism of action.Objective:In this study,the protective effect of wogonoside on the ulceration of UC mice and the reduction of intestinal permeability were demonstrated.The protective effect of baicalin on the intestinal epithelial barrier of UC mice was demonstrated.Fluorescence in situ hybridization was also used.In situ hybridization(FISH)was used to evaluate the effect of baicalin on intestinal epithelial barrier function.Combined with in vitro cell experiments,we observed the regulation of scutellarin on tight junction proteins and explored its mechanism of action on MLCK/pMLC2 signaling pathway.Methods:1.Pharmacodynamic study of wogonoside on UC miceThe UC model was induced by CDSBL/6 mice by DSS oral method.C57BL/6 mice were randomLy divided into 6 groups:control group,3%DSS group,low,medium and high dose group of wogonoside and sulfasalazine group.The model group and the drug-administered group were given free consumption of 3%DSS for 6 days,and the purified water or the corresponding drug was administered by the group at the same time,and the body weight,water intake,food intake and fecal state of the mice were recorded every day.In order to comprehensively evaluate the overall efficacy of the drug,this study examined the proportion of white blood cells in the serum of each group,calculated spleen index and thymus index,statistical colon length and histopathological score.2.Protective effect of wogonoside on intestinal epithelial barrier2.1 Effect of wogonoside on intestinal epithelial permeability and colonic bacterial invasion in UC miceOn the 11th day after the administration of the mice,the mice were given a dose of 20 mg/kg of FITC-dextran(FD4)at a dose of 12 hours after fasting,and the mouse serum was collected 4 hours later to evaluate the intestinal epithelium permeability.Fluorescence in situ technique was used to detect colonic bacterial invasion.2.2 Protective effect of wogonoside on TNF-α-induced monolayer Caco-2 cell injury modelThe modeling concentration and modeling time of TNF-α were screened by detecting the change of transepithelial resistance(TER),and the effect of wogonoside on the proliferation of Caco-2 cells was observed by cell dynamic imaging.The TER and FD4 pass rates were measured by a resistance meter to evaluate the repair effect of the wogonoside on the intestinal epithelial barrier.2.3 Effect of wogonoside on tight junction protein in colonic and monolayer Caco-2 cell injury models in UC miceThe expression of tight junction proteins ZO-1,Occludin and Claudin1 in colon and Caco-2 cells was detected by Western blot(WB),and the effect of wogonoside on its tight junction was observed by immunofluorescence(IF).2.4 Effect of wogonoside on the tight junction morphology of intestinal epithelial cells in colon of UC miceH&E staining was used to observe the repairing effect of the wogonoside on the ulcer part of UC mice,and the tightly connected ultrastructure was observed by electron microscopy.3.Mechanism of repairing intestinal epithelial barrier by inhibiting MLCK/pMLC2 signaling pathwayThe expression level of cytokines in serum of UC mice was determined by ELISA.The effect of wogonoside on the expression of F-actin and localization of Caco-2 cells was detected by IF.Western blotting was used to detect the effects of wogonoside on the expression of MLCK,pMLC2 and MLC2 in Caco-2 cells and colon tissues.Results:1.Therapeutic effect of wogonoside on UC miceThe weight of UC mice treated with 3%DSS was significantly reduced,with less movement contracture,diarrhea and blood in the stool;due to inflammation of the colon,the length of the colon of hyperemia and edema was significantly shortened,and the doses of the wogonoside and the positive drug sulfasalazine were significantly reduced.(Sulfasalazine,SASP)can alleviate the above symptoms to varying degrees,especially in the middle and high doses of Chinese wogonoside and SASP(P<0.01).In addition,wogonoside and SASP reduced the number of leukocytes in peripheral blood of UC mice(P<0.05)and improved inflammation.Pathological scoring results showed that the muscle layer and mucosa of the colon tissue of the model group showed inflammation,the muscle layer was thickened due to edema,the mucosal layer was ulcerated,and many goblet cells and colon glands disappeared and were replaced by infiltrating inflammatory cells(P<0.001).The histopathological scores of UC mice were significantly reduced after treatment with wogonoside or SASP,and the medium and high dose groups and SASP group were close to normal levels(P<0.05 or P<0.001).At the same time,the subject evaluated the spleen and thymus index in UC mice,which can indirectly reflect the level of inflammation in mice.The results showed that the high dose of wogonoside improved the two(P<0.05 or P<0.001),and SASP also significantly restored the thymus index(P<0.001).2.Effect of wogonoside on intestinal permeability in UC miceThe results showed that wogonoside can reduce the FD4 passage rate in the blood of UC mice in a dose-dependent manner(P<0.05 or P<0.01).At the same time,this study found that the bacterial infiltration of colonic mucosa in the model group was more severe than that in the normal group,and was significantly reduced after administration of wogonoside or SASP.3.Effect of wogonoside on TJ protein expression in UC miceCompared with the normal group,the expression of TJ protein in the model group was significantly down-regulated(P<0.05 or P<0.001).After the intervention,there were different degrees of recovery,especially the wogonoside,high-dose group and SASP.Groups were statistically significant(P<0.05,P<0.01 or P<0.001).Consistent with the WB results,IF results showed that the expression of ZO-1 and Occludin in the colon of the model group was decreased and recovered after treatment with wogonoside or SASP.Elevated levels of TNF-α,IL-13,and IFN-γ in colitis can destroy intestinal epithelial TJ,leading to barrier dysfunction.The results of ELISA showed that wogonoside can down-regulate these three cytokines in the serum of UC mice(P<0.05,P<0.01 or P<0.001).H&E staining results showed that different doses of wogonoside and SASP could restore intestinal epithelial cell shedding caused by 3%DSS.In addition,transmission electron microscopy results showed that wogonoside protected the TJ of UC mice.4.Effect of wogonoside on the pass rate of TER and FD4 in TNF-a-induced monolayer Caco-2 cell injury modelAfter 24,48,and 72 h of stimulation with 25,50,and 75 ng/mL TNF-α,the monolayer barrier was disrupted and TER decreased significantly(P<0.01 or P<0.001).Cytotoxicity experiments showed that 25,50 and 100 ng/mL TNF-α and low,medium and high dose groups of wogonoside were not toxic to Caco-2 cells after 24 hours of stimulation.Therefore,using 25ng/mL is close to the reported dose as the stimulation concentration of TNF-α.Oryzanin or MLCK inhibitor Peptide 18 can significantly inhibit TNF-α inductionThe level of TER in the Caco-2 cell injury model was reduced(P<0.05 or P<0.01).Consistent with the results of the TER analysis,TNF-[alpha]increased the cell bypass rate of FD4.After the administration of wogonoside and wogonoside and Peptide 18,the FD4 pass rate decreased significantly(P<0.05 or P<0.01).5.Effect of wogonoside on Caco-2 cytoskeletal protein F-actin and TJ proteinAfter stimulation with TNF-α,the levels of ZO-1,Occludin and Claudin1 proteins were significantly decreased(P<0.05 or P<0.01),and their expression was significantly restored after intervention with wogonoside and Peptide 18,indicating that wogonoside And MLCK inhibitors attenuated TJ barrier integrity damage caused by TNF-α(P<0.05 or P<0.01).This study demonstrates that TNF-α induces changes in the distribution of ZO-1 and Occludin in Caco-2 cells,and some parts are almost discontinuous.The intervention of wogonoside and MLCK inhibitors can alleviate the changes of TNF-α-induced ZO-1 and Occludin distribution,and inhibit TNF-α-induced F-actin disintegration and restore the normal morphology of cells.6.Effect of wogonoside on MLCK/pMLC2 signaling pathway in UC mice and Caco-2 cellsMLC2 phosphorylation can affect F-actin changes,and MLC phosphorylation is regulated by MLCK.The results showed that wogonoside inhibited the activation of MLCK induced by TNF-α,thereby inhibiting the phosphorylation of MLC2(P<0.05 or P<0.01).Consistent with in vivo results,in vitro studies found that the medium and high doses of wogonoside and the MLCK inhibitor Peptide 18 exerted the same inhibitory effect on the MLCK/pMLC2 signaling pathway.Conclusion:1.Wogonoside can repair the damaged intestinal epithelial barrier in ulcerative colitis mice and improve the symptoms of disease;2.Wogonoside has a regulatory effect on intestinal epithelial tight junctions and is associated with its regulation of MLCK/pMLC2 signaling pathway. |
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