Protective Effect And Mechanism Of Vitexin On Liver Injury In Mice With Ulcerative Colitis

Ulcerative colitis is a chronic non-specific intestinal disease that mainly occurs in colonic mucosa and submucosa,which belongs to the category of inflammatory bowel disease.Hepatobiliary disease is a relatively common extraintestinal manifestation of ulcerative colitis.At present,the drugs used for the treatment of ulcerative colitis may have some adverse reactions to the liver.Therefore,it is of great significance to find drugs for the treatment of liver injury in ulcerative colitis for adjuvant treatment of ulcerative colitis.Vitexin is a kind of natural flavonoid carbon glycosides,which comes from the leaves of hawthorn of rosaceae.Studies have shown that vitexin has anti-inflammatory,anti-tumor,anti-virus,liver protection and other biological activities.The purpose of this project is to explore whether vitexin can inhibit inflammation so as to achieve the purpose of treating liver injury in ulcerative colitis.ObjectiveThe purpose of this study was to investigate the protective effects and mechanisms of vitexin on liver injury in acute ulcerative colitis.In vivo,an acute colitis mice model was used to confirm the hepatoprotective activity and the possible mechanisms of vitexin.In vitro,we performed further study,using HepG2 cells,to confirm the molecular mechanisms of vitexin on inflammatory response.MethodsAll male BALB/c mice were randomly divided into 4 groups(n=12/group):control group,model(4%DSS)group,DSS+vitexin(40 and 80 mg/kg)group.The control group was given distilled water every day,and mice in the other groups received DSS added at 4%concentration to the drinking water within 7 consecutive days.For control group and model group,mice were administrated with distilled water,while vitexin groups were treated orally with vitexin at 40 and 80 mg/kg once daily.The daily weight changes and hematochezia of mice in each group were recorded during the experiment.At the end of the experiment,the severities of colitis and liver injury in mice were evaluated,the length of colon was recorded and the liver coefficient was calculated.The levels of ALT,AST,CAT and GSH-PX in the liver were detected by specific detection kits(Jiancheng Bioengineering Institute of Nanjing)according to the manufacturer’s instruction.In addition,the level of LPS in the liver were assayed using quantitative chromogenic tachypleus amebocyte lysate for endotoxin(pyrogen)detection kit.The levels of TNF-α,IL-6 and IL-1β in liver tissues were analyzed by corresponding ELISA kits,F4/80+macrophage infiltration was assayed by immunofluorescence staining.The expression of proteins related to TLR4/NF-kappa B p65 signal pathway(TLR4,p65,p-p65,IκBα and p-IκBα)were detected by western blot technique.In vitro,the cytotoxicity of vitexin for HepG2 cells was detected by MTT assay.Inflammatory cell model was established by LPS.The contents of inflammatory cytokines(TNF-α,IL-6 and IL-1β)were measured by real-time PCR technique.The expressions of TLR4,p65,p-p65 and p-IκBα were detected by western blot.ResultsAnimal experiments showed that compared with the control group,the mice in the model group had the characteristic phenomena of ulcerative colitis,such as increased DAI score and shortened colon.Furthermore,the liver index of mice in model group was obviously increased,DSS could increase the levels of ALT and AST and decrease the activities of CAT and GSH-PX in the liver.After vitexin treatment,the symptoms of colitis in mice were alleviated,the liver coefficient was significantly reduced.And the levels of liver ALT,AST,CAT and GSH-PX were alleviated.In addition,compared with the control group,the levels of LPS and inflammatory factors such as TNF-α,IL-6 and IL-1β in liver tissue of mice in the model group were increased in varying degrees after administration of DSS.The immunofluorescence results showed that the macrophage activation of the model group was significantly higher than that of the control group,meanwhile,the protein expressions of TLR4,p-p65 and p-IκBα were significantly increased in model group.After treatment with vitexin,the levels of LPS and inflammatory cytokines in liver tissue of mice decreased in varying degrees,the expression of F4/80 was significantly reduced and the expressions of TLR4,p-p65 and p-IκBα in liver of mice in the vitexin-treated group were closer to those in the control group.The results of MTT showed that when the concentration is in the range of 5-640 μM,vitexin did not decrease significantly the viability of HepG2 cells compared with the control group after 24-hour incubation.The four drug concentrations of 10,20,40 and 80μM were selected for follow-up experiments.Compared with the control group,the mRNA expression of inflammatory cytokines such as TNF-α,IL-6,IL-1β and the protein expressions of TLR4,p-p65 and p-IκBα were significantly increased in LPS-stimulated HepG2 cells.However,after 24 h treatment with vitexin,these parameters had been decreased in varying degrees.ConclusionIn summary,this study preliminarily confirmed that vitexin can effectively alleviate liver injury and inhibit liver inflammation in mice with acute ulcerative colitis caused by DSS.Its mechanism of action may be related to the regulation of TLR4/NF-κB signaling pathway,suggesting that vitexin can be used as a promising adjuvant treatment strategy for the treatment of liver injury in colitis.