Protective Effect Of Ursolic Acid On Intestinal Mucosal Barrier In Hepatic Fibrosis Rats And Its Mechanism

Background:Intestinal inflammation and NADPH oxidase(NOX)-mediated Oxidative stress play an important role in intestinal mucosal barrier impairment in various disease states.Recent evidence suggests that intestinal mucosal barrier injury is present in hepatic fibrosis rats,but the exact molecular mechanism is still largely unknown.Another research shows that Ursolic acid(UA)protects against ulcerative colitis in mice via anti-inflammatory and antioxidant effects.In liver fibrosis rats induced by carbon tetrachloride(CCl4),whether UA protects the intestinal mucosal barrier by inhibiting intestinal inflammation and NOX-mediated oxidative stress is worth further investigation.Objective:To investigate the effect of UA on intestinal mucosal barrier in CCl4-induced hepatic fibrosis rats and its potential mechanisms.Methods:Male SD rats were randomly divided into 3 groups:control,liver fibrosis group and UA treatment group.Pathological changes in the liver were observed via HE and Sirius red staining.HE staining was performed to observe the pathological changes of the ileum.The protein expressions of tight junction claudin 1 and occludin in the ileum were analyzed with Western blot.Caspase 3 protein expression was detected in ileal epithelial cells by immunohistochemistry.The levels of serum c-reactive protein(CRP),procalcitonin(PCT)and lipopolysaccharide(LPS)were determined with ELISA in each group.The m RNA and protein expressions of tumor necrosis factor-?(TNF-?)were determined by q RT-PCR and ELISA respectively.The levels of ileal malondialdehyde(MDA)and total antioxidant capacity(T-AOC)were analyzed biochemically.The protein expressions of P67 phox and NOX2 in ileum of each group were measured.Results:1.Effect of ursolic acid on the pathological changes of liver tissue in rats with hepatic fibrosis The tissue sections from control group showed normal hepatic lobular structure and little collagen deposition.In liver fibrosis group,the histopathological features of liver included disordered hepatic lobular structure,hepatocyte steatosis and necrosis.Hepatic fibrosis score in liver fibrosis group(2.67�0.36)was significantly higher than control(0.43 � 0.29)(P<0.01).Compared with vehicle-treated fibrotic rats,ameliorative hepatic lobular structure and reduced degrees of hepatocyte degeneration as well as necrosis were observed in UA-treated fibrotic rats.Hepatic fibrosis score was decreased significantly in UA treatment group(1.70 � 0.37)compared with liver fibrosis group(P<0.01).2.Effect of ursolic acid on intestinal mucosal barrier in CCl4-induced hepatic fibrosis rats2.1 Effect of ursolic acid on the pathological changes of ileal tissue in rats with hepatic fibrosis The tissue sections of rats in control group showed normal intestinal structures.The ileal mucosa was observed as intact.Ileal Sections from liver fibrosis group showed mucosal structure disturbance.Mucosal injury score in ileum of liver fibrosis group(2.51�0.34)was significantly higher than control(0.28�0.21)(P<0.01).Compared with vehicle-treated fibrotic rats,ameliorative intestinal structures were observed in UA-treated fibrotic rats.Mucosal injury score of ileum was decreased in UA treatment group(2.04�0.33)compared with liver fibrosis group(P<0.05).2.2 Effect of ursolic acid on the protein expressions of tight junction claudin 1and occludin in the ileum of the hepatic fibrosis rats Compared with control,the protein expressions of tight junction claudin 1 and occludin in the ileum of liver fibrosis group decreased significantly(P<0.01).The increased expressions of claudin 1 and occludin protein were observed in UA treatment group compared with liver fibrosis group(P<0.01).2.3 Effects of ursolic acid on the expression of apoptosis protein caspase 3 in the ileal epithelial cells of rats with hepatic fibrosis Compared with control,the expression of apoptosis protein caspase 3 increased significantly in the ileal epithelial cells of rats in liver fibrosis group(P<0.01).The decreased expression of caspase 3 was observed in UA treatment group compared with liver fibrosis group(P<0.05).2.4 Effect of ursolic acid on serum LPS,PCT and CRP in rats with hepatic fibrosis Compared with control,the increased levels of serum LPS and PCT were observed and serum CRP had no significant change in liver fibrosis group.The levels of serum LPS and PCT in UA treatment group were lower than liver fibrosis group(P<0.01 and P<0.05 respectively).The content of serum CRP was no statistic difference between UA treatment group and liver fibrosis group(P>0.05).3.Effect of ursolic acid on intestinal inflammation in CCl4-induced hepatic fibrosis rats Compared with control,the m RNA and protein expressions of TNF-? in the ileum of Liver fibrosis group increased significantly(P<0.01 and P<0.05 respectively).The decreased expressions of TNF-? m RNA and protein were observed in UA treatment group compared with liver fibrosis group(P<0.05 and P<0.01 respectively).4.Effect of ursolic acid on intestinal oxidative stress in CCl4-induced hepatic fibrosis rats4.1 Effect of ursolic acid on ileum MDA and T-AOC in rats with hepatic fibrosis Compared with control,the increased level of ileal MDA was observed and ileal T-AOC had no significant change in liver fibrosis group.The level of ileal MDA in UA treatment group were lower than liver fibrosis group(P<0.01).The content of ileal T-AOC was no statistic difference between UA treatment group and liver fibrosis group(P>0.05).4.2 Effect of ursolic acid on the protein expression of NOX subunits P67 phox and NOX2 in the ileum of the hepatic fibrosis rats Compared with control,the protein expressions of NOX subunits P67 phox and NOX2 in the ileum of liver fibrosis group increased significantly(P<0.05).The decreased expressions of ileal NOX subunits P67 phox and NOX2 were observed in UA treatment group compared with liver fibrosis group(P<0.05).Conclusions:1.Intestinal inflammation and NADPH oxidase-mediated oxidative stress participates in intestinal mucosal barrier damage in rats with hepatic fibrosis.2.Ursolic acid ameliorates intestinal mucosal barrier injury in rats with hepatic fibrosis,by inhibiting intestinal inflammation and oxidative stress mediated by NADPH oxidase.