Therapeutic Effect And Its Mechanism Of Ursolic Acid In A Rat Model Of Experimental Hepatic Fibrosis

Background:Hepatic fibrosis which is associated with the production and deposition of extracellular matrix is a pathological response to various chronic liver diseases. Although hepatic fibrosis is thought to be a reversible pathological state, there is no established therapy for hepatic fibrosis.Several traditional Chinese herbs have shown a promising future on anti-hepatic fibrosis in some studies.Based on the newest researches on hepatic fibrosis and our prior research basis, we adopted dimethy- nitrosamine(DMN) solution intraperitoneal injection to prepare the SD rat hepatic fibrosis model, then to study the benefit effects of Ursolic acid on experimental animal hepatic fibrosis and the possible mechanisms. It will provide a theory foundation for us to develop a drug to prevent and inhibit hepatic fibrosis.Objective:To observe the effects of Ursolic acid on liver function and histo- morphology; to observe antioxidation and the effects of Ursolic acid on Matrix metalloproteinase-1(MMP-1)mRNA expression,Matrix metalloproteinase inbihitor-1 (TIMP-1) mRNA expression andα-SMA expression; then to approach possible mechanisms.Methods: Hepatic fibrosis model was induced by DMN,1ml/kg body weight, intraperitoneal injection,the control model's animals were simultaneously injected saline water at the same administrating route and dosage for successive three days, successive four weeks. 4 weeks later, the experimental rats were randomly devided into 6 groups:normal control group, model group, colchicine group(0.1mg/kg/d), the low dose group of UA(10mg/kg/d), the mean dose group of UA(20mg/kg/d), the highdose group of UA(40mg/kg/d).All the rats were administrated once per day with corresponding drugs and saline water by intraperitoneal injection for successive 2 weeks, 4 weeks. At the end of this experiments, the liver function and oxidation indexes were observed by measuring the indexes of ALT, AST, ALB, A/G and SOD, MDA in serum.After administrated with corresponding drugs for 4 weeks, hepatic lobules constitution, hepatic cell necrosis and hyperplasia of fibrous tissue were observed via HE dyeing method, hyperplastic degree of collagen fiber was observed via VG dyeing method and judged by semi-determinalion. MMP-1mRNA and TIMP-1mRNA expression levels were measured using RT-PCR. The levels ofα-SMA protein were measured by Westernblot.Results:①Compared with normal control group, the indexes of ALT, AST were higher, the indexes of ALB, A/G were lower in model group.Administration of Ursolic acid and colchicine with hepatic fibrosis rats for successive 2 weeks, 4 weeks,Ursolic acid could significantly cut down the indexes of ALT, AST and step up the indexes of ALB, A/G compared with the model group (P<0.01 or P<0.05); colchicine had no effect on A/G, other were between the low dose group of UA and the mean dose group of UA in no time-dependent manner;②The liver in model group was augmented, the fringe was muticous and the texture was hard through naked eye, the livers in UA and colchicine groups were signicantly improved.In HE dyeing, hepatic lobules constitution was complicated, hepatic cell necrosis, hyperplasia of fibrolast and collagen were obvious and hepatic fibrosis was formed in model group; In VG dyeing, fiber septa was composed of collagen fiber, consecutive and staccato crico-collagen were deposited in sinus hepaticus and hyperplastic degree of collagen fiber was higher than normal control group(P<0.01).Morphologic changes of liver tissue were improved in different extent in UA and colchicine groups; In HE dyeing, UA could improve the constitution of hepatic tissue, recovery hepatic lobules constitution, lessen hepatic cell necrosis and obviously inhibit hyperplasia of fibrous tissue; In VG dyeing, collagen fiber significantly decreased in UA and colchicine groups, the hyperplastic degrees of collagen fiber were lessened in the mean dose group of UA and the high dose group of UA compared with model group and colchicine group (P<0.05, P<0.01).③SOD expression index in model group was (124.73±19.79)U?ml-1, which is lower than normal group(176.79±26.69)U?ml-1 (P<0.01); MDA expression index in model group was (66.44±6.51) nmol?ml-1, which was higer than that in normal group(7.95±0.97)nmol?ml-1(P<0.01);Treatment with UA and colchicine for 4 weeks, SOD expression indexes were(154.29±15.39)U?ml-1,(201.22±3.38)U?ml-1, (245.74±37.24)U?ml-1,(128.45±10.06)U?ml-1, the UA groups were higher than that in model group(124.73±39.79)U?ml-1 (P<0.05,P<0.01) and no signicance was found between colchicine group and model group, whereas MDA expression indexes were(26.69±4.75)nmol?ml-1, (14.38±0.48)nmol?ml-1, (10.50±1.44)nmol?ml-1, (42.02±6.29 )nmol?ml-1, respectively, which were significantly lower than that in the model group(66.44±6.51) nmol?ml-1(P<0.05,P<0.01); Significant difference were found in Ursolic acid groups and colchicine group (P<0.01).④Compared with normal group, MMP-1mRNA expression levels were significantly lower and TIMP-1mRNA expression levels significantly higher in model group(P<0.01);After treating liver fibrosis rats with different doses of Ursolic acid and colchicine for 4 weeks, MMP-1mRNA expression levels were significantly higher than that in model group (P<0.01) and TIMP-1mRNA expression levels were significantly lower (P<0.05,P<0.01);Significant difference were found in Ursolic acid groups and colchicine group(P<0.05,P<0.01).⑤Westernblot also indicatedα-SMA protein expression in model group was higher than that in normal group(P<0.01);α-SMA protein expression in Ursolic acid groups, compared with model group, were significantly lower(P<0.01);NO significant difference were found in model group and colchicine group,significant difference were found in UA groups and colchicine group (P<0.05,P<0.01).Conclusions:①Ursolic acid can improve hepatic fibrosis, such as Ursolic acid can significantly cut down the indexes of ALT,AST and step up the indexes of ALB, A/G and then improve liver function; In HE dyeing, Ursolic acid can significantly improve hepatic lobules constitution, lessen hepatic cell necrosis and inhibit hyperplasia of fibrous tissue , In VG dyeing, Ursolic acid can significantly decrease the deposition of collagen fibers and degrade hyperplastic degree of collagen fiber; the synthetic action of UA is stronger than colchicine and the optimal dose of UA is 40mg/kg/d.②Improvements of liver fibrosis induced by Ursolic acid were associated with several possible mechanisms. First, UA can block oxidative stress and inhibit lipid peroxidation by increasing the expression of SOD and decreasing the expression of MDA; Second, UA can increase ECM degradation and decrease ECM deposition by up-regulating MMP-1mRNA expression levels and down-regulating TIMP-1mRNA; Third, UA can inhibit the activation of HSC by down-regulatingα-SMA protein expression levels.