| Background and Objective:As an important stage that influences the prognostic treatment for chronic liver diseases, hepatic fibrosis is a histopathological change that is commonly seen in the process of many chronic liver diseases developing into hepatic cirrhosis. This pathological process is characterized by the excess disposition of extracellular matrix (ECM) (mainly consisting of collagen) due to the increasing synthesis and relative deficiency in degradation. Hepatic fibrosis is a dynamic process that is a reversible pathological change. However, it becomes irreversible if hepatic fibrosis develops into the structural change of hepatic lobule and the formation of pseudo-lobule (hepatic cirrhosis stage). Up to now, anti-hepatic fibrosis drug with widely approved effect has not been available for clinic treatment. Therefore, the preventive and therapeutic research for hepatic fibrosis will still remain a hot but difficult topic for home and abroad researchers in the future.Bile acid derivative-Ursodeoxychlic acid (UDCA) is a bile acid that is synthesized in the third step of human endogenous synthesis. It can antagonize the cytotoxity of the hydrophobic bile acid, protect the hepatocyte membrane, regulate the immunological function, suppress the apoptosis, eliminate the radicals and have effects such as antioxidation, inflammation suppression and so on. It also has pronounced therapeutic effects on diseases of intrahepatic cholestatic chronic hepatitis, primary biliary cirrhosis of liver.It is reported that UDCA may also have effects on anti-hepatic fibrosis. To further investigate the effect of UCDA on hepatic fibrosis, we employ a classic experimental animal model of hepatic fibrosis -porcine serum induced hepatic fibrosis, choose Colchicin as the control medicine and monitor the therapeutic effect of UCDA on intervening on immuno-damaged hepatic fibrosis in animal entirety. Furthermore, we investigate the effects of UCDA on the stimulation of TGFβ1 to the proliferation of hepatic stellate cell (HSC) and the secretion of collagen type I and the effects of UCDA on signal transduction of TGF|31/Smad. Possible molecular mechanisms of the anti-hepatic fibrosis effect of UDCA are discussed in order to provide preliminary fundamental theory and animal experimental proofs for in clinical prevention and cure of hepatic fibrosis.Methods: 1.120 Wister rats (60 for each sex), are randomized into 6 groups: 20 rats for hepatic fibrosis model group (Group B), and the rat model of hepatic fibrosis model is established by intraperitoneal injection with porcine serum, 0.5ml once, twice per week for 12 weeks; 20 rats for control group (Group A), intraperitoneally injected with equivalent physiological saline; 20 rats for each UDCA group with different doses (Group C, D, E), porcine treatment is same as the model group, and UDCA doses of 30mg·kg-1·d-1,15 mg·kg-1·d-1,7.5mg·kg-1·d-1 (dissolved with normal drink water to concentration of 8 g/L, 4 g/L, 2 g/L) are given to rats in drink water for 12 weeks; 20 rats for colchicine group (Group F), porcine treatment is the same as the model group, and colchicine dose of 0.1mg·kg-1·d-1 is given to the rats once per day for 12 weeks. All the rats are sacrificed after the study and their livers and spleens are examined; HE staining and Masson staining are used to examine the histopathological change of liver and rate the degree of the fibrosis, and random samples are examined for ultra microstructural change. Alanine transaminase (ALT), asparatate transaminase (AST),gamma-glutamyl transferase (GGT), total bile acid (TBA) and Albumin(Alb) are analyzed with an automatic biochemical analyzer; Hyaluronic acid (HA), Laminin (LN), Type IV collagen (C IV), Procollagen type III(PCIII) are analyzed with radioimmunoassay; Expressions of type I and type III collagen and TGF β1 in liver tissue are examined in immunohistochemistry methods, then the mean gray value is analyzed by the graphic semi-quantitative method; Expressions of type I collagen and type III and TGF β1 mRNA in liver tissues are examined by using reverse transcription - polymerase chain reactions (RT-PCR) method, and the ratio between the peak area below the curve and GAPDH mRNA integral value is calculated by graphic analysis.2. 24 hours or 48 hours after HSC-T6 is treated with different concentrations of UDCA (0.25, 0.5, 1.0 mmol/L), 5ng/ml TGFβ1 is added for stimulating. The effect of UDCA on HSC-T6 proliferation is analyzed with MTT colorimetry and cell periodic location is examined in Flow Cytometry (FCM); Expressions of type I collagen and cyclin D, cyclin E protein in each cell group are examined by immunocytochemistry and expressions of type I collagen and mRNA in each cell group are examined with RT-PCR method.3. 24 hours or 48 hours after HSC-T6 is treated with different concentrations of UDCA (0.25, 0.5, 1.0 mmol/L), 5ng/ml TGFβ1 is added for stimulating. Effects of UDCA on the expression of TGFβ1 receptor Type I (TβR (?)), Smad3, Smad4 and Smad7 protein are examined with western blotting. Expressions of Smad 3, Smad 7 and CBP mRNA in each cell group, blank group and control group are examined with RT-PCR method.Results: 1.Compared to model group, the body weight of rats in UDCA groups remarkably increases (P<0.05～0.01), while the liver weight and spleen weight remarkably decrease (P<0.05～0.01); The degree of hepatic fibrosis in the UDCA group significantly improves (P<0.05～0.01); The activity of ALT, AST, GGT and TBA level of rat serum in UDCA groups with each dose significantly decreases compared with the model group (P<0.05～0.01), the Alb level of serum of high dose UDCA increases significantly compared with the model group (P<0.05), The HA, LN, C IV and PC III levels of rat serum in UDCA groups with each dose significantly decrease compared with the model group (P mean< 0.01). The expression of type I collagen and its mRNA in UDCA groups with each dose significantly decrease (P<0.05～0.01) and the decrease is dose dependent. The expression of type III collagen and its mRNA significantly decrease (P<0.05～0.01); The expression of TGF β1 mRNA in UDCA high dose group significantly decrease (P<0.05) and the expression of TGF β1 protein shows no influence (P>0.05); Electron microscopy results show a significant improvement in the rat liver ultra microstructure compared to the model group.2.UDCA can remarkably inhibit the stimulation of TGF β1 to HSC proliferation, and this effect is obviously time and dose dependent. UDCA can induce an increase of HSC in G0/G1 stage (P<0.05～0.01) and effectively retard the HSC cell cycle, and this effect is significantly time and dose dependent. UDCA can remarkably inhibit the expression of type I collagen and its mRNA (P<0.05～0.01), and this effect is significantly time and dose dependent. High dose of UDCA can effectively inhibit the expression of cyclin D and cyclin E protein (P<0.05).3.After exogenetic TGF β1 stimulation, the expressions of TβR I , Smad3, Smad4, Smad7 protein in rat liver HSC all increase significantly (PO.05～0.01) and the expressions of Smad, Smad7mRNA and CBPmRNA also increase remarkably. Meanwhile, UDCA shows no influence on the stimulation of TGFβ1 on the expression of TβR I protein and Smad 4 protein in HSC (P>0.05). UDCA can significantly decrease the stimulation of TGFβ1 on the expressions of Smad 3 protein and its mRNA in HSC (P<0.05～0.01), and this effect is time and dose dependent. UDCA can significantly decrease the expression of CBPmRNA (P<0.05～0.01), and this effect is time and dose dependent. UDCA can significantly increase the expressions of Smad 7 protein and its mRNA(P<0.05～0.01),Conclusions: 1.UDCA can significantly increase the rat body weight with immuno-damaged hepatic fibrosis, decrease the liver weight and spleen weight, improve the pathologic degree of the rat hepatic fibrosis, decrease the level of ALT, AST, GGT, TBA, increase Alb level in rat serum, protect hepatic cells and improve hepatic function; Meanwhile, it significantly decreases haluronic acid (HA), laminin (LN), type IV collagen (C IV) level and procollagen (PC III) level in rat serum and thereby decreases the degree of fibrosis; It also remarkably inhibits the expression of type I collagen in rat liver tissue and this inhibition is dose dependent; It significantly inhibits the expression of type III collagen in rat liver tissue. High dose of UDCA can remarkably inhibit the expression of fibrosis factor TGF β1 mRNA in rat liver tissue and thereby inhibit the development of the fibrosis.2.UDCA can remarkably inhibit the stimulation of TGFβ1 to HSC proliferation, and this inhibition is time and dose dependent; It remarkably inhibits the secretion of type I collagen, and this inhibition is time and dose dependent; high dose of UDCA can effectively inhibit cyclin D and cyclin E protein.3.UDCA has no influence one the stimulation of TGFβ1 to the expression of TβRI protein and Smad4 protein in HSC; UDCA can remarkably decrease the expression of Smad3 protein, and this effect is dose and time dependent; It decreases the CBP expression and this effect is dose and time dependent; It increases Smad7 expression and thereby effectively weakens the signal transduction of TGFβ1, which is possibly due to the fact that it intervenes the signaling pathway of TGFβ1/Smad and exhibits important molecular basis for anti-hepatic fibrosis effects.
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