Effects of oxymatrine on experimental hepatic fibrosis and its mechanism in vivo

Abstract

Aim: Hepatic fibrogenesis has close relation with hepatic stellate cells (HSC) and tissue inhibitors of metalloproteinase (TIMP). Oxymatrine (OM) is a kind of Chinese herb that is found to have some effects on liver fibrosis. We aimed to determine the effects of OM on hepatic fibrosis and explore the possible mechanism.

Methods: Thirty-two rats were randomly divided into four groups; 16 were used to develop hepatic fibrosis by carbon tetrachloride (CCl4) and treated with or without OM, and 16 were used as controls. The expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and alpha-smooth muscle actin (alpha-SMA) in the livers of rats was detected by immunohistochemical assay. Liver pathology was determined by H and E staining and reticulum staining.

Results: In CCl4-injured rats, the normal structure of lobules was destroyed, and pseudolobules were formed. Hyperplasia of fibers was observed surrounding the lobules. While the degree of fibrogenesis in liver tissues was significantly decreased in those rats with OM-treatment compared with those without OM treatment. The pseudolobules were surrounded by strong, multi-layer reticular fibers, which netted into pseudolobules in CCl4-injured rats, however, there was a significant decrease in reticular fibers in OM-treated rats. The expression of TIMP-1 in hepatic cells was weak in control groups, but strong in CCl4-injured groups, however, the expression of TIMP-1 was significantly inhibited by OM (F = 52.93, P<0.05). There was no significant change in the expression of alpha-SMA between CCl4-injured rats with or without OM treatment (F = 8.99, P>0.05).

Conclusion: OM effectively inhibits CCl4-induced fibrogenesis in rat liver tissues, probably by reducing the expression level of TIMP-1.

Figure 1

Pathological changes in rats’ liver by H.E. staining. 10×10 A: (Control group): The structure of liver lobules was normal, the size and shape of hepatic cells were normal and distributed centrifugally; B: (CCl₄ group): The normal structure of lobules was destroyed, and pseudolobules were formed. Liver cells swelled and hepatic lobules were encysted and separated by collagen bundles, and associated with infiltration of inflammatory cells; C: (CCl₄+OM group): The normal structure of lobules was also destroyed, pseudolobules formed, and liver cells swelled at a milder extent. The hepatic lobules were encysted and separated by slimmer collagen bundles, with milder infiltration of inflammatory cells.

Figure 2

Observation on reticular collagen by reticular staining. 10×10. A: (Control group): A few fiber tissues were only seen in portal areas; B: (CCl₄ group): The pseudolobules were surrounded by strong, multi-layer reticular fibers, which netted into pseudolobules; C: (CCl₄+OM group): The pseudolobules were surrounded by slimmer, multi-layer reticular fibers; a significant decrease of reticular fibers was observed in this group, compared with CCl₄ group.

Figure 3

Contents of collagen in livers of rats in different groups.

Figure 4

Expression of TIMP-1antigen by immunohistochemical staining. 10×10 A: (Control group): There was only a weak expression of TIMP-1 antigen in individual liver cells and hepatic stellate cells (HSCs); B: (CCl₄ group): The expression of TIMP-1 was detected in cytoplasm of most hepatic cells; C: (CCl₄+OM group): There was a significant decline of the expression of TIMP-1 in CCl₄+OM group, as compared with that in CCl₄ group.

Figure 5

Expression of -SMA antigen by immunohistochemical staining. 10×10 A: (Control group): α-SMA antigen was detected in myofibroblasts and vascular endothelial cells; B: (CCl₄ group): The expression of α-SMA was significantly stronger than that in the control groups; C: (CCl₄+OM group): The expression of α-SMA was not significantly different from that in CCl₄ group.