| Hepatic fibrosis is a repair response to chronic liver injury. It is a slow pathological process and a common pathological change in chronic liver disease. Hepatic fibrosis is the result of combined effects of a variety of types of cells, oxidative stress, cytokines and growth factors. The change of texture in the liver is abnormal hyperplasia and deposition of liver extracellular matrix (ECM), which may cause severe liver fibrosis or cirrhosis and even lead to liver failure if not treated in time. Liver fibrosis has become a worldwide health problem. If liver fibrosis is prevented, liver cirrhosis and its complications will be effectively avoided. The researches in recent years suggest that hepatic stellate cells (HSC) are the major source of excessive ECM in liver fibrosis. The activated HSC is proliferated in a large quantity and leads to phenotypic changes and secretion of more ECM deposition, which will result in liver fibrosis.Many anti-fibrosis drugs have been developed in recent years, but the effects have not been affirmed yet. It is of significance to find an ideal anti-fibrosis drug and understand the pathogenesis of liver fibrosis.Oxymatrine (OM) is matrine synthesis, a monomer processed by oxidation, purification and extraction, and N-oxides of matrine. OM which possesses a special oxygen structure and whose molecular polarity is increased is more effect than Matrine. Previous studies have confirmed that OM can protect liver cells, resist hepatitis B virus, inflammation and tumor, and can regulate immunity. Recent study demonstrates that OM has a role in the prevention of liver fibrosis. In this experiment, liver fibrosis models induced by carbon tetrachloride were used. The overall effects of OM on hepatic fibrosis in rats were observed, and the inhibition of OM on HSC-T6 proliferation of molecular mechanisms in vitro as well as its influence on the proliferation of HSC-T6 stimulated by TGF-β1 was explored. The cross effects of MAPK signal transduction pathway and TGF-β1/smads signal pathway in the proliferation of HSC-T6 and the mechanism of OM were investigated. This study consists of two parts:I. Protective effect of OM on liver fibrosis induced by CCl4 in ratsCCl4-induced liver fibrosis model was established. The rats were randomly divided into normal control group, liver fibrosis model group, OM(30mg?kg-1, 60mg?kg-1, 120mg?kg-1)treated group and colchicine(0.2 mg?kg-1)treated group.The experiment results demonstrated that OM had obvious protective effects on CCl4-induced liver fibrosis in rats. The serum level of HA, LN, CIV and PCIII, the surrogate markers of liver fibrogenesis, increased significantly in hepatic fibrotic rats, which was significantly suppressed by OM therapy. Hyp contents, another index of liver fibrosis, showed that OM might reduce the Hyp contents significantly. Histological results manifested that OM might inhibit hepatic fibrosis and improve the alteration in the liver texture significantly.OM could also ameliorate the oxidative stress state in hepatic fibrosis rats, decrease the production of MDA and enhance the activities of anti-oxidative enzyme including SOD and GSH-PX. In the experiment, the influence of OM on the expression ofα-SMA, TGF-β1, smad2, smad3, smad7mRNA was observed. RT-PCR result displayed that the contents ofα-SMA, TGF-β1, smad2 and smad3 were decreased in the liver tissue of the model rats after OM therapy, but smad7 was increased.Phosphorylation of p38, JNK and ERK1/2 increased significantly in the model rats, and OM effectively reduced the Phosphorylation of p38, JNK and ERK1/2 in liver fibrosis rats.II. Molecular mechanism of OM on anti-fibrosis1. Inhibition effect of OM on proliferation of HSC-T6 stimulated by TGF-β1Models of HSC-T6 stimulated by TGF-β1 in vitro were established. OM at a concentration of 0.01, 0.1, 1, 10 and 100mg·L-1 could reduce the proliferation of HSC-T6; the inhibition rate was dose-depended related to the concentration of OM.2. Influence of OM on p38MAPK signaling pathway in HSC-T6 stimulated by TGF-β1.Western blot results showed that TGF-β1 stimulaion might cause rapid p38MAPK phosphorylation in HSC-T6. OM could inhibit the p38MAPK activation in HSC-T6 stimulated by TGF-β1, and reduce the level of p38 phosphorylation significantly, which indicated that p38MAPK phosphorylation in HSC-T6 stimulated by TGF-β1 might resist hepatic fibrosis.3. Influence of OM on TGF-β1/smads signaling pathway in HSC-T6 stimulated by TGF-β1The results by western blot showed that the protein expression of TβRI, smad2 and smad3 in HSC-T6 stimulated by TGF-β1 increased in a time dependent manner, while the expression smad7 decreased. OM could significantly upregulate the protein expression of TβRI, smad2 and smad3 in HSC-T6 stimulated by TGF-β1, and downregulate the protein expression of Smad7, suggesting that inhibiting TGF-β1/smads signaling pathway in HSC-T6 stimulated by TGF-β1 is one of the mechanisms of OM on anti-hepatic fibrosis.4. The relations between p38MAPK and TGF-β1/smads signaling pathway in HSC-T6 stimulated by TGF-β1The effect of TGF-β1 Inhibitor decorin on the HSC-T6 was compared between the model group and the control group, and the level of p38 phosphorylation in the model group increased significantly. Compared with the model group, the inhibitor decorin could not inhibit or upgrade the level of p38MAPK, and there were no significant differences between the two groups. It suggested that decorin had no effect on the p38 pathway.When P38 Inhibitor SB203580 was used to HSC-T6, the expression of TβRI, smad2 and smad3 in the model group increased, and the expression of smad7 decreased. There was no significant difference between p38 pathway inhibitor SB203580 group and model group, indicating that SB203580 had no influence on TGF-β1/smads signaling pathway.
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