| Objective:To investigate the effect of thymosin ?4(T?4) against acute liver injury induced by carbon tetrachloride(CCl4), and to explore its possible mechanism.Methods:Male Balb/c mice were randomly divided into normal control group, CCl4 control group, Low dose T?4-treated group(L-T?4 group) and High dose T?4-treated group(H-T?4 group). Acute liver injury models were established by a single intraperitoneal injection of CCl4(1 ml/kg, as a 40% solution in olive oil). Mice in normal control group were carried out an injection of olive oil intraperitoneally. T?4(3 mg/kg and 10 mg/kg) was injected intraperitoneally after 1 h following the CCl4 administration. Mice were sacrificed at 2 d and 5 d after the CCl4 administration.Blood samples were collected from the inferior vena cava and left lobes of livers were isolated. The levels of alanine aminotransferase(ALT) and aspartate aminotransferases(AST) were measured in samples of serum. Liver tissue histopathological changes were analyzed by hematoxylin-eosin(HE) staining.Transcript levels of anti-oxidative stress factors(SOD/CAT/GPx), pro-inflammatory factors(TNF-?/IL-1?/MCP-1/IL-6) and apoptosis related genes(Bcl2/caspase-3)were analyzed by real-time quantitative PCR(qRT-PCR) in samples of liver tissue.Western Blot and immunofluorescence staining were performed to evaluate the protein expressions of ?-smooth muscle actin(?-SMA) and Collagen I(Col-I) in liver tissue, respectively.Results:1. T?4 can reduce the increased levels of serum ALT and AST induced by CCl4.ALT level was significantly decreased in H-T?4 groups compared with L-T?4 groups(P<0.05).2. Results of HE staining showed an improvement of histopathologic changes with acute liver injury in T?4 treated groups compared with CCl4 control groups. The effects in H-T?4 group and 5 d group were more obvious than L-T?4 group and 2 d groups, respectively.3. Results of qRT-PCR showed a decreased mRNA levels of anti-oxidative stress factors(SOD/CAT/GPx), an increased mRNA levels of pro-inflammatory factors(TNF-?/IL-1?/MCP-1/IL-6) and an increased mRNA levels of apoptosis related genes(anti-apoptotic gene, Bcl2; pro-apoptotic gene, caspase-3) in CCl4 control group compared with normal control group(P<0.05). At 2 d after CCl4, T?4 increased transcript levels of anti-oxidative stress factors and Bcl2, and T?4 attenuated transcript levels of pro-inflammatory factors and caspase-3. Effect of high dose T?4was more obvious. Compared to CCl4 control group, TNF-?, IL-1? and MCP-1mRNAs in L-T?4 group and CAT, GPx, TNF-?, IL-1?, MCP-1, IL-6 and caspase-3mRNAs in H-T?4 group were significantly changed(P<0.05). Trends of transcript levels in 5 d group were same with 2 d group except for upregulated IL-6 mRNA, and there was a greater variation. CAT, TNF-?, IL-1? and MCP-1 m RNAs in L-T?4group and SOD, CAT, GPx, TNF-?, IL-1?, MCP-1, IL-6 and caspase-3 mRNAs in H-T?4 group were significantly changed compared to CCl4 control group(P<0.05).4. Compared with normal control group, the protein expression of ?-SMA was increased in CCl4 control group by Western Blot analysis. At both 2 d and 5 d after CCl4, low dose T?4 might increase expression of ?-SMA. High dose T?4 had no effect on the expression of ?-SMA.5. Compared with normal control group, the protein expression of Col-I was increased in CCl4 control group by immunofluorescence staining. At both 2 d and 5 d after CCl4, low dose T?4 might increase expression of Col-I. High dose T?4 had no effect on the expression of Col-I.Conclusion:T?4 could ameliorate CCl4-induced acute liver injury in mice in a dose- and time- dependent manner, by suppressing oxidative stress, inhibiting inflammation response and reducing hepatocellular apoptosis. |
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