| Hepatitis, widely popular in China, is caused by hepatitis b virus(HBV). As intensive study of the pathogenesis of hepatitis B virus infection in the host, scientists come to realize that the immune response of the host to the viruse damage the hepatic cells.Shandougen is the dried roots and rhizomes of Sophora tonkinens Gapnep. In China, it is the preferred treatment of chronic hepatitis clinic at present. After previous studies and research, its main chemical component oxymatrine(OMT) can regulate immunity, protect liver et al. But now the mechanism of protecting liver is not clear. So We use the model of concanavalin(Concanavalin A Con A)-induced liver injury, which is similar to the model of hepatitis B immune-related liver injury, to study the effect of inhibiting liver injury of OMT in terms of immunomodulatory and anti-oxidantion and then provide data to explain the mechanisms of treating HBV of OMT. PartⅠ The immunomodulatory effects of OMT on Con A- induced liver injury.Objective:To investigate the immunomodulatory effect of OMT on on Con Ainduced liver injury.Method:1.The Balb / c mice, 5-6 weeks old, 18-22 g, were randomly divided into the control group, Con A 5 mg·kg-1 group, Con A 10 mg·kg-1 group, Con A 15 mg·kg-1 groups. Every group was composed of six Balb / c mice. The control group was injected saline intravenously, the rest groups injected difference concentration of Con A intravenously. After 8 hours, then take blood by removing eyeball to test the ALT and the liver tissue was sampled to take HE-staining.2.The Balb / c mice, 5-6 weeks old, 18-22 g, were randomly divided into the control group(Group A), Con A 15 mg·kg-1 group(Group B), bicyclol(positive drug) 156 mg·kg-1 treatment group(Group C), OMT 60 mg·kg-1 group(Group D), OMT 120 mg·kg-1 group(Group E). Every group was composed of six Balb / c mice. Group B, Group C, Group D and Group E were injected intravenously Con A of 15 mg·kg-1 to made acute liver failure model, while Group D and Group E were injected OMT intraperitoneally. Group B was received bicyclol by intragastric administration. Group A and Group B made mold were administered separately at the same dose of saline. After 8 hours, then take blood by removing eyeball to test the ALT 、AST, the expression of correlative cytokines and test the SOD、GSH-PX in the liver. Liver tissue was sampled to take HE-staining. The spleen lymphocyte was Isolated and then the Th17 and Treg cells was tested by FCM.Result:1. The establishment of acute hepatic failure model induced by Con A: With the concentration of Con A increasing, the aminopherase in serum gradually increased. Compared with normal group, the injury of liver tissue and gradually increased with HE stain and the mortality of mice gradually increased. When the concentration of Con A was15 mg·kg-1, the level of aminopherase reached up to 1032.08 ± 85.6 2 U·L-1. This concentration was used in the following study.2. The influence of OMT on the acute hepatic failure model : Compared with the control group the aminopherase in serum significantly increased(P <0.01). With the HE stain, hepatocyte was edematous and congestive. The structure of hepatic lobules was disordered with bridging necrosis and patchy necrosis. The liver was infiltrated with inflammation cell. After the injection of OMT inroperitoneally, the aminopherase and T-BIL in serum significantly declined(P <0.01). When the concentration of OMT was 120 mg·kg-1, the aminopherase and T-BIL in serum declined most and the injury of hapar alleviated compared with the model group. The ALT of Positive medicine group declined(P<0.05), but the AST increased(P>0.05).3. The influence of OMT on cytokine: Compared with the control group, the level of IL-17 and IL-10 increased significantly in the model group(P <0.01). After treatment with OMT, the level of IL-17 was significantly decreased(P <0.01), but the IL-10 was significantly increased(P <0.05).4.The influence of OMT on Th17 and Treg cells: Compared with the control group, the percentage of Th17 and Treg cells in the model group was significantly increased(P<0.05); After OMT intervention, the percentage of Th17 cells was significantly decreased in a dose-dependent manner(P<0.05); the percentage of Treg cell were increased in a dose-dependent manner(P <0.05); The OMT 60 mg·kg-1 group, the percentage of Treg cell although increased, but the rate was not obvious and has no significant difference(P > 0.05).Conclusion:OMT can protect the liver cell by inhibiting the proliferation of Th17 cells. OMT can protect the liver cell by promote the proliferation of Treg cells. PartⅡ The anti-oxidation effects of OMT on Con A- induced liver injury.Objective:To investigate the anti-oxidation effects of oxymatrine(OMT) on Con Ainduced liver injury.Method:1. Animal experiment: on the basis of ALT、AST and pathology in PartⅠ, the SOD and GSH-PX in liver was tested to evaluate the effect of anti-oxidation of OMT.2. Cell experiment: Using CCK-8 to detect the cell activation of L02 cells treated by different OMT. L02 cells were damaged by hydrogen peroxide(H2O2) to establish the model of hepatocellular oxidative stress. The apoptosis of L02 cells was detected using Annexin-V/7-AAD apoptosis detection kit.The level of ROS, GSH-PX and SOD was detected to analysis the mechanism of OMT against oxidative stress.Result:1.Animal experiment: Compared with the control group, the SOD and GSH-PX reduced(P<0.05); when treated by OMT, the SOD and GSH-PX rised(P<0.01) especially in the OMT 120 mg·kg-1 group.2.Cell experiment: When the concentration of OMT is between 6.25 μg·ml-1 and 100 μg·ml-1, it can promote the activation of L02 cells detected by CCK-8. The OMT whose concentrations were in this range could reduce the apoptosis rate of L02 cell induced by H2O2 and could protect L02 from H2O2 by reducing ROS and strengthening the activity of SOD and GSH-PX.Conclusion:OMT protect the liver cell by strengthening the activity of GSH-PX and SOD which can help GSH geting rid of the ROS. |
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