| Tripterygium wilfordii Hook. f.(TWHF) belongs to Celastraceae. Its taste is bitter and belongs to liver and kidney channel. It has a relatively good therapeutic effect for many diseases, such as nephrotic syndrome, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, skin diseases and so on. But, the toxicity of TWHF is fierce. Its liver toxicity is in the first place in documents reported about single traditional Chinese medicine, which limits its clinical application to a large extent. But the etiology and pathogenesis of TWHF-induced liver toxicity is not entirely clear. So, this object aims to explore the molecular mechanism of liver toxicity caused by TWHF and the protective effects and mechanism of Xiaoyao San on it by the modern molecular biology techniques, thus to provide theoretical and experimental basis for the prevention and cure of Xiaoyao San on liver toxicity caused by TWHF.Part One Preparation for the acute liver toxicity Model in Rats Induced by TWHF Water DecoctionObjective:To establish acute liver toxicity model in rats by TWHF water decoction. Methods:Rats were given TWHF water decoction by gavage and divided into different groups by doses of2.5,3.75and5.0g·kg-1and time of1,2,3,4,5and6d. The liver index of rats was detected by weighing method. The activities of serum ALT and AST were detected by dry chemical method. Liver tissue pathological changes were analyzed by HE staining and Semi-quantitative analysis was combined. Results:Compared with the control group, the liver index and the activities of serum ALT and AST were increased significantly at the middle and high doses of TWHF water decoction. At the same time, the pathological changes and serious liver damage were found in liver tissue too. With the extension of the intervention time, the toxic effects of TWHF have a increasing trend and arrived the peak after4days. Conclusion: Intervening rats for4days at the middle dose of3.75g.kg-1of TWHF could be considered as relative right methods to induce acute liver toxicity model.Part Two Protective Effect of Xiaoyao San on the TWHF Water Decoction Induced Acute Liver ToxicityObjective:To observe the protective effect of different doses and dosing regimens of Xiaoyao San on the acute liver toxicity induced by the TWHF water decoction. Methods:Rats were given TWHF water decoction by gavage to induce the acute liver toxicity model and were intervented by different doses of3.375,6.75and13.5g. kg-1and dosing regimens of prevention, combat and treatment of Xiaoyao San for4days at the same time. The liver index of rats was detected by weighing method. The activities of serum ALT and AST were detected by dry chemical method. Liver tissue pathological changes were analyzed by HE staining and Semi-quantitative analysis was combined. Results:Compared with the model group, the Liver index, the activities of serum ALT, AST and liver pathological changes in rats of middle and high doses of Xiaoyao San group were reduced significantly and had no significant difference compared with the control group. Compared with the model group, the Liver index, the activity of serum ALT, AST and liver pathological changes in rats of Xiaoyao San prevention group were reduced significantly and had no significant difference compared with the control group. Conclusion:It was showed that interventing rats at the middle dose of Xiaoyao San ahead of TWHF could be considered as relative right methods to protect the acute liver toxicity caused by TWHF. Part Three Study on Mechanism of the TWHF Induced Acute Liver Toxicity and Protective Effect of Xiaoyao SanObjective:To observe the mechanism of the TWHF induced acute liver toxicity and the protective effect of Xiaoyao San. Methods:Rats were respectively given Xiaoyao San by gavage of6.75g.kg-1and Phenobarbital of1010mg·kg-1and ketoconazole of80mg·kg-1by abdomen injection respectively for4days before being given TWHF at the dose of model. The control group and model group were set. The content of CYP450was detected by CO reduction Spectrophotometric method and the content of b5was detected by the same method except without adding CO. The content of protein of CYP3A4, CYP2C19and Cyt-C of liver were detected by Western-immunoblotting. The activity of CYP3A4and CYP2C19of liver were detected by HPLC. The liver index of rats was detected by weighing method. The activities of serum ALT and AST were detected by dry chemical method. Liver tissue pathological changes were analyzed by HE staining and Semi-quantitative analysis was combined. The mRNA levels of Bcl-2, Bax and Caspase-3genes in the liver tissue were detected by Real-Time PCR. The protein expression levels of Bcl-2, Bax and Caspase-3in the liver tissue were detected by immunohistochemistry. The membrane potential of mitochondrial in liver cells was detected by FCM. The apoptosis of liver cells was detected by TUNEL assay. The activities of SOD and GSH-Px and the content of MDA in serum were detected by Chemical colorimetric. The content of CD68and TNF-a in serum were detected by ELISA assay. Results:Compared with the control group, the content of liver CYP450and b5of model group were reduced significantly, both the activities and protein expression levels of CYP3A4and CYP2C19in rats liver of the model group were increased significantly, the expression of Bcl-2mRNA and protein in rats liver of the model group were reduced significantly and that of Bax, Caspase-3mRNA and protein were increased significantly, the ΔΨm level of liver cells of the model group was reduced significantly, the release of Cyt-C level of the model group was increased significantly, the apoptosis rate of liver cells of the model group was increased significantly, the activities of SOD, GSH-Px in serum of rats of the model group were reduced significantly, however, the content of MDA was increased significantly, the content of CD68, TNF-a in serum of rats of the model group were increased significantly. Compared with the model group, the content of liver CYP450of Xiaoyao San prevention group and CYP450induction group were increased significantly, the Liver index and the activities of serum ALT, AST of these groups were reduced too.There were no obvious pathological changes in liver tissue of Xiaoyao San prevention group and CYP450induction group. Compared with the model group, both the activities and protein expression levels of CYP3A4and CYP2C19in rats liver of Xiaoyao San prevention group and CYP450induction group were increased significantly, the expression of Bcl-2mRNA and protein in rats liver were increased significantly and that of Bax, Caspase-3mRNA and protein were reduced significantly of the Xiaoyao San prevention group and CYP450induction group, the ΔΨm level of liver cells was increased significantly, the release of Cyt-C level was reduced significantly, the apoptosis rate of liver cells was reduced significantly, the activities of SOD, GSH-Px in serum of rats of Xiaoyao San prevention group and CYP450induction group were increased significantly, however, the content of MDA was reduced significantly, the content of CD68, TNF-α in serum of rats of Xiaoyao San prevention group and CYP450induction group were reduced significantly. Conclusion:There is some association between CYP450enzymes metabolic disorders and liver toxicity induced by TWHF. The possible protection mechanism of Xiaoyao San against TWHF induced liver toxicity may be related with inducting of CYP450enzymes and further to inhibit excessive apoptosis of liver cells, lipid peroxidation induced liver toxicity and cytokine induced immune liver toxicity. |
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