| HCC(hepatocellular carcinoma, HCC) is one of the common digestive system malignant tumor in the world, every year the world of death from liver cancer reached 700000 cases, which ranked third for the mortality in the world.And HCC was mostly caused by viral hepatitis, alcohol or liver toxic substances. The main means of clinical treatment of liver cancer is surgical resection and chemotherapy, but the chemotherapy drug has a great deal of adverse reactions, poor selectivity and resistance, therefore, the advantages of Chinese medicine treating liver cancer is increasingly prominent and practical. Current research of traditional Chinese medicine in the treatment of liver cancer have been making progress, many traditional Chinese medicine active ingredients have been confirmed with pharmacological activities for the treatment of liver cancer.Gegen Qinlian decoction which consists of puerariae, glycyrrhizae, scutellaria a nd rhizome coptidis, is from Shanghanlun of Zhang Zhongjing in Han dynasty, and a lso it is one of the famous compounds of traditional Chinese medicine.Traditional Chinese medicine compound has distinct characters, that is quantities of ingredients and targets. The effective substances of TCM after absorption into the blood may be the prototype, metabolic product or something new. Based on the characteristics of traditional Chinese medicine compound, the research of TCM compound efficacy should not be confined to examine the effect of compound as a whole, or single higher composition and content only,but the pharmacokinetic characteristics of Chinese traditional medicine compound should be well considered. Thus, the mechanism of the traditional Chinese medicine compound can be illuminate in detail by in vitro and in vivo experiments, serum pharmacological method and so on with the serum containing Chinese medicine compound and its main metabolism. For most of the research on the Gegen Qinlian decoction, they are limited to focus on the whole compound and the main ingredients of quality control and therapeutic effects, and the main composition is not necessarily the best active ingredients. As the relative studies and references on Gegen Qinlian decoction and its metabolism are less, this study will further explore and develop it on this aspect. Thus,it provides a new idea to study the decoction and a new way to select new compounds as a leading drug reasonably.To further investigate the effect and correlation of Gegen Qinlian Decoction and its main metabolites from the rat’s serum on the huh7 cells P38 MAPK signaling pathway,MTT, Real-time fluorescence quantitative PCR and wstern blot method were used.Firstly, to ensure the proper and reasonable drug administrative doses, MTT assay was used, and then Real-time fluorescence quantitative PCR was used to determine the transcriptional levels of P38 MAPK genes and meanwhile,the relevant proteins expression of P38 MAPK were detected by western blot. The Results showed that huh7 cells administrated with Gegen Qinlian decoction had an increasing expression of P38 and JNK proteins,and decreasing expression of ERK protein,and puerarin has a down-regulating effect on the expression of ERK proteins at the dose of 64μ M,but has an up-regulating effect on the JNK proteins, daidzin at the dose of 32μ M has increased the expression of P38 and JNK,Isoflavoues Aglycone group has a up-regulating effect on the P38 protein(P<0.05).All in above, Gegen Qinlian decoction and its main metabolites has different effects on the P38 MAPK signaling pathway,but the overall effects of its main metabolites are in accordance with Gegen Qinlian decoction.This study combined 2-DE with serum pharmacology each other, and then the two 2-DE images of the huh7 cell groups administrated by control rats serum and drug serum were compared and analyzed to look for variable proteins, which can precisely and effectively show the corresponding proteins relating to Gegen Qinlian decoction. It was showed that there were totally 60 dots between the images of the huh7 cells administrated by control rats serum and drug serum, and 5 of them were identified by mass spectrometry, including: fibronectin, Deoxyuridine 5’-triphosphate nucleotidohydrolase, Superoxide dismutase[Cu-zinc], Adenylate cyclase type1, GDP dissociation inhibitory factor(Rho GDP dissociation inhibitor, GDI). Among them, fibronectin, Deoxyuridine 5’-triphosphate nucleotidohydrolase, Superoxide dismutase[Cu-zinc] had down-rugulating expressions, otherwise adenylate cyclase 1 and GDI had up-regulating expressions. In this study, the new discovered 5 proteins has provided a theoretical and reasonable basis to further illuminate much more mechanisms of the Chinese traditional formula Gegen Qinlian decoction. |
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