The Research Of Mechanism Of "Qingzhi Hugan Prescription" And Src Inhibitors PP2 In NASH

Background:Nonalcoholic fatty liver disease refers to a pathology syndrome of no history of heavy drinking and other clear liver damage factors,it also can be accompanied with liver parenchyma cell fatty degeneration, necrosis, inflammatory cells infiltration and fat deposition. NASH refers to a pathology of hepatocyte steatosis, and lobular inflammation and/or portal areas.It also can be accompanied with formation of Mallory body ang liver fibrosis. NASH is accounted for 30%～50% in NAFLD.It can lead to nonalcoholic fat liver cirrhosis and hepatocellular carcinoma and related complications, It will change the prognosis of NAFLD if we can block this link. Therefore the research of mechanism of "qingzhi hugan prescription" and Src inhibitors PP2 in NASH can provide certain theoretical basis for clinical treatment of NAFLD.Objective:To observe the expression of liver function, blood lipids, inflammatory factor (IL-1, IL-6, TNF-α) of NASH rats and Src family related kinases (Hck, Lyn, Fgr)/SSeCKS in rat liver tissues and kupffer cells after useing Qingzhi hugan prescription and Src inhibitors PP2, to explore the mechanism of "qingzhi hugan prescription" and Src inhibitors PP2 in NASH.Methods:In body level research:Selection of The Third people’s Hospital of Nantong from 2012 to 2014 diagnosed as liver tissue of NAFLD wax blockl2cases, and 10 cases of normal liver tissue, deceted the expression of Src family kinase (Hck,Lyn,Fgr)/SSeCKS by Immunohistochemistry. In cell experiment:q RT-PCR::Well growing kupffer cells are cultivate to extract the RNA of kupffer cell.Then we transcribe it into cDNA,and using real time PCR to detect the expression of mRNA of Src family kinase (Hck,Lyn,Fgr)/SSeCKS; Well growing kupffer cells are cultivate to extract the RNA of kupffer cells after stimulating with 60% CCL4 liquid damage for 4 hours and trainning with 20% Qingzhi Hugan Prescription drug serum or Src inhibitors PP2 for 48 h.Then we transcribe it into cDNA,and using real time PCR to detect the expression of mRNA of Src family kinase (Hck,Lyn,Fgr)/SSeCKS. ICC:Well growing kupffer cells are stimulated with 60% CCL4 liquid damage for 4 hours and then trainning with 20% Qingzhi Hugan Prescription drug serum or Src inhibitors PP2 for 48 h to detect the expression of Src family kinase (Hck,Lyn,Fgr)/SSeCKS by Immunocytochemistry. Western Blot:Well growing kupffer cells are cultivate to extract the albumen of kupffer cell, and using Western Blot to detect the expression of Src family kinase (Hck,Lyn,Fgr)/SSeCKS. In animal experiments: The normal SD rats are treated with high-fat (80.5% Ordinary feed and 2% cholesterol,12% lard, 5% egg yo lk powder,0.5% sodiumcholic acid) for 8 weeks and the models were established. The models were randomized into model group, "Qingzhi Hugan Prescription" groups (large,medium and small dose),PP2 group.The model group were gavaged with 0.9% NaCl solution. The "Qingzhi Hugan Prescription" groups were gavaged with decotions(2.43g/kg/d,12.15g/kg/d, 24.3g/kg/d) for 6 weeks.The PP2 group were injected withlOmmol/L Src inhibitors PP2 0.1 ml intraperitoneal. The control group were injected with saline volume. The mouse changes in body weight and behaviors were recorded.The next day after the end of the intervention, all rats were sacrificed and the serum and livers were obtained. we can observe the livermorphology and detect the biochemical ang blood lipids indexes of serum ALT、AST and so on.The liver tissue sections are using HE staining and the expression of Src family kinase (Hck,Lyn,Fgr)/SSeCKS in the rat liver tissue were deceted by Immunohistochemistry. The expression of IL-1、IL-6、TNF-α were deceted by enzyme-linked immuno sorbent assay.Results:In body level research:the IHC results showed low expression of Hck,Lyn in normal liver tissues and high expression with inflammatory liver tissues(P< 0.05), but there were no obvious expression of Fgr and SSeCKS in normal liver tissue or inflammatory liver tissues.In cell experiment:q RT-PCR:the expression of Hck, Lyn in Kuffer cells abundance to meet the requirements on reducing experiment, the expression of SSeCKS in Kuffer cells abundance was low, did not meet the requirements on reducing experiment,but it has no expression of Fgr in Kuffer cells. The expression of Hck, Lyn increased significantly (P<0.05) after stimulating with 60% CCL4 liquid damage, SSeCKS mRNA expression decreased, but without statistical significance (P> 0.05); The expression of Hck, Lyn was reduced after useing Qingzhi hugan prescription and Src inhibitors PP2; the expression of SSeCKS slightly increased, whereas it has no expression of Fgr. The ICC results showed that the Hck was expressed in kupffer cells, tan grains, and was positively associated with the degree of inflammation, after training with drug serum and Src inhibitors PF2, the expression of Hck was reduced,tan particles change money, less obviously, a scattered distribution.But it has no expression of Lyn、Fgr and SSeCKS express in kuffer cells.Western Blot:Hc、Lyn and SSeCKS have expression in kuffer cells, especially the expressoin of Hck and SSeCKS was obvious and it has no obvious expression of Fgr in rat kupffer cells. In animal experiments:the IHC results showed low expression of Lyn, Hck in normal liver tissues and high expression with inflammatory stimuli (positive correlation), low expression of SSeCKS in normal liver tissue and decreased expression with inflammatory stimuli (negative correlation), extremely low expression of Fgr in normal liver tissue and almost no expression with inflammatory stimuli. After useing Qingzhi hugan prescription and Src inhibitors PP2 in NASH rats,we found that whatever the large、medium and small dose of the Chinese medicine groups and PP2 groups,especially the large dose of Qingzhi Hugan Prescription" group,the positive expression of Hck and Lyn were decreased markedly (P<0.01); The expression of SSeCKS in model group was decreased markedly;In the Chinese medicine groups,the SSeCKS were expressed between the model group and normal group; It showed a trend of decrease along with the rise of the expression of Lyn and Hck in the rat liver tissuewith nonalcoholic fatty liver disease;Compared with model group,effect of "Qingzhi Hugan Prescription" on the expression of Fgr was insignificant (P>0.05); The ELISA results showed that:compared with normal group,the expression of IL-1、IL-6 in model group in NASH was significant (P<0.01),but the expression of TNF-α was insignificant (P>0.05); Compared with model group, the expression of IL-1、IL-6 in medium and high dose group was significant (P< 0.01), especially the high dose group best,and the expression of TNF-α was insignificant (P>0.05).Conclusion:When inflammation stimulation, the expression of Lyn, Hck and SSeCKS were negatively correlated and the expression of Lyn was positively correlated with Hck. which indicates that there’s some kind of upstream and downstream inhibition between SSeCKS and Src family related kinase (Lyn, Hck),but the reduced Fgr expression is in accordance with the SSeCKS is mainly considered to be the result of the competitive inhibition between Fgr and family members Lyn and Hck. It suggests that Src family kinase (Lyn, Hck, Fgr)/SSeCKS are involved in the occurrence and development of NASH, that can be used as a potential target of NAFLD inflammation. The expression of Hck、Lyn and SSeCKS were regulated by "Qingzhi Hugan Prescription", which reduced inflammation of the liver.lt can be used as one of the mechanisms for thetreatment of NAFLD.