The Research Of Mechanism Of Src Family Related Kinase Hck In NAFLD After Using Qingzhi Hugan Prescription

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" in NASH can provide certain theoretical basis for clinical treatment of NAFLD.To observe the expression of liver function, blood lipids, inflammatory factor (IL-1, IL-6, TNF-?) of NASH rats and Src family related kinases Hck in rat liver tissues and kupffer cells after useing Qingzhi hugan prescription, to explore the mechanism of "qingzhi hugan prescription" in NASH.In body level research:Selection of The Third people's Hospital of Nantong from 2012 to 2014 diagnosed as liver tissue of NAFLD wax block 12cases, and 10 cases of normal liver tissue, deceted the expression of Src family kinase Hck 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 Hek; 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 dreg serum for 48 h.Then we transcribe it into cDNA,and using real time PCR to detect the expression of mRNA of Src family kinaseHck. 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 for 48 h to detect the expression of Src family kinase Hck 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. In animal experiments:The normal SD rats are treated with high-fat (80.5% Ordinary feed and2% cholesterol,12% lard,5% egg yo 1k 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).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 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 in the rat liver tissue were deceted by Immunohistochemistry. The expression of IL-1?IL-6?TNF-? were deceted by enzyme-linked immuno sorbent assay.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 in Kuffer cells abundance to meet the requirements on reducing experiment,. The expression of Hck increased significantly (P< 0.05) after stimulating with 60% CCL4 liquid damage; The expression of Hck was reduced after useing Qingzhi hugan prescription. 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, the expression of Hck was reduced,tan particles change money, less obviously, a scattered distribution.Western Blot:Hck have expression in kuffer cells. In animal experiments: the IHC results showed low expression of Hck in normal liver tissues and high expression with inflammatory stimuli (positive correlation),After useing Qingzhi hugan prescription in NASH rats,we found that whatever the large?medium and small dose of the Chinese medicine groups,especially the large dose of "Qingzhi Hugan Prescription" group,the positive expression of Hck were decreased markedly (P<0.01); It showed a trend of decrease along with the rise of the expression of Hck in the rat liver tissuewith nonalcoholic fatty liver disease; 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-? 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-? was insignificant (P>0.05)When inflammation stimulation, the expression of Hck were negatively correlated.It suggests that Src family kinase Hck are involved in the occurrence and development of NASH, that can be used as a potential target of NAFLD inflammation. The expression of Hck were regulated by "Qingzhi Hugan Prescription", which reduced inflammation of the liver.It can be used as one of the mechanisms for thetreatment of NAFLD.