Study Of Risk Factors Of Non-alcoholic Fatty Liver Disease And The Functions And Mechanisms Of Xingjiang Coreopsis Tinctoria In Protecting Liver Damage

Objective: With the improvement of living standards and the change of lifestyle, the prevalence of non-alcoholic fatty liver disease(NAFLD) is on rise year by year. 1) This study aims to survey the clinical characteristics and risk factors of NAFLD in Urumqi region and to assess the relationship between liver stiff measure and metabolic parameters in patients with NAFLD. 2) To examine the inhibitory effect of extract of coreopsis tinctoria total flavonoids(CTTFE) on NAFLD in mice and investigate the potential mechanisms. 3) To examine the protective effect of CTTFE, Huang Nuoma glycosides(CT01) and Marein(CT02) on liver LO2 cell damage induced by hydrogen peroxide(H2O2) and investigate the potential mechanisms. These all try to provide more theoretical basis for the prevention and treatment of NAFLD. Methods: Part one A total of 2574 cases were included in this study. The patients were divided into two groups(NAFLD and non-NAFLD) and compared their clinical characteristics, biochemical index and related diseases prevalence. Using multiple logistic regression analyze risk factors of NAFLD. Then, the patients with NAFLD were divided into two groups including elevated LSM and normal LSM. Compared the two groups in their clinical characteristics, biochemical index and related diseases prevalence. Using multiple logistic regression analyze risk factors of NAFLD with high LSM levels. Part two Male mice were used and randomly divided into the control, model, CTTFE100, and 200 mg/kg groups, and positive drug lipanthyl 40 mg/kg group. A mouse model with NAFLD was induced by orally feeding high-fat milk by gavage when they were simultaneously treated with drug for 8 weeks. Thereafter, the diet condition and weight were observed, the liver weight and liver weight index were calculated, the liver and blood were taken and the liver function, the serum lipid levels, the liver lipid levels and antioxidant ability were measured. The hepatic histological changes were examined under a light microscope. The protein expressions of hepatic adipose differentiation- related protein(ADRP) were examined by Western blot methods. Part three Using liver LO2 cell line, control group, H2O2 model group(concentration is 100μmol/L), different concentration groups(25、50、100μmol/L) of CTTFE and its main ingredient CT01 and CT02 were established according to experimental requirements. The cell viability was measured by MTT assay. And, the cell apoptosis rate was monitored by TUNEL staining. Then the active of caspase-3 was investigated by fluorescence in situ expression and the level of nitric oxide was measured by kit. Data were analyzed by SPSS 17.0 software. Result: Part one 930 cases were diagnosed NAFLD so that the overall prevalence rate was 36.13%. The prevalence rate of male and female is 42.38% and 30.13% respectively, the prevalence rate between men and women was statistically significant(P<0.001). Total prevalence rate of 60~69 group is highest of 51.6%, male prevalence rate of 40~49 group is highest of 52.3% and female prevalence rate of 70~group is highest of 57.5%. In NAFLD group, age, body metabolism index, systolic blood pressure, diastolic blood pressure, waist circumference, hip circumference, waist hip ratio, alanine aminotransferase, uric acid, fasting blood glucose, glycosylated hemoglobin, triglycerides, total cholesterol, low density lipoprotein cholesterol are higher than control group(P<0.001). Compared with control group, hyper-total cholesterol, hypertriglycerides, low high-density lipoprotein cholesterol hyperlipidemia, high low-density lipoprotein cholesterol hyperlipidemia, hypertension, impaired fasting blood glucose regulate or diabetes, obesity, hyperuricemia and abnormal liver function prevalence rate in NAFLD group is higher(P<0.001). The multiple logistic regression analysis show the following seven factors: sex, age, SBP, ALT, FBG, UA, TC and HDL-C enter into regress equation, all the regress coefficients are positive. It show that the seven factors are the independent risk factor of NAFLD. 930 cases with NAFLD were divided into high LSM and normal LSM group. In high LSM group, age, BMI, SBP, DBP, WC, HC, WHC, AST, Hb A1 C and TG is higher than normal LSM group(P<0.05 or P<0.001) and hyper-total cholesterol, hyper triglycerides, low high-density lipoprotein cholesterol hyperlipidemia, high low-density lipoprotein cholesterol hyperlipidemia, hypertension, obesity, hyperuricemia and abnormal liver function prevalence rate is higher than normal LSM group in which the prevalence rate of hypertension, blood glucose metabolic disorder and obesity between two groups was statistically significant(P<0.05). The multiple logistic regression analysis show the following four factors: age, SBP, TG and LSM enter into regress equation, all the regress coefficients are positive. It show that the four factors are the independent risk factor of NAFLD with high LSM. Part two According to the hepatic tissue pathology of mice, we succeeded in replicating the models of mice with NAFLD after orally feeding high-fat milk by gavage for 8 weeks. After treatment with CTTFE 100-200 mg/kg for 8 weeks, no adverse effect was observed in animal food intake and weight growth, but the degree of hepatic steatosis in mice was significantly decreased in the high and low dosage CTFFE-treated mice. Compared with model group, hepatic weight decreased in the high and low dosage CTFFE-treated mice but no significantly statistical difference was observed(P>0.05), liver index decreased(P<0.05), TC in hepatic tissue decreased(P<0.05), TG in hepatic tissue decreased but no significantly statistical difference was observed(P>0.05), no significant change in AST, ALT and ALP(P>0.05), serum TG decreased in the low dosage CTFFE-treated mice(P<0.05), serum TG decreased in the high dosage CTFFE-treated mice(P<0.01), serum TC decreased in the high and low dosage CTFFE-treated mice but no significantly statistical difference was observed(P>0.05), serum HDL-C decreased in the high and low dosage CTFFE-treated mice and significantly statistical difference was observed in the high dosage group(P > 0.05), serum LDL-C increased in the high and low dosage CTFFE-treated mice and no significantly statistical difference was observed(P>0.05), MDA in hepatic tissue decreased in in the high and low dosage CTFFE-treated mice(P<0.05), GSH-PX in hepatic tissue increased in the high and low dosage CTFFE-treated mice but no significantly statistical difference was observed(P>0.05), western-blot result show that the protein expression of ADRP decreased in the high and low dosage CTFFE-treated mice(P<0.05). Part three Compared with control group, cell viability significantly decreased(P<0.05), cell apoptosis rate, activity of caspase-3 and NO level significantly increased(P<0.05) in LO2 cell group induced H2O2. Compared with H2O2 model group, cell viability significantly increased as well as cell apoptosis rate, activity of caspase-3 and NO level significantly decreased in groups with different dosage(25, 50, 100μmol/L) CTTFE, CT01 and CT02. Significantly statistical difference was observed in moderate and high dosage group(P<0.05). Conclusion: 1) The higher prevalence of NAFLD in urumqi is related to these metabolic disorders including obesity, dyslipidemia, glucose metabolism disorders, hypertension, hyperuricemia etc. Therefore, the early prevention and treatment of NAFLD such as controlling glucolipid metabolic disorders, hypertension, hyperuricemia etc, show important for preventing the further development of NAFLD. 2) we succeeded in replicating the models of mice with NAFLD after orally feeding high-fat milk by gavage for 8 weeks. CTFFE can inhibit NAFLD formation induced by high-fat milk with regulating lipid metabolism disorder at some extent. The mechanisms might be related to regulate lipid metabolism disorder, increase hepatic antioxidation and decrease expression of ADRP protein. 3) All of three flavonoids extracted from Coreopsis tinctoria can increase cell viability, decrease the amount of apoptosis cell for LO2 cell damage induced by H2O2. CTTFE, CT01 and CT02 can protect hepatic cell and its mechanism is related to inhibit cell apoptosis,Caspase-3 activity and NO releasing.