Study Of T Anshinone ⅡA On Improving Nonalcoholic Fatty Liver Disease Induced By High Fat Diet

In recent years,Nonalcoholic fatty liver disease(NAFLD)has shown a trend toward younger age and increased prevalence.Its etiology is complex and diverse.It is known to be mainly related to factors such as insulin resistance,inflammation and adipokines.At present,there is a lack of ideal therapeutic drugs and programs,and long-term medical treatment is prone to toxic and side effects.Screening biologically active substances from functional foods is extremely important for the prevention and treatment of NAFLD.Salvia miltiorrhiza,as one of the most commonly used traditional Chinese medicines in China,has the effects of activating blood circulation and removing blood stasis,protecting the liver and cholestasis,and has been included in the health food catalog.Tanshinone ⅡA(TAN-ⅡA)is one of the main ingredients in Salvia miltiorrhiza,which is often used to treat cardiovascular diseases,but there are few studies on liver diseases.Therefore,this study explored the improvement of TAN-ⅡA on NAFLD and the possible molecular mechanism through in vivo animal experiments and in vitro cell experiments.The main research contents are as follows:(1)Explored the therapeutic effect of TAN-IIA on mouse NAFLD in animal model.Six weeks male SPF C57BL/6 mice were randomly divided into two groups: Normal Control;High Fat Diet.After 14 weeks of feeding,the success of the model was determined by measuring the weight,fasting blood glucose,glucose tolerance and insulin resistance.After excluding abnormal individuals,the mice were regrouped to start treatment.They were divided into 4 groups: normal control group;high-fat diet group;TAN-IIA treatment group(50 mg/kg);metformin treatment group(200 mg/kg),metformin was used as a positive control,administered by gavage every day for 5 weeks.The oral glucose tolerance test(OGTT)and insulin tolerance tests(ITT)in mice were conducted during the last week,and the metabolic rate and other indicators were measured using a comprehensive monitoring system.After the experiment,mice’s plasma,liver,epididymal white fat and other tissues were collected,and the relevant indicators of plasma lipids in plasma were measured using an automatic biochemical analyzer and kit.Hematoxylin-Eosin staining was used to evaluate the pathological morphological changes of liver tissues,and western blotting was used to detect the changes of protein expression levels related to fat metabolism in liver tissues.(2)Explored the possible molecular mechanism and target of TAN-IIA in cell model.To determine the appropriate concentration of administration,the cytotoxicity of TAN-IIA on HepG2 cells was measured.The possible molecular mechanism was explored through pharmacological suppression and gene silence methods.Specifically,the changes in the expression levels of fat metabolism related proteins were detected by western blotting,the intracellular calcium ion concentration was determined by fluorescent probe method,intracellular Cyclic adenosine monophosphate(cAMP)level was measured by kit,and finally molecular target was verified through pull-down assay.The results showed that:(1)In animal experiments,TAN-IIA treatment reduced the weight of obese mice,improved fasting blood glucose,glucose tolerance and insulin resistance and other abnormal glucose metabolism;liver and epididymis white fat weight were significantly reduced;plasma lipid index was improved significantly;pathological section of liver tissue showed that liver cell lipid deposition and steatosis were significantly reduced;respiratory quotient,heat production and spontaneous exercise of mice increased significantly.These results indicated that TAN-IIA had a significant improvement effect on NAFLD.(2)In cell experiments,TAN-IIA could activate AMP-activated protein kinase-Acetyl CoA carboxylase(AMPK-ACC)signaling pathway in human hepatocellular carcinoma HepG2,and it had a dose and time dependence;it’s found that TAN-IIA could increase intracellular calcium ion levels and was calmodulin-dependent with the help of calcium ion fluorescent probes;through pharmacological inhibition and gene level silence of β2-adrenergic receptors(β2-AR),it was found that HepG2 cells were less sensitive to TAN-IIA.These results indicated that TANIIA might activate downstream signaling pathways through β2-AR,promoting liver cell fat oxidation,and thereby reducing liver fat accumulation.Our research found that TAN-IIA was a functional factor with liver-protective effects and revealed its possible molecular mechanisms,which provided new ideas for the future development of functional foods.