Effects And Mechanism Of Radix Hedysari Polysaccharide On Rats With Nonalcoholic Fatty Liver Disease

Objective:The rat model with nonalcoholic fatty liver disease (NAFLD) induced by high-fat diet was established. The intervention effect of Radix Hedysari polysaccharide (RHP) on weight, liver index, hepatocyte steatosis, liver function, glucolipid metabolism of NAFLD model rats was observed. The possible pharmacological action mechanism of RHP treating NAFLD was discussed preliminarily.Methods:After adaptive breeding of44male SD (Sprague Dawley) rats for a week, the experimental animals was divided into normal control group (10rats) and high-fat diet (HFD) model group (34rats) randomly. Normal control group was fed with regular diet and HFD model group was fed with high-fat diet. In the end of8th week,2rats were took randomly from control group and HFD model group and liver pathology was examined to confirm the formation of NAFLD lesion. After the HFD model establishing, the32experimental animals in model group was divided randomly into model group, low level of RHP group, high level of RHP group, and rosiglitazone (positive control) group (8rats in each group). All these32rats were feeding with high-fat diet and the rats in normal control group were feeding with regular diet continually. The rats in normal control and model groups were given an oral gavage of physiological saline solution (10ml/kg/d). The low level of RHP group and high level of RHP group were given oral gavages of50mg/kg and150mg/kg RHP liquid, respectively. Rosiglitazone group was given oral gavages of3mg/kg/d rosiglitazone liquid. All of the rats were treated once daily for8weeks. In the end of16th week, the rats were executed and serum and liver tissue samples were collected. The weight of rat body and liver were weighed to calculate liver index. The degree of liver steatosis was assessed by observe the general morphology of rat liver and pathological examination. Serum alanine amino transferase (ALT), aspartate amino transaminase (AST), total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c) were examined by automatic biochemical analyzer. The level of liver TG was examined by kit, rat fasting plasma glucose (FPG) was examined by the enzyme method, and fasting insulin (FINS) were measured by radio-immunity assay. Expression level of liver inflammation factors, transcription factors of lipid metabolism, and downstream genes of lipid metabolism were determined by real time PCR and semi-quantitative RT-PCR. The phosphorylation level and content of AMPK and downstream molecules associated with glycolipid metabolism signaling pathway in liver tissue were assessed by immunoblotting. The difference in protein maps of rat liver total protein was compared by two-dimensional gel electrophoresis in normal, model, and RHP (150mg/kg/d). The difference proteins were determined by MALDI-TOF/TOF mass spectrum and verified by western blot.Results:1. Compared with model group, the weight, liver index, and liver TG content of RHP group rats was decreased obviously. The degree of hepatocyte steatosis and inflammation were improved by hepatic pathology examination. The levels of serum ALT and AST in rats of high level of RHP group were decreased obviously. The levels of serum ALT in rats of low level of RHP and rosiglitazone group were reduced and the levels of AST have no changed. The levels of serum TG, TC, and LDL-c in RHP groups were all reduced and the levels of HDL-c have no obvious effects.2. Compared with normal control group, the levels of fasting plasma glucose (FPG) and insulin (FINS) in model group were increased remarkably. Administration of RHP and rosiglitazone could reduce the levels of FPG and FINS, lower the insulin resistance index (HOMA-IR) in model group. However, the effects of RHP are not as good as rosiglitazone.3. Compared with normal control group, the expression levels of inflammation factors of liver TNFa and IL-1p were upregulated. Administration of RHP and rosiglitazone could downregulated expression of TNFa and IL-1β. The effects in high level of RHP group are better than that of low level of RHP group, but have no significant difference with rosiglitazone group.4. Compared with normal control group, expression levels of most lipolysis genes (FASN and SCD-1) were downregulated and lipogenesis genes (CPT1and ATGL) were upregulated. Administration of RHP could upregulated expression of FASN and SCD-1, downregulated expression of CPT1and ATGL.5. Compared with normal control group, the phosphorylation levels of AMPK and ACC were decreased. The level of transcription factor PPAR a was downregulated and SREBP-1c was upregulated. Administration of RHP could increase the phosphorylation levels of AMPK and ACC, upregulated expression of PPAR a and downregulated expression of SREBP-lc.6.11proteins expressed different in HFD and RHP interfered liver tissue were verified by proteomics analysis. The trends of PPIA, Reguculcin, and Prohibitin were in accordance with that of proteomics by western blot.Conclusions:1. RHP could reduce the weigh and liver index in NAFLD rats, alleviate hepatocyte steatosis effectively.2. RHP could correct disorder of lipid metabolism in NAFLD rats, protect liver and lower liver enzyme.3. RHP could ameliorate the status of insulin resistance in in NAFLD rats.4. RHP could lower the expression level of inflammatory factors in NAFLD rats.5. To reduce the accumulation of fat in the liver, RHP could inhibit expression of lipid biosynthesis and strengthen expression of lipidolysis by enhance the phosphorylation levels of AMPK, activate PPARα and ACC, and inhibit SREBP-lc.6.11difference expression proteins were identified after HFD and RHP interference. They were involved with functions of oxidative stress, protein modification, and energy metabolism. Some of them maybe become the therapeutic targets of RHP on NAFLD.