| In recent years,with the influence of Western eating habits on China,the consumption of high-fat and high-calorie foods has increased significantly.Epidemiological data show a sharp rise in the incidence of non-alcohol fatty liver disease(NAFLD).NAFLD has become the largest chronic liver disease in most western countries.The majority of NAFLD lesions are reversible in the early stage.Therefore,the study on the pathogenesis and therapeutic targets of NAFLD has become the focus of prevention and treatment of diabetes and metabolic diseases in China.NAFLD refers to a group of clinical pathological syndromes characterized by hepatic steatosis and fat accumulation,with a daily alcohol intake of less than 10 g and a fat deposition of more than 5% of the liver,NAFLD is closely related to insulin resistance and genetic susceptibility.Relatedly,the disease spectrum includes a series of liver damage such as nonalcoholic simple fatty liver,nonalcoholic steatohepatitis,progressive cirrhosis and hepatocellular carcinoma.NAFLD is closely related to metabolic syndrome,and one of the important pathological basis is insulin resistance(IR).Therefore,IR or lipid deposition caused by high-fat diet is a common model for studying its mechanism and seeking treatment.Previous studies in our group found that high-fat diet feeding can induce liver lipid deposition and IR in rats and mice,so high-lipid-induced IR model was selected as the experimental model in this study.Oxymatrine(OMT),also known as oxymatrine,is an alkaloid extracted from traditional Chinese medicines such as Sophora flavescens,Soybean root and Sophora alopecuroides.It has the functions of diuretic,anti-infection,hypolipidemic,anti-fibrosis,anti-tumor,protection of liver cells and immune regulation.It is mainly used for the treatment of liver damage such as chronic hepatitis and fatty liver.Studies have found that oxymatrine can improve insulin resistance induced by high fructose and reduce liver lipid deposition,but the specific mechanism of action is still unclear,which needs to be further studied.There are 22 members in the fibroblast growth factor(FGF)family,which has various physiological functions such as promoting cell proliferation,body development and wound repair.FGF21 is an atypical member of the FGF family,and it has been found that the main biological function of FGF21 is to regulate glucose and lipid metabolism.FGF21 is mainly expressed in the liver,and can also be expressed in skeletal muscle,adipose tissue and islet β cells.Both clinical studies and animal experiments have shown that serum FGF21 levels can be increased in the condition insulin resistance,which could regulate the synthesis of glycogen and the production of ketone,and then regulate insulin resistance.Related studies have found that oxymatrine can serve to regulate FGF21.However,whether Oxymatrine can improve liver lipid deposition and insulin resistance induced by high fat by regulating fibroblast growth factor 21 is not much studied at home and abroad.Research reports the level of FGF21 in patients with type 2 diabetes mellitus with non-alcoholic fatty liver is associated with the abnormal lipid metabolism and insulin resistance.In this study,we first observed the population with different lipid metabolism to understand the correlation between triglycerides and FGF21,and observed the correlation between FGF21 and non-alcoholic fatty liver at the same time.Next we aimed to induce insulin resistance in rats by high-fat induction,and to observe the changes of insulin signaling pathway,fatty acid metabolism and FGF21 expression in liver tissue after oxymatrine intervention.In the end,we explored whether oxymatrine can improve high lipid-induced lipid deposition and insulin resistance in HepG2 cells by FGF21 at the cellular level,and further to reveal the role of oxymatrine and FGF21 in the development of nonalcoholic fatty liver,providing a theoretical basis of looking for new therapeutic targets for nonalcoholic fatty liver.Part One A Correlation Study between FGF21 and non-alcoholic fatty liver disease in population with different lipid metabolismObjective: To compare the expression of serum FGF21 before and after fat meal in population with different lipid metabolism,and to explore the relationship between FGF21 and non-alcoholic fatty liver.Methods: The study was conducted in Endocrinology Department of Hebei Province General Hospital from June,2017.Eventually,a total of 93 volunteers were enrolled in the study.All subjects provided written informed consent and took an oral fat test.Venous blood samples of fasting and postprandial at 4-hour were taken.Non-alcoholic fatty liver diagnosis based on ultrasound according to guidelines for diagnosis and treatment of non-alcoholic fatty liver disease in 2010.The subjects were divided into 3 groups: normal fat tolerance(NFT),impaired fat tolerance(IFT),hypertriglyceridemia(HTG).Results:1.Comparison of BMI in NFT group、IFT group and HTG group: Compared with NFT group,the BMI in IFT group and HTG group were increased(P<0.05).2.Comparison of fasting blood glucose,TC,TG and LDL-C in NFT group、IFT group and HTG group:Compared with NFT group,the fasting blood glucose、TC、TG and LDL-C in IFT group and HTG group were increased(P<0.05).3.Comparison of the incidence of NAFLD in NFT group、IFT group and HTG group:Compared with NFT group,the incidence of NAFLD in IFT group and HTG group were increased(P<0.05).According to serum FGF21 quartiles,volunteers were divided into four groups.From Q1 to Q4 group,the incidence of non-alcoholic fatty liver was 4.0%,15.8%,30.4% and46.2%.Compared with Q4 group,the incidence of fatty liver in Q1、Q2、Q3 group was statistically significant(P<0.05).4.Comparison of the FGF21 levels of fasting and postprandial at 4-hour in NFT group、IFT group and HTG group:Compared with NFT group,the fasting FGF21 levels in IFT group and HTG group were increased(P<0.05).the postprandial FGF21 levels at 4-hour were all increased in 3 groups(P<0.05).Compared with the fasting FGF21 levels,the postprandial FGF21 levels at 4-hour were all increased in each group(P<0.05).Summary:1.The level of serum FGF21 is closely related to the level of serum triglyceride.As the level of serum triglyceride increases,the level of FGF21 increases.2.The level of serum FGF21 is closely related to the occurrence of non-alcoholic fatty liver.As the level of FGF21 increases,the incidence of non-alcoholic fatty liver increases.Part Two The effect of oxymatrine on liver lipid deposition in rats with insulin resistance induced by high-fat dietObjective: To explore the effects of oxymatrine on liver lipid deposition and insulin resistance in rat induced by high-fat diet.Methods: Forty-eight healthy cleaning grade male SD rats(180-220 g)of 8 weeks old were randomly divided into two groups after 1 weeks of adaptive feeding: normal control(ND)12 and high fat diet group(HFD)36.The ND group was fed with normal diet and the HFD group was fed with high fat diet.The mice in each group were fed with isocaloric feed every day,their daily intake was recorded,their free intake of water was free,and their fasting weight was measured at fixed time every week.After 8 weeks of feeding,6 rats were randomly selected from ND group and HFD group respectively,and were sacrificed after anaesthesia with 3% pentobarbital sodium.Liver tissue was retained,and transmission electron microscopy specimens were routinely prepared to confirm whether the liver lipid deposition model was established.After successful modeling,the remaining 6 rats in the ND group were unchanged.The remaining 30 rats in the HFD group were randomly divided into 5 groups: high fat group(HFD)and low dose oxymatrine group(OMT-L),medium dose group oxymatrine(OMT-M),high dose oxymatrine group(OMT-H)and metformin group(Met).The rats in each group were continued to be fed and the corresponding drugs were given for 10 weeks.OMT-L,OMT-M,and OMT-H groups were given oxymatrine 50 mg/kg,100 mg/kg,and 150 mg/kg,respectively,once a day.Met group was given metformin 500mg/kg intragastrically once a day.The ND group and the HFD group were intragastrically administered with 2 ml/kg of normal saline once a day.The drug was administered for 10 weeks,and the corresponding dose was calculated based on the body weight value every week.At the end of 8 weeks of high-fat feeding and 10 weeks of drug intervention respectively,the rats to be tested were fasted for 12 hours overnight,the intraperitoneal glucose tolerance test(IPGTT)was performed to detect the blood glucose level and the area under the curve(AUCglu)at each time point and the high insulin-positive glucose clamp test in awake state was utilized to calculate the glucose infusion rate(GIR).Serum samples were collected for fasting blood glucose(FBG),total cholesterol(TC),triglycerides(TG),alanine aminotransferase(ALT),aspartate aminotransferase(AST).Serum fasting insulin(FINS)and free fatty acid(FFA)levels were measured by ELISA.The liver tissue samples were collected for hepatic triglyceride content determination,and the ultrastructure of rat liver was observed by electron microscope.Results:1.After a week of acclimation feeding,no change were observed in body weight and average food intake per day between Con group and HFD group(P>0.05).2.Establishment of liver insulin resistance and lipid deposition model induced by high-fat diet1)Comparison of general biochemical parameters between the two groups: Compared with the ND group,the body weight,FBG,FINS,TC,ALT,TG and FFA of the HFD group were increased(P<0.05).There was no significant difference in serum AST between the two groups(P>0.05).2)Comparison of glucose tolerance results between the two groups: IPGTT results showed that blood glucose level in the HFD group were significantly higher than that in the ND group at 0 min,30 min,60 min,and 120 min(P<0.05).The AUC glu in the HFD group also increased significantly(P<0.05).3)Comparison of glucose infusion rates between the two groups: Compared with the ND group,the GIR of the HFD group was significantly lower(P<0.05).4)Ultrastructural comparison of liver cells in two groups of rats: Under electron microscope,there were no lipid droplets in the cytoplasm of hepatocytes in ND group,mitochondria and endoplasmic reticulum were abundant,and the endoplasmic reticulum was neatly arranged.Lipid droplets were visible in liver cells of HFD group,mitochondria were blurred or absent,and endoplasmic reticulum was fused,blurred or degranulated,all suggesting lipid deposition and organelle damage in liver cell3.The alteration of various indicators after 10 weeks of drug intervention1)Comparison of general indexes of rats in each group: The body weight,FBG,FINS,TC,TG,AST,ALT and FFA of HFD group were higher than those of ND group(P<0.05).The oxymatrine groups and the metformin group were lower than the HFD group(P<0.05).2)Comparison of glucose tolerance results in each group: IPGTT results showed that blood glucose levels in the HFD group were higher than those in the ND group at 0 min,30 min,60 min,and 120 min(P<0.05).The oxymatrine groups and metformin group were lower than the HFD group(P<0.05).The AUCglu in the HFD group was higher than that in the ND group(P<0.05).The oxymatrine groups and the metformin group were lower than the HFD group(P<0.05).3)Comparison of glucose infusion rate in each group: Compared with ND group,GIR was significantly decreased in HFD group(P<0.05).The oxymatrine groups and metformin group were higher than the HFD group(P<0.05).4)Comparison of ultrastructure of liver cells in each group: Under electron microscope,there were no lipid droplets in the cytoplasm of ND group,mitochondria and endoplasmic reticulum were abundant,and the endoplasmic reticulum was neatly arranged.Lipid droplets were observed in liver cells of HFD group,mitochondria are ambiguous or absent,and the endoplasmic reticulum has fusion,ambiguity or degranulation,all suggesting lipid deposition and organelle damage in liver cells.After the intervention of oxymatrine,the lipid droplets of HFD+OMT group were decreased compared with the HFD group,and the mitochondrial swelling and endoplasmic reticulum loss were alleviated.Summary:1.High-fat diet can induce liver lipid deposition and insulin resistance in rats.2.Oxymatrine can improve liver lipid deposition,glucose tolerance and insulin resistance caused by high-fat diet.Part Three The effect of oxymatrine on the expression of FGF21、insulin signaling pathway and fatty acid metabolism in the liver of rats with high fat-induced insulin resistanceObjective: To detect the expression of FGF21 after oxymatrine intervention.To observe the expression of insulin signaling pathway and fatty acid metabolism pathway at the same time.The aim is to explore whether FGF21 can serve as a downstream target involved in improved liver glucose metabolism abnormalities and liver mitochondrial dysfunction and fatty acid β oxidation by oxymatrine.Methods: Animal grouping and specimen collection were the same as part one.The expression of FGF21,PI3 K,IRS-1,AKT and GLUT2 were examined by QRT-PCR.Western blot analysis was used to detect the expression of FGF21,IRS-1,p-IRS-1,Akt,p-Akt,p-AMPK,AMPK,PGC1α,CD36 and CPT1 in liver tissue.Results:1.The mRNA expression of FGF21 protein in liver tissue of rats in each group: Compared with ND group,the levels of FGF21 protein in liver tissue of rats in HFD group were increased(P<0.05).The oxymatrine groups and the metformin group were significantly higher than the HFD group(P<0.05).2.The expression of FGF21 protein in liver tissue of rats in each group: Compared with ND group,the levels of FGF21 protein in liver tissue of rats in HFD group were increased(P<0.05).The oxymatrine groups and the metformin group were significantly higher than the HFD group(P<0.05).3.The levels of TG in liver tissue of rats in each group: Compared with ND group,the levels of TG in liver tissue of rats in HFD group were increased(P<0.05).The oxymatrine groups and the metformin group were significantly lower than the HFD group(P<0.05).4.The mRNA expression of PI3 K,IRS-1,Akt and GLUT2 were decreased(P<0.05).The levels of IRS-1,PI3 K,Akt and GLUT2 mRNA expression were significantly higher in the oxymatrine groups and the metformin group than in the HFD group(P<0.05).5.Phosphorylation levels of Akt and IRS-1 in liver of each group.Compared with the ND group,the level of p-Akt in the liver tissue of the HFD group was decreased(P<0.05).The levels of p-Akt in liver tissue were significantly higher in the oxymatrine groups and the metformin group than in the HFD group(P<0.05).The level of p-IRS-1 in the liver tissue of the HFD group was increased(P<0.05).The levels of p-IRS-1 in liver tissue were significantly lower in the oxymatrine groups and the metformin group than in the HFD group(P<0.05).6.Phosphorylation level of upstream factor AMPK and protein level of PGC1α、 CPT1 and CD36 in liver fatty acid metabolism of rats in each group.Compared with ND group,the level of p-AMPK in liver tissue of rats in HFD group decreased,while the expression of PGC1α and CPT1 decreased and the expression of CD36 increased(P<0.05).Compared with the HFD group,the levels of p-AMPK 、PGC1α and CPT1 in the oxymatrine groups and the metformin group increased(P<0.05).the expression of CD36 decreased(P<0.05).Summary:1.Oxymatrine can increase the expression of FGF21 in the liver.Oxymatrine can up-regulate PI3 K,IRS-1,AKT and GLUT2.Suggesting that oxymatrine can regulate the FGF21/IRS-1/Akt/GLUT2 pathway,thereby improving liver insulin resistance and glucose metabolism disorders.2.Oxymatrine can up-regulate p-AMPK and PGC1,suggesting that oxymatrine can regulate the p-AMPK/PGC1 pathway and enhance the oxidation process of fatty acids,thereby improving liver lipid deposition.Part Four To explore the mechanism of oxymatrine in improving hyper-lipidemia-induced disorder of glucose and lipid metabolism in hepatocytes by regulating the expression of fibroblast growth factor 21Objective: To establish a model of insulin resistance in HepG2 cells using high fat(containing 0.25 mmol/L palmitic acid medium).The transfection technique was used to target the expression of FGF21,and the changes of lipid deposition in HepG2 cells were observed,the relationship between oxymatrine and FGF21 in the process of insulin signaling pathway and lipid deposition was investigated,whether oxymatrine regulates glucose metabolism and fatty acid metabolism in hepatocytes by FGF21/Akt and FGF21/AMPK pathway to improve insulin resistance and lipid deposition were observed,and the possible mechanism by which oxymatrine regulates glycolipid metabolism was explored.Methods: Small interfering RNA targeting fibroblast growth factor 21 was transfected into HepG2 cells cultured with palmitic acid and oxymatrine,which were divided into the following six groups: normal control group(N),high fat group(Pa),high fat+ low dose oxymatrine group(Pa+OMT-L),high fat + high dose oxymatrine group(Pa+OMT-H),high fat + FGF21 small interfering RNA group(Pa+S),high fat+over expression FGF21 plasmid group(Pa+P).The level of TG in HepG2 cells was measured to observe the condition of cell lipid deposition.The protein expression of FGF21,the protein expression of p-IRS-1,p-Akt,and the expression of fatty acid oxidation pathways protein p-AMPK,CPT1 and CD36 were detected by western blot.Results:1.Establishment of insulin resistance in HepG2 cells: After 24 hours culture of HepG2 cell,the glucose concentration in the Pa group was higher than that in the ND group,suggesting the successful establishment of insulin resistance in vitro model.2.The effect of targeted intervention of FGF21 on the expression of FGF21 in HepG2 cells: The protein expression of FGF21 in Pa+S group was lower than that in Pa+OMT groups(P<0.05).There was no significant difference between Pa+S and Pa group(P>0.05).The expression of FGF21 protein in Pa+P group was significantly higher than that in Pa group after transfection of HGFG2 cells cultured with palmitic acid and oxymatrine(P<0.05).There was no significant difference between Pa+P group and Pa+OMT groups(P>0.05).3.The effect of targeted intervention of FGF21 on lipid deposition in HepG2 cells: It was found that the TG in the Pa group was higher than that in the ND group(P<0.05).The TG level in Pa+S group was higher than that in Pa+OMT groups(P<0.05).There was no significant difference between Pa+S group and Pa group(P>0.05).The TG level in Pa+P group was lower than that in Pa group(P<0.05).There was no significant difference between Pa+P group and Pa+OMT groups(P>0.05).4.Effect of targeted intervention of FGF21 on insulin signaling pathway in HepG2 cells: The expression of p-IRS-1 protein in the insulin signaling pathway of Pa group was increased,the protein expression of p-Akt was decreased,The expression of p-IRS-1 protein was decreased in Pa+OMT groups,the protein expression of p-Akt was increased(P<0.05).The protein expression of p-IRS-1 was increased and the expression of p-Akt protein was increased in Pa+S group(P<0.05).There was no significant difference between Pa+ S group and Pa group(P>0.05).The protein expression of p-IRS-1 was decreased and the protein expression of p-Akt was increased in Pa+ P group.There was no significant difference between Pa+P group and Pa+OMT groups(P>0.05).5.Effect of targeted intervention of FGF21 on fatty acid oxidation pathway in HepG2 cells: The expression of p-AMPK and CPT1 protein in the fatty acid oxidation pathway of Pa group was decreased,the protein expression of CD36 was increased,The expression of p-AMPK and CPT1 protein was increased in Pa+OMT groups,the protein expression of CD36 was decreased(P<0.05).The protein expression of p-AMPK and CPT1 was increased and the expression of p-Akt protein was decreased in Pa+S group,(P<0.05).There was no significant difference between Pa+S group and Pa group(P>0.05).The protein expression of p-AMPK and CPT1 was increased and the protein expression of CD36 was decreased in Pa+P group.There was no significant difference between Pa+P group and Pa+OMT groups(P>0.05).Summary:1.Oxymatrine can improve the lipid deposition of HepG2 cells induced by high fat by regulating the expression of insulin signaling pathway IRS-1 and Akt,fatty acid oxidation pathway AMPK and CPT1.2.Oxymatrine can increase the level of FGF21 and regulate the expression of FGF21/IRS-1/Akt and FGF21/AMPK pathway.FGF21 is a key factor for oxymatrine to improve insulin resistance and hepatic glycolipid metabolism.Conclusions:1.High triglycerides can increase FGF21 secretion,the level of FGF21 is closely related to the occurrence of non-alcoholic fatty liver.2.FGF21 increases glucose utilization,promotes fatty acid oxidation.Thereby improves insulin resistance and lipid deposition.3.Oxymatrine can regulate the level of FGF21 before and after high fat intervention,thereby regulate the expression of FGF21/IRS-1/Akt and FGF21/AMPK pathway to improve insulin resistance and hepatic glucose and fatty acid metabolism. |
PDF Full Text Request