The Role Of Liraglutide On Cholesterol Efflux In HepG2 Cells Under High Glucose Condition And Effects Of Berberine On Lipid Metabolism In ApoE^-/-Mice With High-fat Diet

Background and objective1.Compared with patients with simple coronary heart disease,coronary heart disease combined with type 2 diabetes is more serious,and the risk of cardiovascular incident is higher.One of the reasons is that hyperglycemia will cause blood lipid metabolism disorder,and accelerate the process of atherosclerosis.Many studies have shown that effective control of blood glucose levels in patients with type 2 diabetes can reduce the risk of adverse cardiovascular events.Liraglutide,as a glucagon like peptide-1 analogue,is a new type of diabetes drug,which can stimulate the secretion of insulin by glucose dependence by binding and activating the GLP-1 receptor.Besides the hypoglycemic function,Liraglutide peptide also plays a regulatory role in blood lipid metabolism.The decrease of cholesterol efflux induced by abnormal reverse cholesterol transport pathway plays an important role in the metabolism of lipids.At present,there are few studies on the effect of Liraglutide on RCT.Therefore,this study aims to explore the effect of Liraglutide on the cholesterol efflux of HepG2 cells under high glucose conditions,and to elucidate the changes in the expression of related proteins and the signaling pathway.2.Cardiovascular disease is a frequently-occurring disease threatening human health and atherosclerosis(AS)is one of its common causes.Atherosclerosis is the pathological basis of coronary heart disease,and lipid metabolism disorder,excess lipid deposition on the blood vessel wall and gradually form plaque,making the vascular endothelium thickened and hardened,is an important reason for the formation of AS.ApoE-/-mice with high fat diet can induce hyperlipidemia,which is a common tool to study atherosclerosis.Berberine is the main active ingredient of Coptis chinensis,which has many functions such as lipid regulation,anti-inflammatory,hypoglycemic and anti-tumor.Therefore,the aim of this study was to investigate the effect of berberine on lipid metabolism in high-fat diet ApoE-/-mice.Methods1.HepG2 cell experiments(1)HepG2 cells were cultured in MEM medium containing 10%FBS,100kU/L penicillin and lOg/L streptomycin,incubating in 37℃,5%C02 saturated humidity incubator.After 80%-90%fusion,the cells 12h was starved by serum-free medium.Cells were treated with different concentrations of glucose(0,5,25,and 50 mmol/L)for 24 hours for following tests.The cell activity was detected by CCK8,and the intracellular cholesterol efflux was detected by BODIPY-cholesterol fluorescence labeling.Real-time PCR was used to detect the mRNA expression of ABCA1,ABCG1 and SR-B1 in cells after stimulated by different concentrations of glucose.Western blot was used to detect the protein expression of ABCA1,ABCG1 and SR-B1 after the stimulation.(2)The cells were cultured for 24 hours under different concentrations(0,10,100,1000,2000nmol/L)of Liraglutide.The cell activity was detected by CCK8,and select the most suitable concentration for subsequent experiments.The cells were cultured under the condition of high glucose(50mmol/L glucose)and Liraglutide(1000nmol/L),and the intracellular cholesterol efflux was detected by BODIPY-cholesterol fluorescence labeling.The expression of ABCA1,ABCG1 and SR-B1 in the cells was detected by Western blot.(3)Under the condition with/without ERK1/2 inhibitor U0126,cells were cultured under the condition of high glucose(50mmol/L glucose)and Liraglutide(1000nmol/L),which was used for the pathway study.2.Animal experimentsThere were 7 male C57BL/6J mice at 8 weeks old(1 group)and 35 male ApoE-/-mice aged 8 weeks.After 1 weeks of normal feeding,ApoE-/-mice were randomly divided into 5 groups.2 group were fed with normal diet,3 group were fed with high-fat diet,4 group were fed with of high-fat diet and intragastric administration with 50mg/kg/d berberine,5 group were fed with of high-fat diet and intragastric administration with 100mg/kg/d berberine,6 group were fed with of high-fat diet and intragastric administration with 10mg/kg/d atorvastatin,which as a positive control.After 16 weeks,the mice were killed and four blood lipids were detected by automatic biochemical analyzer.The liver lesions were detected by oil red O and HE staining.PCR and Western blot were used to detect the expression of ABCA1,ABCG1,SR-B1,PCSK9 and LDLR in the liver of mice.Results1.Liraglutide can promote cholesterol efflux in HepG2 cells under high glucose condition(1)Glucose can inhibit the cholesterol efflux in HepG2 cells in a concentration dependent manner and reduce the expression of cholesterol transporter ABCA1.(2)Liraglutide could promote the cholesterol efflux of HepG2 cells under high glucose,increase the expression of ABCA1,activate the ERK1/2 pathway and increase the expression of p-ERK1/2 protein in a time dependent manner.U0126(ERK1/2 inhibitor)could significantly reduce the increase of ABCA1 induced by the liraglutide.2.Berberine improves lipid metabolism in high fat diet ApoE-/-miceAfter 16 weeks,the blood lipids and pathological changes of ApoE-/-mice were significant.The results are as follows:(1)Compared with the wild type,the serum TC,TG and LDL-C of the ApoE-l-mice in the normal diet and the high fat diet group increased significantly,and the HDL-C level decreased significantly.Pathological staining showed that the liver and the aorta were diseased,such as lipid droplet like structure and vacuoles of the liver,large amounts of lipid droplets in the liver and the membrane and intima of aorta and the aorta lipid foam like macrophages and vacuolated fibroid cells.They were more significant in the high fat diet group.The Western blot results showed that the expression of ABCA1,ABCG1,SR-B1 and LDLR in the liver of ApoE-/-mice decreased significantly,and the level of PCSK9 increased significantly.(2)Compared with high fat diet ApoE-/-mice,low dose berberine(50mg/kg/d)and high dose berberine(100mg/kg/d)significantly decreased the level of TC,TG and LDL-C in serum,increased the concentration of HDL-C,improved the liver and aorta lesions of ApoE-/-mice,and significantly increased the ABCA1,ABCG1,SR-B1 and LDLR expression of the liver in the ApoE-/-mice liver,and reduced the PCSK9 level.Conclusions1.High glucose inhibits the cholesterol efflux of HepG2 cells.Liraglutide can promote cholesterol efflux in HepG2 cells under high glucose condition,the mechanism may be associated with the up-regulation of ABCA1,and the ERK pathway may be involved in it.2.Berberine can significantly improve the level of blood lipid and lipid deposition in the liver tissue of the high fat diet ApoE-/-mice,change the expression of lipid metabolism related protein in the liver of mice,and reduce the degree of atherosclerosis.