Effects Of Liraglutide On Cholesterol Efflux In Db/Db Mice With High-Fat Diet And The Role Of Diallyl Disulfide On Lipid Metabolism In HepG2 Cells And Its Molecular Mechanism

Background and objective1.Type 2 diabetes mellitus(T2DM)is one of the most common chronic diseases.Coronary heart disease(CHD)refers to the heart disease caused by coronary artery atherosclerosis(AS)disease,which leads to stenosis or occlusion of the vascular lumen and myocardial ischemia,hypoxia or necrosis.Diabetic patients with coronary heart disease are more serious than those with coronary heart disease alone,and the risk of cardiovascular events is greater.The reason is that hyperglycemia could cause dyslipidemia and accelerate the development of atherosclerosis.A glucagon-like peptide-1 analogue(lilalutide)is a drug to treat diabetes in clinic,it can not only reduce blood glucose,but also regulate the metabolism of blood lipid.Reverse cholesterol transport(RCT)is an important mechanism of heart protection.The decrease of cholesterol outflow can lead to abnormal blood lipid metabolism.At present,there is little research on the RCT of liraglutide.This study aims to find the effect and mechanism of liraglutide on the reverse effect of cholesterol in high-fat diet db/db mice.2.The most common pathological basis of coronary heart disease is atherosclerosis(AS),which can be caused by abnormal blood lipid,diabetes,and other risk factors.The disorder of lipid metabolism,the accumulation of excess lipid on the wall of blood vessel,and the thickening and hardening of endothelium are the important cause of AS.Diallyl disulfide(DADS)is an effective component of volatile sulfide in garlic,which can reduce cholesterol and improve blood lipid metabolism.At present,about the effect of DADS on the lipid metabolism in HepG2 cells is few.Therefore,this study aims to explore the effect of DADS on the lipid metabolism in HepG2 cells under the condition of lipopoly saccharide,and to elucidate the changes of its related protein expression and its molecular mechanism.Methods1.Animal experimentsThere were 48 male db/db mice(aged 6 weeks)and 12 male C57BL/6J mice(6 weeks old).All of the mice were randomly divided into 5 groups after one week of adaptive feeding with normal feeding:wild type+normal diet(n=12),db/db+normal diet(n=12),db/db+high-fat diet(n=12),db/db+high-fat diet+liraglutide(n=12),db/db+high-fat diet+atorvastatin(n=12).Mice were administered either liraglutide(200 μg/kg)or equivoluminal saline subcutaneously,twice daily for 8 weeks and body weight was measured every week.After the 8-week treatment,the blood was collected for lipid evaluation and liver was obtained from the mice for hematoxylin-eosin(HE)staining,red O staining and Western blotting.Cholesterol efflux was assessed by measuring the radioactivity in the plasma and feces after intraperitoneal injection of 3H-labeled cholesterol.Then checked the ABCA1,ABCG1 and SR-B1 expression in db/db mice.2.HepG2 cell experiments(1)HepG2 cells were cultured in DMEM medium with 10%FBS,10 g/L streptomycin and 10 g/L penicillin at 37℃ and 5%CO2.The density of the cells was observed under microscope.After the fusion degree reached 80%-90%,the cells were starved for 12 hours with DMEM medium without serum.CCK8 was used to detected the cell activity.Real-time PCR was used to detect the mRNA expression of PCSK9、LDLR、SREBP2 and HNF1α,Western blot was used to detect the protein expression of lipid metabolism related genes PCSK9、LDLR、SREBP2 and HNF1α after the treatment.(2)Cells were cultured under different concentrations of DADS(0,20,40,80,160μg/ml)for 24 h.CCK8 was used to detect the cell activity,and the appropriate concentration was selected for subsequent experiments.Real-time PCR was used to detect the mRNA expression of PCSK9、LDLR、SREBP2 and HNF1α,Western blot was used to detect the protein expression of lipid metabolism related genes LDLR、PCSK9、SREBP2、HNF1α after the stimulation.The cells were cultured under the condition of LPS(1000 ng/ml)and different concentrations of DADS(0、20、40、80 μg/ml),and the Dil-LDL uptake assay was used to examine the LDL uptake.The expression of PCSK9、LDLR、SREBP2 and HNF1α in the cells was detected by Real-time PCR and Western blot.(3)Under the condition with/without PI3K/Akt inhibitor LY294002,cells were cultured under the condition of LPS(1000 ng/ml)and DADS(80 μg/ml),which was used for the pathway study.(4)In addition,we examined the effect of the combination of DADS and atorvastatin on PCSK9 expression.Results1.liraglutide promotes the reversal of cholesterol transport in high fat diet db/db mice After 8 weeks of treatment,db/db mice had significant changes in blood lipids and pathology.The results are as follows:(1)The serum TC,TG and LDL-C levels of db/db mice in the common diet and high-fat diet groups were significantly higher than those in wild-type mice;pathological staining results showed that liver tissue showed pathological changes,such as the structure of lipid droplets and vacuoles,there are a lot of lipid droplets in the inner membrane.They were more significant in the high fat diet group.The serum and fecal 3H cholesterol content of db/db mice with normal diet and db/db mice with high-fat diet decreased significantly.Western blot results indicated that the expression of ABCA1 in db/db mice was significantly reduced.(2)Compared with high fat diet db/db mice,Liraglutide significantly decreased blood glucose,serum total cholesterol(TC),triglyceride(TG)and low-density lipoprotein cholesterol(LDL-C).It also reduced liver lipid deposition in db/db mice fed with HFD.Moreover,the movement of 3H-cholesterol from macrophages to plasma and feces was significantly enhanced in db/db mice fed with HFD after liraglutide adminstration.Western blot showed that lilalutide increased the expression of ABC A1 protein in the liver.2.DADS decreases the expression of PCSK9 through PI3K/Akt-SREBP2 signaling pathway and promotes the uptake of LDL in HepG2 cells(1)Lipopolysaccharide increases the expression of PCSK9 and SREBP2 in HepG2 cells,thus reducing the expression of LDLR.(2)DADS can increase the expression of LDLR under LPS condition,thus promoting the uptake of LDL in HepG2 cells,and activate the PI3K/Akt-SREBP2 signal pathway,increasing the expression of p-AKT protein,and reduce the expression of PCSK9 in HepG2 cells.(3)DADS can reduce PCSK9 expression in HepG2 cells induced by atorvastatin.Conclusions1.Liraglutide could improve lipid metabolism and hepatic lipid accumulation in db/db mice fed with HFD by promoting reversal of cholesterol transport.2.DADS could significantly attenuated PCSK9 expression in a dose-dependent manner induced by LPS and increased the LDLR expression in HepG2 cells,which was associated with the activation of PI3K/Akt-SREBP2 signaling pathway.It provides a new perspective for understanding the lipid metabolism and anti-atherosclerosis regulated by DADS.