Mechanism Of Atorvastatin Promoting Reverse Cholesterol Transport Through Apolipoprotein M

Background:In recent decades,with the improvement of living standards and the arrival of an aging society,the incidence of coronary atherosclerotic heart disease has been increasing.Hypertension,diabetes mellitus and hyperlipidemia are the main factors inducing coronary heart disease,among which hyperlipidemia and its related atherosclerosis is one of the research focuses.The lipid metabolic imbalance theory of macrophages has become the consensus of the academic community.Macrophages consume oxidized low-density lipoprotein in the case of excessive lipid load,causing cholesterol to accumulate in the macrophages and eventually transform the macrophages into foam cells.Cholesterol accumulation in the macrophages is one of the important links in the formation of atherosclerosis.Inhibiting the foam cells formation and the lipid deposition in the vascular endothelium are of great clinical significance in the prevention and treatment of atherosclerosis.At present,the treatment of atherosclerosis mainly focuses on inhibiting lipid inflow and promoting lipid efflux.Currently,the application of statin is the main method to inhibit lipid inflow,and it plays an important role in the treatment of atherosclerosis.Previous studies have shown that statins can reduce plasma low density lipoprotein(LDL)and total cholesterol,in addition to elevating high density lipoprotein(HDL),stabilizing atherosclerotic plaques and improving vascular endothelial function.To facilitate the flow of lipids from macrophages,namely cholesterol reverse transport(RCT),is the process of transporting the cholesterol from macrophages to liver,transforming it into bile salts and then excluding it from the body.More and more researchers focus on cholesterol reverse transport.Whether statins can promote cholesterol reverse transport is still a point of debate in the academic community.Apolipoprotein M(ApoM),a recently discovered new type of apolipoprotein,has been shown to be closely associated with cholesterol reverse transport mediated by high-density lipoprotein,which is associated with the formation of pre-βHDL,a key step in cholesterol reverse transport.Studies have reported that statins promote cholesterol efflux by increasing the HDL receptors such as ATP-binding transporter A1(ABCA1)and scavenger receptor type B(SRBI),and also regulate the expression of ApoM in HepG2 cells.Therefore,we speculated that the mechanism of statins’ anti-atherosclerosis may be related to ApoM,ATP binding transporter and scavenger receptor.Atorvastatin was used to stimulate HepG2 cells and THP 1 macrophage derived foam cell.The supernatant fluid from HepG2 cells after atorvastatin stimulation was put together with foam cells.Then we observed the cholesterol efflux of co-culture system and explored the influence atorvastatin on promoting reverse cholesterol transport and the role of ApoM in reverse cholesterol transport.We aim to provide theoretical basis for statins to promote cholesterol reverse transport.Part I Study on the effect of atorvastatin on cholesterol reverse transport in hyperlipidemia miceThe first section Establishment of hyperlipemia model in ApoE deficient miceObjective:C57BL/6 mice and Apo E-/-mice were used to establish hyperlipidemia model through high-fat diet.We aim to provide experimental basis for further study on the therapeutic effect of atorvastatin on hyperlipidemia.Methods:Male C57BL/6 mice and Apo E-/-mice with age of 8 weeks and weight of 20-25 grams were separately fed on standard synthesis diet or high fat diet(4%cholesterol,0.5%sodium cholate,10%lard,0.2%propyl thiouracil)for 8 weeks.They were fed in SPF grade standard polycarbonate cage with 12 hours of regular light day and night.The study was divided into four groups as follows,control group:C57BL/6 mice with standard synthesis diet;Apo E-/-mice with standard synthesis diet;Apo E-/-mice with high fat diet.Serum lipids were measured after 8 weeks.Results:There was no significant difference in the initial weight of the mice in each group.After 8 weeks of feeding,the weight of the Apo e-/-mice with standard synthesis diet group and the Apo e-/-mice with high fat diet group were significantly increased.Serum LDL,TC and TG in normal diet group of Apo E-/-mice were significantly higher than those in control group.Serum LDL,TC and TG in normal diet group of Apo E-/-mice were significantly higher than those in control group,while In the Apo E-/-mice,the lipid content in the high fat diet group was significantly higher than that in the standard synthesis diet group.Conclusions:The hyperlipidemia model of mice established by this method is similar to the pathophysiological process of hyperlipidemia caused by overeating high protein and high fat food,which provides a good experimental basis for further study of cholesterol reverse transport promoted by atorvastatin.The second section Effect of atorvastatin on cholesterol reverse transport in hyperlipidemic miceObjective:We aim to study the effects of atorvastatin on reverse cholesterol transport in hyperlipidemia by observing the expression of genes and proteins related to RCT in vivo.Methods:12-week-old male C57BL/6 mice and hyperlipidemia ApoE-/-mice established in the first section were divided into four groups:C57BL/6 mice with standard synthesis diet;Apo E-/-mice with standard synthesis diet;hyperlipidemia Apo E-/-mice with high fat diet and normal saline;hyperlipidemia Apo E-/-mice with high fat diet and atorvastatin.Each group had 5 mice.Atorvastatin treatment group was given gastric lavage at a dose of 10mg/kg for 2 weeks.The saline treatment group was given gastric lavage daily with a dose of 0.1 ml/kg for 2 weeks.C57BL/6 mice and ApoE-/-mice with standard synthesis diet group did not deal with it.Serum lipids were measured at the end of treatment,and the aortic plaque specimens were taken after execution.The pathological changes were observed by oil red O staining.The liver expressions of ABCA1,ABCG1 SC ARBI and apoM were detected by RT-PCR and Western-blot.Results:The serum LDL in atorvastatin group was significantly lower than that in normal saline group(P<0.01),while the levels of HDL and Preβ-HDL were significantly higher(P<0.01).There was no significant difference in serum levels of TC and TG between mice in atorvastatin group and saline group.The expression levels of ABCA1 and SCARB1 genes and proteins in liver of Atorvastatin group were significantly higher than those of normal saline group(P<0.01).ApoM gene expression level in liver of hyperlipidemia ApoE-/-mice was lower than that of C57BL/6 mice and Apo E-/-mice with standard synthesis diet group,but there was no significant difference.ApoM expression level in atorvastatin group was significantly higher than that in normal saline group(P<0.001).Conclusions:Atorvastatin can not only reduce serum LDL and increase HDL,but also promote reverse cholesterol transport in ApoE-/-mice.Part II Effect of atorvastatin on cholesterol efflux in THP-1 macrophage derived foam cellsObjective:To explore the mechanism of atorvastatin on promoting reverse cholesterol transport and the role of ApoM in reverse cholesterol transport.Methods:First,THP-1 macrophage derived foam cell model was established.Atorvastatin was used to intervene foam cells.Five groups were set up including control group(normal THP-1 macrophages),foam cell group and three atorvastatin groups with different concentration(0.1,1,10μmol/L).Cholesterol reverse transport related indicators were detected after disposing.Secondly,atorvastatin intervened HepG2 cells,which were divided into four groups:control group and atorvastatin groups with different concentration(0.1,1,10μmnol/L).Last,the supernatant fluid from HepG2 cells after atorvastatin stimulation was co-cultured with foam cells.Grouping is as follows:control group(pure foam cells),foam cells with HepG2 supernatant(DMSO),foam cells with atorvastatin,foam cells with HepG2 supernatant(atorvastatin).Then cholesterol reverse transport related tests were performed after intervention.Results:1 The THP-1 macrophage-derived foam cell that we established was irregular with round and transparent small vacuoles in the cytoplasm.Some cells were enlarged and the nucleus was pushed aside by lipid droplets,which accorded with the foam cell morphology.After oil red O staining,a large number of red fat droplets were observed in the cytoplasm under inverted fluorescence microscope.The foam cell model was successfully established.Compared with the control group,the expression levels of CD36 mRNA and CD68 mRNA increased significantly in foam cells(P<0.01).2 Compared with the foam cell group,the expression levels of SCARB1 mRNA and protein in the foam cells treated with atorvastatin were significantly increased(P<0.05),but the expression levels of ABCA1,ABCG1 and ApoM were not significantly different.There was no significant difference in intracellular TC,FC and CE contents between the two groups(P>0.05).There was no significant difference in the content of Pre-beta HDL between the control group,the foam cell group and the Atorvastatin group by ELISA(P>0.05).Compared with normal THP-1-derived macrophages,the cholesterol efflux rate of foam cell group increased significantly(P<0.05),however,compared with the foam cell group,three different concentrations of atorvastatin did not significantly increase the cholesterol efflux rate of foam cells.3 Compared with the control group,atorvastatin significantly increased the expression of apoM mRNA and protein in HepG2 cells(P<0.05).4 The expression levels of SCARB1 mRNA and protein in co-culture(atorvastatin)group and foam cell with atorvastatin group were significantly higher than those in control group and co-culture(DMSO)group(P<0.01).There was no significant difference in the expression of ABCA1 and SC ARBI mRNA and protein between foam cell with atorvastatin group and co-culture(atorvastatin)group(P>0.05).The expression of ABCG1 in the co-culture group was significantly higher than that in the foam cell with atorvastatin group(P<0.05).The contents of TC and CE in the co-culture(atorvastatin)group were significantly lower than those in the other three groups(P<0.05).The content of Pre-beta HDL in the co-culture(atorvastatin)group was significantly higher than that in the other three groups(P<0.05).The cholesterol efflux rate in the co-culture(atorvastatin)group was significantly higher than that in the other three groups(P<0.05).Oil red O staining showed that after the intervention of atorvastatin alone,a large number of lipid accumulation was still observed in the foam cells and no significant changes was found to be different with the untreated foam cells.However,after co-culture with HepG2,the cell volume was reduced,and the red stained lipid droplets were reduced.Conclusions:Atorvastatin could up-regulate the expression of SC ARBI in THP-1 macrophage-derived foam cells,but could not increase the cholesterol efflux from foam cells.Atorvastatin can increase the expression of ApoM gene and protein in HepG2 cells.After co-culturing the supernatant of HepG2 cells treated with atorvastatin and foam cells,the contents of ABCG1 and Pre-beta HDL increased,and the reverse cholesterol translocation was enhanced.Part Ⅲ Gene chip analysis of apoM overexpression in HepG2 cells and the role of TLR2 in atorvastatin-induced reverse cholesterol transport in foam cellsThe first section Gene chip analysis of apoM overexpression in HepG2 cellsObjective:To analyze the target genes of ApoM by microarray expression profiles after overexpression of ApoM in HepG2 cells and indentify the genes involved in reverse cholesterol transport.Methods:Affymetrix gene chip was used to analyze the gene expression profiles of ApoM lentivirus overexpression group and control group.The differentially expressed genes were identified and analyzed by bioinformatics analusis.Results:Through the analysis of differentially expressed genes and their targeting function,five possible target genes including Toll-like receptor 2(TLR2),diacylglycerol fatty acid enzyme alpha(DAGLA),ATP-binding transporter B4(ABCB4),Tribbles homolog 1(Trib1)and uridine diphosphate glucuronosyltransferase 1A1(UGT1A1)were seclected.TLR2 and DAGLA were significantly downregulated in the chip,and ABCB4,Tribl and UGT1A1 were significantly up-regulated in the chip.Further,RT-PCR confirmed that significant differences were observed after atorvastatin intervention in HepG2 cells.Conclusions:As the target genes of apoM,TLR2,DAGLA,ABCB4,Tribl and UGT1A1,may be involved in the reverse cholesterol transport regulation by ApoM.The second section Role of TLR2 in process of reverse cholesterol transport in foam cells that atorvastatin promotedObjective:To identify that atorvastatin is involved in the reverse cholesterol transport in foam cells through apoM and its target gene TLR2 signaling pathway at cell levels.Methods:TLR2 agonist Pam3CSK4 was used to up-regulate the expression of TLR2,and the effect of TLR2 agonist Pam3CSK4 on the reverse cholesterol transport mediated by atorvastatin in foam cells was observed.Grouping is as follows:control group(pure foam cells),foam cells with HepG2 supernatant(DMSO),foam cells with atorvastatin,foam cells with HepG2 supernatant(atorvastatin),foam cells with HepG2 supernatant(atorvastatin+Pam3CSK4).Results:After adding TLR2 agonist Pam3CSK4,there was no significant difference in the expression of ABCA1 and SC ARBI mRNA between the co-culture group and the non-agonist group(P>0.05),but the expression of ABCG1 was significantly down-regulated in Pam3CSK4 group(P<0.05),and the change of protein level was consistent with the gene level.The contents of TC and CE in the cells increased significantly in Pam3CSK4 group(P<0.05),and there was no significant difference in FC content in both group(P>0.05).There was no significant difference in the content of Pre-beta HDL between the agonist group and the non-agonist group by ELISA(P>0.05).The cholesterol efflux rate increased significantly in the co-culture(atorvastatin)group(P<0.05)and decreased significantly after the intervention of Pam 3CSK4(P<0.05).Oil red O staining showed that the volume of foam cells in the co-culture(atorvastatin)group was reduced and the lipid droplets in the cells were reduced,however,the red staining in the cells was increased and the foam cells were significantly increased after TLR2 intervention.Conclusions:TLR2 plays a key role in promoting reverse cholesterol transport in foam cells by atorvastatin.As a target gene of ApoM,TLR2 is down-regulated by ApoM,which promotes the expression of ABCG1 and enhances the reverse cholesterol transport.