The Effect Of Heat Shock Protein 70 On The Expression Of ABCA1 And ABCG1 In Macrophages And The Progression Of Atherosclerosis

[Background and Objective]:The foam cells formation from the macrophages was one of the main reasons to accelerate atherosclerosis.Therefore,it is an important strategy to prevent atherosclerosis by inhibiting the foam cells formation.ATP-binding cassette transporter A1(ABCA1)and ATP binding cassette transporter G1(ABCG1)are critical plasma-membrane receptors for participation in cholesterol efflux and RCT,and they play a key role in preventing cholesterol from accumulation in macrophage.And ABCA1 and ABCG1 promotes cholesterol efflux to lipid-poor apolipoprotein A-Ⅰ(apo A-Ⅰ)and nascent high-density lipoprotein(n HDL)to maintain cholesterol homeostasis of macrophages and prevent foam cell formation.Therefore,it is significance to explore the mechanism of the expression of ABCA1 and ABCG1,and to promote the cholesterol efflux from macrophages foranti-atherosclerosis.Heat Shock Proteins(HSPs)are a kind of highly conservative stress proteins.HSPs is an essential molecular chaperone for the maintenance of cellular functions in several important cell processes,for example,protein folding and refolding and anti-apoptosis.Research evidence suggests that HSP70 plays an important role in anti-apoptosis and immune response to maintain and stabilize intracellular environment,and the HSP70 levels are higher in plasma of patients and atherosclerotic plaques,suggesting that HSP70 may be one of risk factors closely associated with atherosclerosis.It was reported that HSP70 activates the NF-κB pathway by TLRs and induces the expression of E-selectin,intercellular cell adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1)and interleukine-6(IL-6)to facilitate the inflammatory response,even accelerate atherosclerosis.However,it is largely unknown whether and how HSP70 plays a role in RCT.Therefore,our study is to explore the role of HSP70 on atherosclerosis and the potential mechanism from the RCT.Our results show that HSP70 can inhibit the expression of ABCA1 and ABCG1 by JNK/Elk-1 pathway to reduce the cholesterol efflux and promote the foam cells formation.In summary,these dates can help to clarify the role of HSP70 in RCT and As,and it may provide new targets and theoretical basis for the drugs to atherosclerosis.[Methods]:The HSP70 was transfected with adenovirus,and thetransfection efficiency was detected by Western Blot,and the following series of experiments were carried out.First,oil red O staining and HighPerformance Liquid Chromatography(HPLC)were used to detect the lipid content of macrophages after HSP70 over-expression.The effect of HSP70 on the expression of cholesterol transporter ABCA1 and ABCG1 was detected using q PCR and Western Blot.The liquid scintillation counting meter was used to detect the cholesterol efflux while HSP70 was over-expression.The effects of HSP70 on the combination of transcription factors Elk-1 with ABCA1 and ABCG1 promoter were detected by luciferase reporting gene and Chromatin Immunoprecipitation.When the cells were treated by Elk-1over-expression plasmid and Anisomycin,which is the activation agent of JNK,the levels of ABCA1,ABCG1,p Elk-1 and p JNK were tested by q PCR or Western to verify whether HSP70 influenced on the expression of ABCA1 and ABCG1 through the JNK/Elk-1 pathway.Finally,[3H]labeled cholesterol was used to assess the capacity of cholesterol efflux and reverse cholesterol transport(RCT);Oil Red O,hematoxylin-eosin,and Masson staining were performed to evaluate atherosclerotic plaque in apo E-/-mice fed the Western type diet.Moreover,immunostaining was employed to detect the expression of relative proteins in aortic sinus.[Results] : Western Blot showed that HSP70 was significant increased after transfection with adenovirus,indicating the macrophageswith HSP70 of over-expression was successful.The results of Oil Red O staining and high-performance liquid chromatography(HLPC)showed that HSP70 could increase the content of total cholesterol,cholesterol esters and free cholesterol in macrophages.Then analysis of the ABCA1 and ABCG1 and cholesterol efflux while HSP70 was over-expression in macrophages,the dates showed HSP70 can significantly inhibit ABCA1 and ABCG1 expression and reduce ABCA1-and ABCG1-mediated cholesterol efflux.After that,we explored the mechanisms of HSP70 inhibition ABCA1 and ABCG1 expression further.the transcription factor Elk-1 was found to promote ABCA1 and ABCG1 expression with the promoter of ABCA1 and ABCG1.The results of the luciferase report gene and Chromatin Immunoprecipitation showed that the activity of ABCA1 and ABCG1 promoter were decreased and and the capacity of combination of Elk-1 and ABCA1 and ABCG1 promoter were reduced when HSP70 was over-expression.Other dates suggested that HSP70 can significantly inhibit the activity of Elk-1,but over-express Elk-1 can increased the expression of ABCA1 and ABCG1 while HSP70 was also over-expression.It indicates that the Elk-1 is a key factor to take part in the HSP70 regulates ABCA1 and ABCG.Elk-1 belongs on MAPKs signaling pathways and was the downstream of JNK.Further to test that HSP70 influences on JNK,the results showed that the HSP70 can prevent the activation of JNK.And after the macrophages was treated with theAnisomycin,which is the activation agent of JNK,the levels of p Elk-1,ABCA1 and ABCA1 were increased while HSP70 was over-expression to prove that the HSP70 inhibits ABCA1 and ABCG1 through the JNK/Elk-1 pathway.HSP70 reduced RCT from macrophages to plasma,liver,and feces and increased lipid accumulation in arteries and promoted the formation of atherosclerotic lesion in apo E-/-mice.After that the levels of ABCA1 and ABCG1 expression were also reduced in the peritoneal macrophages and the aorta from apo E-/-mice in response to HSP70.[Conclusion]:1.HSP70 prevents the Elk-1 binding to the promoter of ABCA1 and ABCG1 by inhibiting the phosphorylation of JNK to down-regulate the expression of ABCA1 and ABCG1,reducing the cholesterol efflux and promoting the lipid accumulation in macrophages.2.HSP70 inhibits the expression of ABCA1 and ABCG1,reducs the levels of HDL and the RCT to accelerate the progression of atherosclerosis in apo E-/-mice.