同型半胱氨酸通过氧化物酶增殖物激活受体α-肝脏X核受体α通路导致动脉粥样硬化的机制研究
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摘要
目的研究同型半胱氨酸(HCY)是否通过过氧化物酶增殖物激活受体α(PPARα)-肝脏X核受体α(LXRα)通路导致动脉粥样硬化(AS)。方法 RAW264. 7源性巨噬细胞经氧化型低密度脂蛋白(ox-LDL)诱导为泡沫细胞,采用油红"O"染色进行泡沫细胞的鉴定。用不同浓度梯度(空白对照,0. 5,1. 0,2. 0,5. 0 mmol/L) HCY干预泡沫细胞24 h,油红"O"染色观察细胞内脂滴数量。Western blot和实时定量PCR检测PPARα、LXRα及三磷酸腺苷结合盒转运体A1(ABCA1)、三磷酸腺苷结合盒转运体G1(ABCG1)、卵磷脂胆固醇酰基转移酶(LCAT)的蛋白及mRNA表达,液态闪烁计数法分析细胞内胆固醇流出率,高效液相色谱分析细胞内胆固醇酯含量。结果与空白对照组比较,HCY干预组PPARα、LXRα、ABCA1、ABCG1、LCAT的表达量均降低(P <0. 05),细胞内的胆固醇含量均增高(P <0. 05),细胞内胆固醇流出率降低(P <0. 05)。随HCY浓度增加,PPARα、LXRα、ABCA1、ABCG1、LCAT的表达量逐渐降低(P <0. 05),细胞内的胆固醇含量逐渐增高(P <0. 05),细胞内胆固醇流出率逐渐降低(P <0. 05)。结论 HCY通过抑制PPARα-LXRα途径降低ABCA1、ABCG1、LCAT的表达,降低胆固醇逆转运效率,致脂质聚集增加,进而导致动脉粥样硬化(AS)。
Objective To investigate whether homocysteine causes atherosclerosis through the PPARα-LXRα pathway. Methods RAW264. 7-derived macrophages were induced to foam cells by oxidized low density lipoprotein( ox-LDL),and oil red "O"staining was used to identify foam cells. After the foam cells were treated with different concentrations of HCY( blank control,0. 5,1,2,5 mmol/L) for 24 h,the oil red"O"staining was used to observe the number of intracellular lipid droplets. Western blot and real-time quantitative PCR were used to detect the protein and gene expression of PPARα,LXRα and ATP-binding cassette transporter A1( ABCA1),ATP-binding cassette transporter G1( ABCG1),lecithin cholesterol acyltransferase( LCAT). The liquid scintillation counting was used to analyze the cholesterol efflux rate,and high performance liquid chromatography was used to analyze the intracellular cholesterol ester content. Results Compared with blank control group,the expression of PPARα,LXRα,ABCA1,ABCG1 and LCAT decreased in HCY intervention group( P < 0. 05),the intracellular cholesterol content increased( P < 0. 05),and the intracellular cholesterol efflux rate decreased( P < 0. 05). With the increase of HCY concentration,the expression of PPARa,LXRa,ABCA1,ABCG1 and LCAT was decreased( P < 0. 05),the intracellular cholesterol content was increased( P < 0. 05),and the intracellular cholesterol outflow rate was decreased( P < 0. 05). Conclusion HCY may reduce the expression of ABCA1,ABCG1 and LCAT by inhibiting PPARα-LXRαpathway,decrease the efficiency of cholesterol reverse transport and increase lipid aggregation,which leads to atherosclerosis( AS).
Objective To investigate whether homocysteine causes atherosclerosis through the PPARα-LXRα pathway. Methods RAW264. 7-derived macrophages were induced to foam cells by oxidized low density lipoprotein( ox-LDL),and oil red "O"staining was used to identify foam cells. After the foam cells were treated with different concentrations of HCY( blank control,0. 5,1,2,5 mmol/L) for 24 h,the oil red"O"staining was used to observe the number of intracellular lipid droplets. Western blot and real-time quantitative PCR were used to detect the protein and gene expression of PPARα,LXRα and ATP-binding cassette transporter A1( ABCA1),ATP-binding cassette transporter G1( ABCG1),lecithin cholesterol acyltransferase( LCAT). The liquid scintillation counting was used to analyze the cholesterol efflux rate,and high performance liquid chromatography was used to analyze the intracellular cholesterol ester content. Results Compared with blank control group,the expression of PPARα,LXRα,ABCA1,ABCG1 and LCAT decreased in HCY intervention group( P < 0. 05),the intracellular cholesterol content increased( P < 0. 05),and the intracellular cholesterol efflux rate decreased( P < 0. 05). With the increase of HCY concentration,the expression of PPARa,LXRa,ABCA1,ABCG1 and LCAT was decreased( P < 0. 05),the intracellular cholesterol content was increased( P < 0. 05),and the intracellular cholesterol outflow rate was decreased( P < 0. 05). Conclusion HCY may reduce the expression of ABCA1,ABCG1 and LCAT by inhibiting PPARα-LXRαpathway,decrease the efficiency of cholesterol reverse transport and increase lipid aggregation,which leads to atherosclerosis( AS).
引文
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[14]Kunnen S,Van Eck M.Lecithin:cholesterol acyltransferase:old friend or foe in atherosclerosis?[J].J Lipid Res,2012,53(9):1783-1799.
[15]Ge CX,Yu R,Xu MX,et al.Betaine prevented fructose-induced NAFLD by regulating LXRα/PPARαpathway and alleviating ER stress in rats[J].Eur J Pharmacol,2016,770:154-164.
[16]Rubic T,Lorenz RL.Downregulated CD36 and ox LDL uptake and stimulated ABCA1/G1 and cholesterol efflux as anti-atherosclerotic mechanisms of interleukin-10[J].Cardiovasc Res,2006,69(2):527-535.
[17]Zeng Y,Peng Y,Tang K,et al.Dihydromyricetin ameliorates foam cell formation via LXR-ABCA1/ABCG1-dependent cholesterol efflux in macrophages[J].Biomed Pharmacother,2018,101:543-552.
[18]吕湛,陈运贞.过氧化物酶体增殖体活化受体γ、肝脏X受体α、视黄酸X受体α活化对THP-1细胞ATP结合盒转运蛋白和CD36mRNA表达的影响[J].中国病理生理杂志,2004,20(8):1381-1384.
[2]Lin NN,Qin SC,Luo SH,et al.Homocysteine induces cytotoxicity and proliferation inhibition in neural stem cells via DNA methylation in vitro[J].FEBS J,2014,281(8):2088-2096.
[3]He XW,Yu D,Li WL,et al.Anti-atherosclerotic potential of baicalin mediated by promoting cholesterol efflux from macrophages via the PPARγ-LXRα-ABCA1/ABCG1 pathway[J].Biomed Pharmacother,2016,83:257-264.
[4]Norum KR.The function of lecithin:cholesterol acyltransferase(LCAT)[J].Scand J Clin Lab Invest,2017,77(4):235-236.
[5]Palermo FA,Cocci P,Mozzicafreddo M,et al.Tri-m-cresyl phosphate and PPAR/LXR interactions in seabream hepatocytes:Revealed by computational modeling(docking)and transcriptional regulation of signaling pathways[J].Toxicol Res(Camb),2015,5(2):471-481.
[6]胡大一.中国血脂异常与动脉粥样硬化性心血管疾病防控的新证据和新指南[J].中华心血管病杂志,2016,44(10):826-827.
[7]Kojoglanian SA,Jorgensen MB,Wolde-Tsadik G,et al.Restenosis in Intervened Coronaries with Hyperhomocysteinemia(RICH)[J].Am Heart J,2003,146(6):1077-1081.
[8]Zahra T,Hemen MS,Massoud S,et al.Cholesterol transporters ABCA1 and ABCG1 gene expression in peripheral blood mononuclear cells in patients with metabolic syndrome[J].Cholesterol,2015,2015:1-6.
[9]王义勇,麻芳,黄晖,等.同型半胱氨酸对泡沫细胞中LXRα-ABCA1信号通路的影响[J].临床心血管病杂志,2016,32(7):726-730.
[10]Glomset JA.The mechanism of the plasma cholesterol esterification reaction:plasma fatty acid transferase[J].Biochim Biophys Acta,1962,65(1):128-135.
[11]Lucero D.Svidirov D.Freeman L,et al.Increased cholesterol efflux capacity in metabolic syndrome:Relation with qualitative alterations in HDL and LCAT[J].Atherosclerosis,2015,242(1):236-242.
[12]Tosheska TK,Topuzovska S.High-density lipoprotein metabolism and reverse cholesterol transport:strategies for raising HDLcholesterol[J].Anatol J Cardiol,2017,18(2):149-154.
[13]Ly H,Francone OL,Fielding CJ,et al.Endotoxin and TNF lead to reduced plasma LCAT activity and decreased hepatic LCATmRNA levels in Syrian hamsters[J].J Lipid Res,1995,36(6):1254-1263.
[14]Kunnen S,Van Eck M.Lecithin:cholesterol acyltransferase:old friend or foe in atherosclerosis?[J].J Lipid Res,2012,53(9):1783-1799.
[15]Ge CX,Yu R,Xu MX,et al.Betaine prevented fructose-induced NAFLD by regulating LXRα/PPARαpathway and alleviating ER stress in rats[J].Eur J Pharmacol,2016,770:154-164.
[16]Rubic T,Lorenz RL.Downregulated CD36 and ox LDL uptake and stimulated ABCA1/G1 and cholesterol efflux as anti-atherosclerotic mechanisms of interleukin-10[J].Cardiovasc Res,2006,69(2):527-535.
[17]Zeng Y,Peng Y,Tang K,et al.Dihydromyricetin ameliorates foam cell formation via LXR-ABCA1/ABCG1-dependent cholesterol efflux in macrophages[J].Biomed Pharmacother,2018,101:543-552.
[18]吕湛,陈运贞.过氧化物酶体增殖体活化受体γ、肝脏X受体α、视黄酸X受体α活化对THP-1细胞ATP结合盒转运蛋白和CD36mRNA表达的影响[J].中国病理生理杂志,2004,20(8):1381-1384.