基于miR-33a调控ABCA1表达探讨血管软化丸抗动脉粥样硬化的作用机制
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摘要
目的:通过观察血管软化丸是否通过调控miR-33a,进而影响下游信号ABCA1的表达,促进巨噬细胞内胆固醇流出,研究血管软化丸抗动脉粥样硬化的作用机制。方法:通过病理学检测观察血管软化丸对动脉粥样硬化的影响,通过体外实验建立THP-1巨噬细胞源性泡沫细胞,然后用血管软化丸含药血清处理,Real-Time PCR技术检测细胞miR-33a表达。细胞随机分为空白对照组、血管软化丸含药血清处理组、血管软化丸含药血清+miR-33a mimic处理组,RT-PCR和Western blotting法检测细胞ABCA1 mRNA和蛋白表达,油红O染色和高效液相色谱法检测细胞内脂质含量,[3H]法检测细胞内胆固醇流出。结果:体内实验显示,各中药组血管各层结构正常,排列整齐,局部有小灶性的钙化颗粒物沉积,病变轻,斑块小,泡沫细胞和脂质减少,弹力板基本完整,病变程度明显低于空白对照组。与空白对照组相比,血管软化丸高、中、低三个剂量组小鼠主动脉的miR-33a表达量降低、ABCA1基因和蛋白相对表达量均升高,差异有统计学意义(P<0.05)。体外实验显示,在不影响细胞活性状况下,血管软化丸含药血清呈浓度和时间依赖性下调miRNA33a表达;血管软化丸含药血清能明显上调ABCA1 mRNA和蛋白的表达,但能被转染miRNA33mimic抑制;血管软化丸含药血清可减少THP-1巨噬细胞源性泡沫细胞中的脂质蓄积,但能被细胞中转染miRNA33 mimic所弱化;EGCG减少细胞内胆固醇蓄积是与其促进细胞内胆固醇流出有关,细胞中转入过量miRNA33a可以抑制胆固醇流出。结论:血管软化丸可通过减少miRNA33a的生成,进而上调ABCA1表达,促进巨噬细胞中胆固醇流出,这可能是血管软化丸抗动脉粥样硬化作用的分子机制之一。
Objective:To study the anti-atherosclerosis mechanism of Vascular Softening Pill by observing whether it can regulate miR-33 a to influence the expression of ABCA1,and promote cholesterol efflux from macrophagocyte.Methods:Pathological examination was used to observe the effects of Vascular Softening Pill on atherosclerosis.THP-1 macrophage-derived foam cells were established by in vitro experiment.The cells were intervened with serum containing Vascular Softening Pill.RT-PCR was applied to detect the expression of miR-33 a.The cells were randomly divided into three groups,i.e.group A(blank control),group B(serum containing Vascular Softening Pill),and group C(serum containing Vascular Softening Pill +miR-33 a mimic treatment).Protein and mRNA expressions of ABCA1 were tested by RT-PCR and Western blotting method;intracellular lipid content was detected by Red O staining and HPLC;intracellular cholesterol efflux was examined by [3H] method.Results:In vivo experiment,blood vessels were normally and neatly arranged in all TCM groups,with focal deposition of calcified particles;the lesions were mild,the patches were small,the foam cells and the lipids were reduced,the elastic plates were basically complete,and the pathological changes were less than those of the control group.The expression of miR-33 a in the aorta was significantly decreased;however,protein and mRNA expressions of ABCA1 were significantly increased in the Vascular Softening Pill groups of high-dose,medium-dose,and low-dose,compared to those of the control group(P<0.05).In vitro experiments,serum containing Vascular Softening Pill down-regulated the expression of miRNA33 a in a dose-and-time-dependent manner;it significantly up-regulated protein and mRNA expressions of ABCA1,which could be inhibited by transfected miRNA33 mimic;it reduced lipid accumulation of THP-1 macrophage-derived foam cells,which could be weakened by transfected miRNA33 mimic;EGCG reducing the accumulation of intracellular cholesterol was related to its promotion of intracellular cholesterol efflux,which could be inhibited by excessive transfected miRNA33 a.Conclusion:Vascular Softening Pill can reduce the generation of miRNA33 a to up-regulate ABCA1 expression,to promote cholesterol efflux from macrophagocyte,which may be one of the molecular mechanisms of anti-atherosclerosis.
Objective:To study the anti-atherosclerosis mechanism of Vascular Softening Pill by observing whether it can regulate miR-33 a to influence the expression of ABCA1,and promote cholesterol efflux from macrophagocyte.Methods:Pathological examination was used to observe the effects of Vascular Softening Pill on atherosclerosis.THP-1 macrophage-derived foam cells were established by in vitro experiment.The cells were intervened with serum containing Vascular Softening Pill.RT-PCR was applied to detect the expression of miR-33 a.The cells were randomly divided into three groups,i.e.group A(blank control),group B(serum containing Vascular Softening Pill),and group C(serum containing Vascular Softening Pill +miR-33 a mimic treatment).Protein and mRNA expressions of ABCA1 were tested by RT-PCR and Western blotting method;intracellular lipid content was detected by Red O staining and HPLC;intracellular cholesterol efflux was examined by [3H] method.Results:In vivo experiment,blood vessels were normally and neatly arranged in all TCM groups,with focal deposition of calcified particles;the lesions were mild,the patches were small,the foam cells and the lipids were reduced,the elastic plates were basically complete,and the pathological changes were less than those of the control group.The expression of miR-33 a in the aorta was significantly decreased;however,protein and mRNA expressions of ABCA1 were significantly increased in the Vascular Softening Pill groups of high-dose,medium-dose,and low-dose,compared to those of the control group(P<0.05).In vitro experiments,serum containing Vascular Softening Pill down-regulated the expression of miRNA33 a in a dose-and-time-dependent manner;it significantly up-regulated protein and mRNA expressions of ABCA1,which could be inhibited by transfected miRNA33 mimic;it reduced lipid accumulation of THP-1 macrophage-derived foam cells,which could be weakened by transfected miRNA33 mimic;EGCG reducing the accumulation of intracellular cholesterol was related to its promotion of intracellular cholesterol efflux,which could be inhibited by excessive transfected miRNA33 a.Conclusion:Vascular Softening Pill can reduce the generation of miRNA33 a to up-regulate ABCA1 expression,to promote cholesterol efflux from macrophagocyte,which may be one of the molecular mechanisms of anti-atherosclerosis.
引文
[1]RAYNER K J,SUAREZ Y,DAVALOS A,et al.MiR-33 contributes to the regulation of cholesterol homeostasis[J].Science,2010,328(5985):1570-1573.
[2]MAO M,LEI H,LIU Q,et al.Effects of miR-33a-5P on ABCA1/G1-mediated cholesterol efflux under inflammatory stress in THP-1macrophages[J].PLo S One,2014,9(10):e109722.
[3]杨红霞,高亚,蒋恒波,等.EGCG通过miR-33a上调ABCA1表达减少巨噬细胞脂质蓄积[J].中国药理学通报,2016,32(9):1279-1283.
[4]秦合伟,刘萍.冠心康抗动脉粥样硬化作用机制与SDF-1/CX-CR4生物轴失衡相关性的研究[J].中华中医药杂志,2014,29(5):1558-1562.
[5]张志鑫,李彦杰,秦合伟,等.基于P13K/Akt/m TOR信号通路调控巨噬细胞自噬探讨黄芪甲苷抗动脉粥样硬化的作用机制[J].中草药,2017,48(17):3575-3581.
[6]SACCO J,ADELI K.MicroRNAs:emerging roles in lipid and lipoprotein metabolism[J].Curr Opin Lipidol,2012,23(3):220-225.
[7]RAMíREZ C M,GOEDEKE L,FERNáNDEZ-HERNANDO C.“Micromanaging”metabolic syndrome[J].Cell Cycle,2011,10(19):3249-3252.
[8]NOVáJ,BIENERTOVáVA!üJ,KáA T,NOVáM.MicroRNAs involved in the lipid metabolism and their possible implications for atherosclerosis development and treatment[J].Mediators Inflamm,2014:275-867.
[9]DáVALOS A,GOEDEKE L,SMIBERT P,et al.miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling[J].Proc Natl Acad Sci U S A,2011,108(22):9232-9237.
[10]CHEN W J,ZHANG M,ZHAO GJ,et al.MicroRNA-33 in atherosclerosis etiology and pathophysiology[J].Atherosclerosis,2013,227(2):201-208.
[11]RAYNER KJ,SUáREZ Y,DáVALOS A,et al.MiR-33 contributes to the regulation of cholesterol homeostasis[J].Science,2010,328(5985):1570-1573.
[12]周岚,李杨,汪典,等.利用基因芯片分析活血中药、破血中药对Apo E基因敲除小鼠动脉粥样硬化模型的差异表达基因[J].疑难病杂志,2017,16(1):18-22.
[13]刘建东,秦合伟.血管软化丸治疗急性脑梗死合并颈动脉粥样硬化斑块60例[J].中国民族民间医药杂志,2017,26(18):88-91.
[14]秦合伟,李彦杰,任锟,等.基于TLR9介导巨噬细胞极化效应探讨血管软化丸抗动脉粥样硬化的作用机制[J].中医药信息,2018,35(4):46-51.
[2]MAO M,LEI H,LIU Q,et al.Effects of miR-33a-5P on ABCA1/G1-mediated cholesterol efflux under inflammatory stress in THP-1macrophages[J].PLo S One,2014,9(10):e109722.
[3]杨红霞,高亚,蒋恒波,等.EGCG通过miR-33a上调ABCA1表达减少巨噬细胞脂质蓄积[J].中国药理学通报,2016,32(9):1279-1283.
[4]秦合伟,刘萍.冠心康抗动脉粥样硬化作用机制与SDF-1/CX-CR4生物轴失衡相关性的研究[J].中华中医药杂志,2014,29(5):1558-1562.
[5]张志鑫,李彦杰,秦合伟,等.基于P13K/Akt/m TOR信号通路调控巨噬细胞自噬探讨黄芪甲苷抗动脉粥样硬化的作用机制[J].中草药,2017,48(17):3575-3581.
[6]SACCO J,ADELI K.MicroRNAs:emerging roles in lipid and lipoprotein metabolism[J].Curr Opin Lipidol,2012,23(3):220-225.
[7]RAMíREZ C M,GOEDEKE L,FERNáNDEZ-HERNANDO C.“Micromanaging”metabolic syndrome[J].Cell Cycle,2011,10(19):3249-3252.
[8]NOVáJ,BIENERTOVáVA!üJ,KáA T,NOVáM.MicroRNAs involved in the lipid metabolism and their possible implications for atherosclerosis development and treatment[J].Mediators Inflamm,2014:275-867.
[9]DáVALOS A,GOEDEKE L,SMIBERT P,et al.miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling[J].Proc Natl Acad Sci U S A,2011,108(22):9232-9237.
[10]CHEN W J,ZHANG M,ZHAO GJ,et al.MicroRNA-33 in atherosclerosis etiology and pathophysiology[J].Atherosclerosis,2013,227(2):201-208.
[11]RAYNER KJ,SUáREZ Y,DáVALOS A,et al.MiR-33 contributes to the regulation of cholesterol homeostasis[J].Science,2010,328(5985):1570-1573.
[12]周岚,李杨,汪典,等.利用基因芯片分析活血中药、破血中药对Apo E基因敲除小鼠动脉粥样硬化模型的差异表达基因[J].疑难病杂志,2017,16(1):18-22.
[13]刘建东,秦合伟.血管软化丸治疗急性脑梗死合并颈动脉粥样硬化斑块60例[J].中国民族民间医药杂志,2017,26(18):88-91.
[14]秦合伟,李彦杰,任锟,等.基于TLR9介导巨噬细胞极化效应探讨血管软化丸抗动脉粥样硬化的作用机制[J].中医药信息,2018,35(4):46-51.
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