白桦脂酸调节AMPK/mTOR/ULK1信号通路对冠心病大鼠血管平滑肌细胞自噬和凋亡的影响

Impacts of betulinic acid on autophagy and apoptosis of vascular smooth muscle cells in rats with coronary heart disease by regulating AMPK/mTOR/ULK1 signaling pathway

目的探究白桦脂酸通过调节腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)/Unc-51样自噬激活激酶1(ULK1)信号通路对冠心病大鼠血管平滑肌细胞自噬和凋亡的影响。方法原代培养冠心病大鼠血管平滑肌细胞,将其随机分为正常组(Control组)、模型组(Model组)、低浓度(20μmol/L)白桦脂酸组(BA-L组)、高浓度(40μmol/L)白桦脂酸组(BA-H组)和高浓度(40μmol/L)白桦脂酸+AMPK抑制剂(10μmol/L)Compound C组(BA-H+CC组)。CCK-8法检测大鼠血管平滑肌细胞的细胞活力。流式细胞术检测细胞凋亡。mRFP-GFP-LC3双荧光体系示踪大鼠血管平滑肌细胞的自噬小体。实时荧光定量PCR(RT-qPCR)实验检测大鼠血管平滑肌细胞中AMPK、mTOR、ULK1 mRNA的表达。Western blot实验检测大鼠血管平滑肌细胞中AMPK、mTOR、ULK1、LC3、Bax、Bcl-2、Beclin-1蛋白表达水平。结果与Control组相比,Model组血管平滑肌细胞的细胞活力(48 h、72 h)、Bcl-2蛋白表达、mTOR mRNA和蛋白表达显著上升(P<0.05),细胞凋亡率、Bax、LC3Ⅱ/LC3Ⅰ、Beclin-1蛋白表达、AMPK及ULK1 mRNA和蛋白表达显著下降(P<0.05)。与Model组相比,BA-H组血管平滑肌细胞的细胞活力(48 h、72 h)、Bcl-2蛋白表达、mTOR mRNA和蛋白表达显著下降(P<0.05),细胞凋亡率、Bax、LC3Ⅱ/LC3Ⅰ、Beclin-1蛋白表达、AMPK及ULK1 mRNA和蛋白表达显著上升(P<0.05)。Compound C减弱了白桦脂酸对血管平滑肌细胞自噬和凋亡的促进作用(P<0.05)。结论白桦脂酸可能通过激活AMPK/mTOR/ULK1信号通路促进冠心病大鼠血管平滑肌细胞的自噬和凋亡。

Objective To investigate the impacts of betulinic acid on autophagy and apoptosis of vascular smooth muscle cells in rats with coronary heart disease by regulating adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)/Unc-51-like kinase 1(ULK1)signaling pathway.Methods The vascular smooth muscle cells of coronary heart disease rats were primarily cultured and randomly divided into normal group(Control group),Model group,low-concentration(20μmol/L)betulinic acid group(BA-L group),high-concentration(40μmol/L)betulinic acid group(BA-H group),and high-concentration(40μmol/L)betulinic acid+AMPK inhibitor(10μmol/L)Compound C group(BA-H+CC group).The cell viability of rats′vascular smooth muscle cells was detected by CCK-8 method.Apoptosis was detected by flow cytometry.MRFP-GFP-LC3 double fluorescence system was used to trace autophagosomes of rats′vascular smooth muscle cells.The expression of AMPK,mTOR and ULK1 mRNA in rats′vascular smooth muscle cells were detected by real-time fluorescent quantitative PCR(RT-qPCR).Western blot was used to detect the protein expression levels of AMPK,mTOR,ULK1,LC3,Bax,Bcl-2 and Beclin-1 in vascular smooth muscle cells.Results Compared with the Control group,the cell viability(48 h,72 h),the expression of Bcl-2 protein,and the mRNA and protein expression of mTOR in vascular smooth muscle cells in Model group were obviously higher(P<0.05),while the apoptosis rate,the protein expression of Bax,LC3Ⅱ/LC3Ⅰand Beclin-1,and the mRNA and protein expression of AMPK and ULK1 were obviously lower(P<0.05).Compared with Model group,the cell viability(48 h,72 h),the expression of Bcl-2 protein,and the mRNA and protein expression of mTOR in vascular smooth muscle cells in the BA-H group were obviously lower(P<0.05),while the apoptosis rate,the protein expression of Bax,LC3Ⅱ/LC3Ⅰand Beclin-1,and the mRNA and protein expression of AMPK and ULK1 were obviously higher(P<0.05).Compound C weakened the promotion of BA on autophagy and apoptosis of vascular smooth muscle cells(P<0.05).Conclusion Betulinic acid can promote autophagy and apoptosis of vascular smooth muscle cells in rats with coronary heart disease by activating the AMPK/mTOR/ULK1 signaling pathway.