螺内酯对缺氧/复氧处理的人心肌细胞HO-1/Nrf2/SIRT3信号通路及自噬的影响

The effects of spironolactone on HO-1/Nrf2/SIRT3 signaling pathway and autophagy in hypoxia/reoxygenation treated human cardiomyocytes

目的探讨螺内酯对缺氧/复氧(H/R)处理的人心肌细胞血红素加氧酶1(HO-1)/核因子E2相关因子2(Nrf2)/沉默信息调节因子3(SIRT3)信号通路及自噬的影响。方法体外培养人心肌细胞AC16,设置对照组(AC16细胞正常培养)、H/R组(AC16细胞经H/R处理)、低剂量螺内酯组(AC16细胞经H/R处理+0.5μmol/L螺内酯)、中剂量螺内酯组(AC16细胞经H/R处理+1μmol/L螺内酯)和高剂量螺内酯组(AC16细胞经H/R处理+1.5μmol/L螺内酯)。流式细胞仪检测各组AC16细胞凋亡情况;相应试剂盒检测各组AC16细胞中超氧化物歧化酶(SOD)、丙二醛(MDA)水平;实时荧光定量PCR(qRT-PCR)法检测各组AC16细胞中HO-1、Nrf2、SIRT3 mRNA表达情况;蛋白印迹(Western blot)法检测各组AC16细胞中HO-1、Nrf2、SIRT3、微管相关蛋白1轻链3(LC3)Ⅰ、LC3Ⅱ蛋白表达情况。结果与对照组比较,H/R组AC16细胞凋亡率、MDA含量、LC3Ⅰ蛋白表达水平及LC3Ⅰ/LC3Ⅱ比值明显升高(P<0.05),SOD活性,HO-1、Nrf2、SIRT3 mRNA及蛋白表达水平和LC3Ⅱ蛋白表达水平明显降低(P<0.05);随螺内酯剂量的升高,AC16细胞凋亡率、MDA含量、LC3Ⅰ蛋白表达水平及LC3Ⅰ/LC3Ⅱ比值明显降低(P<0.05),SOD活性,HO-1、Nrf2、SIRT3 mRNA及蛋白表达水平和LC3Ⅱ蛋白表达水平明显升高(P<0.05)。结论螺内酯可能通过激活HO-1/Nrf2/SIRT3信号通路及自噬,减少经H/R处理后的人心肌AC16细胞凋亡,发挥心肌保护作用。

Objective To investigate the effects of spironolactone on heme oxygenase-1( HO-1)/nuclear factor erythroid 2 related factor 2( Nrf2)/silent information regulator 3( SIRT3)signaling pathway and autophagy in hypoxia/reoxygenation( H/R) treated human cardiomyocytes.Methods AC16 cells were cultured in vitro and were set as control group( AC16 cells cultured normally),H/R group( AC16 cells treated with H/R),low-dose spironolactone group( AC16 cells treated with H/R + 0. 5 μmol/L spironolactone),medium dose spironolactone group( AC16 cells treated with H/R + 1 μmol/L spironolactone) and high-dose spironolactone group( AC16 cells treated with H/R + 1. 5 μmol/L spironolactone). The apoptosis of AC16 cells was detected by flow cytometry;the levels of superoxide dismutase( SOD) and malondialdehyde( MDA) in AC16 cells were detected by corresponding kits;real-time quantitative PCR( qRT-PCR) was used to detect the mRNA expressions of HO-1,Nrf2 and SIRT3 in AC16 cells;and Western blot( WB) was used to detect the protein expressions of HO-1,Nrf2,SIRT3,microtubule-associated protein 1 light chain 3( LC3) and LC3Ⅱin AC16 cells. Results Compared with those in the control group,the apoptosis rate,MDA content,LC3Ⅰ protein expression level and LC3Ⅰ/LC3Ⅱ value of AC16 cells in H/R group were significantly higher( P < 0. 05),and the SOD activity,HO-1,Nrf2,SIRT3 mRNA and protein expression levels,LC3Ⅱ protein expression level were significantly lower( P < 0. 05);with the addition of spironolactone and the increase of dosage,the apoptosis rate,MDA content,LC3 Ⅰ protein expression level and LC3Ⅰ/LC3Ⅱ value of AC16 cells were significantly lower( P < 0. 05),and the SOD activity,HO-1,Nrf2,SIRT3 mRNA and HO-1,Nrf2,SIRT3 protein expression levels,LC3Ⅱ protein expression level were significantly higher( P < 0. 05). Conclusions Spironolactone can reduce the apoptosis of human cardiac AC16 cells after H/R treatment by activating HO-1/Nrf2/SIRT3 signaling pathway and autophagy,thus plays a myocardial protective role.