替米沙坦调控miR-495-3p/心肌细胞增强因子2A通路减轻缺氧诱导的神经细胞PC12损伤

Protective effect of telmisartan on hypoxia-induced PC12 cell injury by regulating the miR-495-3p/myocyte enhancer factor 2A pathway

目的探讨替米沙坦(TM)调控miR-495-3p/心肌细胞增强因子2A(MEF2A)通路对缺氧诱导PC12细胞损伤的影响和机制。方法将PC12细胞进行正常培养(对照),缺氧(缺氧24 h),缺氧+0.1、1、10μg/mL TM、缺氧+miR-NC(转染miR-NC),缺氧+miR-495-3p(转染miR-495-3p模拟物),缺氧+TM+anti-miR-NC(转染anti-miR-NC,10μg/mL TM)和缺氧+TM+anti-miR-495-3p(转染anti-miR-495-3p,10μg/mL TM)处理。酶联免疫吸附实验(ELISA)检测乳酸脱氢酶(LDH)漏出量和超氧化物歧化酶(SOD)的活性,流式细胞术检测细胞凋亡,实时荧光定量PCR(RT-qPCR)和Western blot检测miR-495-3p、心肌细胞增强因子2A(MEF2A)表达水平。两组间比较采用t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果与对照比较,缺氧处理的PC12细胞LDH漏出率[(9.46±0.97)﹪比(45.69±4.31)﹪]、细胞凋亡率[(5.36±0.54)﹪比(28.36±2.41)﹪]、MEF2A mRNA(1.00±0.08比2.74±0.26)和MEF2A蛋白表达水平(0.39±0.037比0.87±0.06)均升高;SOD活性[(12.24±1.13)比(5.13±0.52)U/mL]和miR-495-3p表达水平(1.00±0.08比0.35±0.03)均降低,差异有统计学意义(P均<0.05)。与缺氧处理比较,缺氧+0.1、1、10μg/mL TM干预的PC12细胞LDH漏出率[(45.69±4.31)﹪比(32.14±3.84)﹪、(23.54±3.17)﹪、(16.87±1.46)﹪]、细胞凋亡率[(28.36±2.41)﹪比(22.46±2.31)﹪、(17.14±1.65)﹪、(10.23±1.12)﹪]、MEF2A mRNA(2.74±0.26比2.26±0.23、1.87±0.19、1.34±0.18)和MEF2A蛋白表达水平(0.87±0.06比0.75±0.05、0.63±0.04、0.46±0.03)均降低;SOD活性[(5.13±0.52)比(6.87±0.69)、(8.01±0.81)、(10.12±1.02)U/mL]、miR-495-3p表达水平(0.35±0.03比0.49±0.04、0.61±0.06、0.83±0.07)均升高,差异有统计学意义(P均<0.05)。与缺氧+miR-NC比较,缺氧+miR-495-3p的PC12细胞LDH漏出率[(46.87±4.28)﹪比(19.65±1.87)﹪]和细胞凋亡率[(28.38±2.44)﹪比(12.36±1.25)﹪]均降低,SOD活性[(5.15±0.51)比(9.67±0.97)U/mL]升高,差异有统计学意义(P均<0.05)。与缺氧+TM+anti-miR-NC比较,缺氧+TM+anti-miR-495-3p的PC12细胞LDH漏出率[(17.64±1.79)﹪比(32.69±3.57)﹪]和细胞凋亡率[(10.98±1.75)﹪比(22.64±2.13)﹪]均升高,SOD活性[(12.63±1.27)比(7.32±0.71)U/mL]降低,差异有统计学意义(P均<0.05)。结论TM通过上调miR-495-3p/MEF2A通路对缺氧诱导的PC12细胞损伤具有保护作用。

Objective To investigate the effect of telmisartan(TM)on hypoxia-induced PC12 cell injury by regulating miR-495-3p/myocyte enhancer factor 2A(MEF2A)pathway and its mechanism.Methods PC12 cells were divided into control group(cells in normal culture),hypoxia group(hypoxia 24 h),TM+hypoxia-low dose(L)group(0.1μg/mL TM,hypoxia for 24 h),TM+hypoxia-medium dose(M)group(1μg/mL TM,hypoxia for 24 h),TM+hypoxia-high dose(H)group(10μg/mL TM,hypoxia for 24 h),hypoxia+miR-NC group(transfected with miR-NC,hypoxia for 24 h),hypoxia+miR-495-3p group(transfected with miR-495-3p mimics,hypoxia for 24 h),hypoxia+TM+anti-miR-NC group(transfected with anti-miR-NC,hypoxia for 24 h),and hypoxia+TM+anti-miR-495-3p group(transfected with anti-miR-495-3p,hypoxia for 24 h).Enzyme-linked immunosorbent assay(ELISA)was used to detect the leakage of LDH and the activity of SOD in each group.Apoptosis was detected by flow cytometry.The expression levels of miR-495-3p and cardiomyocyte enhancer factor 2A(MEF2A)were detected by real-time quantitative PCR(qRT-PCR)and Western blotting.The comparison between the two groups was performed by t test,and the comparison between multiple groups was performed by one-way ANOVA and LSD-t test.Results Compared with the control group,the LDH leakage rate[(9.46±0.97)﹪vs(45.69±4.31)﹪],apoptosis rate[(5.36±0.54)﹪vs(28.36±2.41)﹪],MEF2A mRNA(1.00±0.08 vs 2.74±0.26)and protein(0.39±0.03 vs 0.87±0.06)expression of PC12 cells were increased in the hypoxia group,and SOD activity[(12.24±1.13)vs(5.13±0.52)U/mL],miR-495-3p expression(1.00±0.08 vs 0.35±0.03)were decreased,and the difference was statistically significant(P<0.05).Compared with the hypoxia group,the LDH leakage rate[(45.69±4.31)﹪vs(32.14±3.84)﹪,(23.54±3.17)﹪,(16.87±1.46)﹪],apoptosis rate[(28.36±2.41)﹪vs(22.46±2.31)﹪,(17.14±1.65)﹪,(10.23±1.12)﹪],MEF2A mRNA(2.74±0.26 vs 2.26±0.23,1.87±0.19,1.34±0.18)and protein(0.87±0.06 vs 0.75±0.05,0.63±0.04,0.46±0.03)expression of PC12 cells in the low,medium and high dose TM intervention group were decreased,SOD activity[(5.13±0.52)vs(6.87±0.69),(8.01±0.81),(10.12±1.02)U/mL],miR-495-3p expression(0.35±0.03 vs 0.49±0.04,0.61±0.06,0.83±0.07)were increased,the difference was statistically significant(P<0.05).Compared with the hypoxia+miR-NC group,the LDH leakage rate[(46.87±4.28)﹪vs(19.65±1.87)﹪]and the apoptosis rate[(28.38±2.44)﹪vs(12.36±1.25)﹪]of PC12 cells were reduced in the hypoxia+miR-495-3p group,and SOD activity[(5.15±0.51)vs(9.67±0.97)U/mL]was increased,and the difference was statistically significant(P<0.05).Compared with the hypoxia+TM+anti-miR-NC group,the LDH leakage rate[(17.64±1.79)﹪vs(32.69±3.57)﹪]and cell apoptosis rate[(10.98±1.75)﹪vs(22.64±2.13)﹪]were decreased,SOD activity[(12.63±1.27)vs(7.32±0.71)U/mL]was increased,the difference was statistically significant(P<0.05).Conclusion Telmisartan has a protective effect on hypoxia-induced PC12 cell injury by activating the miR-495-3p/MEF2A pathway.