舒芬太尼通过miR-199a-5p减轻缺氧复氧H9C2细胞凋亡自噬的机制研究

Mechanism of Sufentanil Alleviating the Apoptotic and Autophagy of Hypoxia Reoxygenation H9c2 Cells Through miR-199a-5p

目的:观察舒芬太尼对缺氧复氧(H/R)心肌细胞H9C2凋亡、自噬的影响,并探究其作用机制。方法:建立H/R H9C2细胞损伤模型,用10μmol/L舒芬太尼处理H9C2细胞再行H/R。采用脂质体法将H/R+SUF+antagomiRNA组(转染antagomiRNA,用10μmol/L舒芬太尼处理)、H/R+SUF+antagomiR-199a-5p组(转染antagomiR-199a-5p,用10μmol/L舒芬太尼处理)转染H9C2细胞并行H/R处理。采用细胞计数试剂盒(CCK8)检测培养12、24和48 h的细胞活力。采用膜联蛋白V-异硫氰酸荧光素/碘化丙锭双染法、蛋白质印迹法检测细胞凋亡率、半胱氨酸蛋白酶-3(Caspase-3)、波形蛋白(survivin)、线粒体自噬相关蛋白(PINK1)、自噬标记物(LC3-Ⅱ/Ⅰ)和细胞色素C(Cyt C)的蛋白表达。结果:与H9C2组相比,H/R组细胞在48、72 h的活力均显著降低;与H/R+ddH_(2)O组相比,H/R+SUF组细胞在48、72 h的细胞活力显著升高,差异均有统计学意义(P<0.05),因此选用培养48 h的细胞用于后续研究。与H9C2组相比,H/R组H9C2细胞凋亡率显著升高,Caspase-3、PINK1、LC3-Ⅱ/Ⅰ和细胞质Cyt C蛋白表达水平显著升高,survivin、线粒体Cyt C蛋白表达水平显著降低,差异均有统计学意义(P<0.05);与H/R+ddH_(2)O组相比,H/R+SUF组H9C2细胞凋亡率显著降低,Caspase-3、PINK1、LC3-Ⅱ/Ⅰ和细胞质Cyt C蛋白表达水平显著降低,survivin、线粒体Cyt C蛋白表达水平显著升高,差异均有统计学意义(P<0.05)。H/R组miR-199a-5p的表达水平显著高于H9C2组,H/R+SUF组miR-199a-5p的表达水平则显著低于H/R+ddH_(2)O组,差异均有统计学意义(P<0.05);特异性抑制miR-199a-5p的表达明显增强了舒芬太尼对H/R H9C2细胞的凋亡自噬抑制作用。结论:舒芬太尼可抑制H/R心肌细胞的凋亡和自噬,其作用机制与调控miR-199a-5p的表达水平有关。

OBJECTIVE:To observe the effects of sufentanil on apoptosis and autophagy of hypoxia reoxygenation(H/R)cardiomyocytes H9C2,and to explore its mechanism.METHODS:H/R H9C2 cell injury model was established,the H9C2 cells were treated with 10μmol/L sufentanil and followed by H/R.The liposome method was used to transfect the H/R+SUF+antagomiRNA group(transfected antagomiRNA with 10μmol/L sufentanil)and the H/R+SUF+antagomiR-199a-5p group(transfected antagomiR-199a-5p with 10μmol/L sufentanil)with H9C2 cells and treated with H/R.Cell viability was detected by the cell counting kit(CCK8)at 12,24 and 48 h of culture.The cell apoptosis rate,protein expression of cysteine protease-3(Caspase-3),survivin,mitochondrial autophagy-associated protein(PINK1),autophagy marker(LC3-Ⅱ/Ⅰ)and cytochrome C(Cyt C)were measured by membrane linked protein V-isothiocyanate fluorescein/propidium iodide double staining method and Western blotting.RESULTS:Compared with the H9C2 group,the cell viability at 48 h and 72 h was significantly lower in the H/R group;compared with the the H/R+ddH_(2)O group,the cell viability at 48 h and 72 h was significantly higher in the H/R+SUF group,with statistically significant differences(P<0.05),the cells cultured for 48 h were selected for further studies.Compared with the H9C2 group,the apoptosis rate of H9C2 cells was significantly higher,the protein expression of Caspase-3,PINK1,LC3-Ⅱ/Ⅰand cytoplasmic Cyt C were significantly higher,the protein expression of survivin and mitochondrial Cyt C were significantly lower in the H/R group,with statistically significant differences(P<0.05).Compared with the H/R+ddH_(2)O group,the apoptosis rate of H9C2 cells was significantly lower,the protein expression of Caspase-3,PINK1,LC3-Ⅱ/Ⅰand cytoplasmic Cyt C were significantly lower,the protein expression of survivin and mitochondrial Cyt C were significantly higher in the H/R+SUF group,with statistically significant differences(P<0.05).The miR-199a-5p expression was significantly higher in the H/R group than that in the H9C2 group,and the miR-199a-5p expression was significantly lower in the H/R+SUF group than that in the H/R+ddH_(2)O group,with statistically significant differences(P<0.05).Specific inhibition of miR-199a-5p expression can significantly enhanced the inhibitory effect of sufentanil on the apoptosis and autophagy of H/R H9C2 cells.CONCLUSIONS:Sufentanil can inhibit the apoptosis and autophagy of H/R cardiomyocytes,its mechanism is related to the regulation of miR-199a-5p expression level.