TNFR2激活NF-κB信号通路调控线粒体融合蛋白OPA1在心力衰竭中的作用-东南大学学报医学版

TNFR2激活NF-κB信号通路调控线粒体融合蛋白OPA1在心力衰竭中的作用

单位：1. 南京医科大学附属淮安第一医院心内科, 江苏淮安 223000;
2. 南京医科大学附属淮安第一医院检验科, 江苏淮安 223000

关键词：心力衰竭肿瘤坏死因子受体-2 线粒体动力学核转录因子-κB 视神经萎缩蛋白1

分类号：R541.6

出版年·卷·期（页码）：2020·39·第一期（17-22）

摘要：

目的：探讨肿瘤坏死因子受体（TNFR）2通过调节视神经萎缩蛋白1（OPA1）促进线粒体融合在慢性心力衰竭中可能的保护机制。方法：构建SiRNA靶向沉默TNFR1（TNFR1-KD）的心肌细胞H9c2。肿瘤坏死因子-α（TNF-α）（0.5 ng·ml^-1）刺激TNFR1-KD心肌细胞H9c2和靶向沉默OPA1的TNFR1-KD心肌细胞H9c2，电镜下测定线粒体形态和数量，荧光素酶-荧光素法测定ATP酶活性。蛋白质印迹法测定PGC1α、OPA1、Mfn1、Mfn2、Drp1的蛋白表达水平；ELISA检测转移到细胞核内的p65蛋白、IL-1β和IL-6含量。结果：TNFR1-KD心肌细胞H9c2的TNFR1表达量在转染48 h最低。在TNFR1-KD心肌细胞H9c2，与安慰剂组比较，TNF-α刺激组的线粒体数量下降24%，平均线粒体面积增加28%，ATP酶活性增加35%，差异均具有统计学意义（均P<0.05）。TNF-α刺激组的OPA1蛋白表达水平明显升高，差异具有统计学意义（P<0.05）。靶向沉默OPA1的TNFR1-KD心肌细胞H9c2组（OPA1-siRNA组）较空白对照组（NC-siRNA组）中的线粒体数量增加40%，平均线粒体面积下降32%，ATP酶活性下降25%，差异均具有统计学意义（均P<0.05）。在TNFR1-KD心肌细胞H9c2中，PDTC可显著抑制核转录因子κB（NF-κB）通路活化，细胞核内的NF-κB p65蛋白含量降低，IL-1β、IL-6表达下降，线粒体OPA1蛋白含量下降。结论：TNFR2激活NF-κB信号通路调控OPA1促进线粒体融合。

Objective: To study how tumor necrosis factor receptor(TNFR)2 activation mediates the effects of optic atrophy1(OPA1) on mitochondria fusion. Methods: Rat myocardial cell line H9c2 treated with siRNA targeting TNFR1(TNFR1-KD) were used to study the effects of TNFR2 activation on mitochondrial function. H9c2 cells with TNFR1-KD and TNFR1-KD cells transfected with OPA1-siRNA were treated with tumor necrosis factor α (TNFα,0.5 ng·ml^-1). The electron microscopic examination was done to analyze the mitochondrial morphology number of mitochondria. The intracellular ATP activity was detected by firefly luciferase reaction. Western blot was used to examine the expression of PGC1α, OPA1, Mfn1, Mfn2 and Drp1 and ELISA was used to examine the expression of p65, IL-1β and IL-6. Results: The expression of TNFR1 in TNFR1-KD H9c2 cells was the lowest at 48 h after transfection. Compared with placebo-treated cells, TNFR1-KD H9c2 cells of TNF-α treatment group exhibited increases in mitochondrial fusion, a change that was associated with a 24% decrease in mitochondrial number, a 28% increase in mitochondrial area and a 35% increase in intracellular ATP levels (all P<0.05). The expression of OPA1 protein was significantly increased in TNF-α group. TNFR1-KD H9c2 cells transfected with OPA1-siRNA exhibited a 40% increase in mitochondrial number, a 32% decrease in mitochondrial area, and a 25% decrease in intracellular ATP levels compared with untransfected cells after TNFR2 stimulation (all P<0.05). The levels of p65 protein in the nucleus, IL-1β, IL-6 and OPA1 were significantly lower in TNFR1-KD H9c2 cells of PDTC treatment group than placebo-treated group. Conclusion: TNFR2 activation promotes mitochondrial fusion by upregulating OPA1 expression via NF-κB signaling pathway.

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