Overexpression of IκBα in cardiomyocytes alleviates hydrogen peroxide-induced apoptosis and autophagy by inhibiting NF-κB activation
Overexpression of IκBα in cardiomyocytes alleviates hydrogen peroxide-induced apoptosis and autophagy by inhibiting NF-κB activation
复制标题
心肌细胞中 IκBα 的过度表达通过抑制 NF-κB 激活来减轻过氧化氢诱导的细胞凋亡和自噬
DOI：
10.1186/s12944-020-01327-2
复制
发表时间：
2020-06-24
影响因子：
4.5
通讯作者：
Ma, Yi-Tong
中科院分区：
文献类型：
--
作者：
Han, Min;Chen, Xiao-Cui;Ma, Yi-Tong
背景炎症和氧化应激在缺血/再灌注 (I/R) 损伤的发生和进展中起主要作用，其中核因子 kappa B (NF-kappa B) 是关键介质。据推测，kappa B α (I kappa B α) 基因抑制剂的过度表达通过抑制 NF-kappa B 途径，对受到过氧化氢 (H2O2) 作用的心肌细胞的凋亡和自噬具有保护作用。方法在H(2)O(2)治疗之前通过腺相关病毒血清型9(AAV9)递送将IκBα(S32A，S36A)基因转染到新生大鼠心室心肌细胞(NRVM)中。 NRVM 分为对照组、H2O2、GFP + H2O2、I kappa B α+H2O2 和吡咯烷二硫代氨基甲酸酯 (PDTC) + H(2)O(2) 组。通过免疫荧光和蛋白质印迹评估 NF-κ B p65 亚基的核转位。通过 Cell Counting Kit-8 测定评估细胞活力。测量上清液乳酸脱氢酶 (LDH) 和细胞内丙二醛 (MDA) 以鉴定 H2O2 刺激的细胞毒性。通过Annexin V-PE/7-AAD染色测定细胞凋亡，通过JC-1染色检测线粒体膜电位（Delta psi m）。蛋白质印迹用于检测凋亡和自噬相关蛋白。结果 I kappa B α 转染显着增加了暴露于 H2O2 的心肌细胞中的细胞活力和 Delta psi m，但降低了上清液 LDH 和细胞 MDA 水平。同时，I kappa B α过表达通过上调 Bcl-2/Bax 比率来减少 H2O2 诱导的细胞凋亡，并通过下调 Beclin-1 的表达和 LC3-II/LC3-I 比率来减少自噬。这些效应部分解释了 I kappa B α 抑制 NF-kappa B 信号通路的能力，p65 磷酸化和核易位的减少证明了这一点。事实上，用特异性抑制剂 PDTC 灭活 NF-kappa B 信号传导的效果类似于 H(2)O(2) 刺激期间 I kappa B α 的心脏保护作用。结论 I kappa B α过表达可以通过抑制 NF-kappa B 信号通路来改善 H2O2 诱导的细胞凋亡、自噬、氧化损伤和 Delta psi m 损失。这些发现表明IκBα转染可以通过抑制NF-κB激活成功抵抗氧化应激诱导的损伤，这可能为预防心肌I/R损伤提供潜在的治疗靶点。
Background Inflammation and oxidative stress play predominant roles in the initiation and progression of ischaemia/reperfusion (I/R) injury, with nuclear factor kappa B (NF-kappa B) serving as a crucial mediator. Overexpression of the inhibitor of kappa B alpha (I kappa B alpha) gene is hypothesized to have protective effects against apoptosis and autophagy in cardiomyocytes subjected to hydrogen peroxide (H2O2) by inhibiting the NF-kappa B pathway. Methods The I kappa B alpha(S32A, S36A)gene was transfected via adeno-associated virus serotype 9 (AAV9) delivery into neonatal rat ventricular cardiomyocytes (NRVMs) prior to H(2)O(2)treatment. NRVMs were divided into control, H2O2, GFP + H2O2, I kappa B alpha+H2O2, and pyrrolidine dithiocarbamate (PDTC) + H(2)O(2)groups. Nuclear translocation of the NF-kappa B p65 subunit was evaluated by immunofluorescence and Western blotting. Cell viability was assessed by Cell Counting Kit-8 assay. Supernatant lactate dehydrogenase (LDH) and intracellular malondialdehyde (MDA) were measured to identify H2O2-stimulated cytotoxicity. Apoptosis was determined by Annexin V-PE/7-AAD staining, and the mitochondrial membrane potential (Delta psi m) was detected by JC-1 staining. Western blotting was used to detect apoptosis- and autophagy-related proteins. Results I kappa B alpha transfection significantly increased cell viability and Delta psi m but decreased the supernatant LDH and cellular MDA levels in cardiomyocytes exposed to H2O2. Meanwhile, I kappa B alpha overexpression decreased H2O2-induced apoptosis by upregulating the Bcl-2/Bax ratio and reduced autophagy by downregulating the expression of Beclin-1 and the LC3-II/LC3-I ratio. These effects partly accounted for the ability of I kappa B alpha to inhibit the NF-kappa B signalling pathway, as evidenced by decreases in p65 phosphorylation and nuclear translocation. Indeed, the effects of inactivation of NF-kappa B signalling with the specific inhibitor PDTC resembled the cardioprotective effects of I kappa B alpha during H(2)O(2)stimulation. Conclusion I kappa B alpha overexpression can ameliorate H2O2-induced apoptosis, autophagy, oxidative injury, and Delta psi m loss through inhibition of the NF-kappa B signalling pathway. These findings suggest that I kappa B alpha transfection can result in successful resistance to oxidative stress-induced damage by inhibiting NF-kappa B activation, which may provide a potential therapeutic target for the prevention of myocardial I/R injury.