Inflammation and oxidative stress play predominant roles in the initiation and progression of ischaemia/reperfusion (I/R) injury, with nuclear factor kappa B (NF-κB) serving as a crucial mediator. Overexpression of the inhibitor of κB alpha (IκBα) gene is hypothesized to have protective effects against apoptosis and autophagy in cardiomyocytes subjected to hydrogen peroxide (H2O2) by inhibiting the NF-κB pathway. The IκBαS32A, S36A gene was transfected via adeno-associated virus serotype 9 (AAV9) delivery into neonatal rat ventricular cardiomyocytes (NRVMs) prior to H2O2 treatment. NRVMs were divided into control, H2O2, GFP + H2O2, IκBα+H2O2, and pyrrolidine dithiocarbamate (PDTC) + H2O2 groups. Nuclear translocation of the NF-κB 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 (ΔΨm) was detected by JC-1 staining. Western blotting was used to detect apoptosis- and autophagy-related proteins. IκBα transfection significantly increased cell viability and ΔΨm but decreased the supernatant LDH and cellular MDA levels in cardiomyocytes exposed to H2O2. Meanwhile, IκBα 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 IκBα to inhibit the NF-κB signalling pathway, as evidenced by decreases in p65 phosphorylation and nuclear translocation. Indeed, the effects of inactivation of NF-κB signalling with the specific inhibitor PDTC resembled the cardioprotective effects of IκBα during H2O2 stimulation. IκBα overexpression can ameliorate H2O2-induced apoptosis, autophagy, oxidative injury, and ΔΨm loss through inhibition of the NF-κB signalling pathway. These findings suggest that IκBα transfection can result in successful resistance to oxidative stress-induced damage by inhibiting NF-κB activation, which may provide a potential therapeutic target for the prevention of myocardial I/R injury.

中文翻译：

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心肌细胞中IκBα的过表达通过抑制NF-κB的活化来减轻过氧化氢诱导的细胞凋亡和自噬。

炎症和氧化应激在缺血/再灌注（I / R）损伤的发生和发展中起主要作用，其中核因子κB（NF-κB）是关键的介质。据推测，κBα（IκBα）基因抑制剂的过表达通过抑制NF-κB途径具有抗过氧化氢（H2O2）的心肌细胞凋亡和自噬的保护作用。在H2O2处理之前，通过腺相关病毒血清型9（AAV9）将IκBαS32A，S36A基因转染到新生大鼠心室心肌细胞（NRVM）中。将NRVM分为对照组，H2O2，GFP + H2O2，IκBα+ H2O2和吡咯烷二硫代氨基甲酸酯（PDTC）+ H2O2组。NF-κBp65亚基的核易位通过免疫荧光和蛋白质印迹进行了评估。细胞活力通过Cell Counting Kit-8测定法评估。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。通过膜联蛋白V-PE / 7-AAD染色确定细胞凋亡，并通过JC-1染色检测线粒体膜电位（Δ（m）。蛋白质印迹用于检测凋亡和自噬相关蛋白。IκBα转染显着提高了暴露于H2O2的心肌细胞的细胞活力和ΔΨm，但降低了上清液的LDH和细胞MDA水平。同时，IκBα的过表达通过上调Bcl-2 / Bax比值来减少H2O2诱导的凋亡，并通过下调Beclin-1的表达和LC3-II / LC3-I的比例来减少自噬。这些作用部分地解释了IκBα抑制NF-κB信号通路的能力，如p65磷酸化和核易位减少所证明。实际上，用特异性抑制剂PDTC灭活NF-κB信号的作用类似于在H2O2刺激过程中IκBα的心脏保护作用。IκBα的过量表达可通过抑制NF-κB信号通路来减轻H2O2诱导的细胞凋亡，自噬，氧化损伤和ΔΨm丢失。这些发现表明，IκBα转染可通过抑制NF-κB激活而成功地抵抗氧化应激诱导的损伤，这可能为预防心肌I / R损伤提供了潜在的治疗靶点。用特异性抑制剂PDTC灭活NF-κB信号的作用类似于在H2O2刺激过程中IκBα的心脏保护作用。IκBα的过量表达可通过抑制NF-κB信号通路来减轻H2O2诱导的细胞凋亡，自噬，氧化损伤和ΔΨm丢失。这些发现表明，IκBα转染可通过抑制NF-κB激活而成功地抵抗氧化应激诱导的损伤，这可能为预防心肌I / R损伤提供了潜在的治疗靶点。用特异性抑制剂PDTC灭活NF-κB信号的作用类似于在H2O2刺激过程中IκBα的心脏保护作用。IκBα的过表达可以通过抑制NF-κB信号通路来减轻H2O2诱导的细胞凋亡，自噬，氧化损伤和ΔΨm丢失。这些发现表明，IκBα转染可通过抑制NF-κB激活而成功地抵抗氧化应激诱导的损伤，这可能为预防心肌I / R损伤提供了潜在的治疗靶点。