Department of Cardiovascular Medicine, Ningbo First Hospital, No. 59, Liuting Street, Haishu District, Ningbo, 315000, Zhejiang, People's Republic of China.
Department of Cardiovascular Medicine, The Second Affiliated Hospital of University of South China, Hengyang, 315000, Hunan, People's Republic of China.
Cardiovasc Eng Technol. 2022 Oct;13(5):651-661. doi: 10.1007/s13239-021-00589-w. Epub 2021 Dec 2.
Oxidative stress and the inflammatory response contribute to the progression of cardiovascular disease. The present study aimed to investigate whether the mitochondrial-derived peptide MOTS-c could alleviate HO-induced oxidative stress and inflammatory status in H9c2 cells through activation of nuclear factor erythroid 2-related Factor 2 (Nrf2)/antioxidative response element (ARE) and inhibition of the NF-κB pathway.
Rat H9c2 cardiomyocytes were obtained, and 10, 20 or 50 μM MOTS-c was pretreated for 24 h before treatment with HO Then, the cell was treated with 100 μM HO for 1 h to induce oxidative stress. An inhibition model of sh-Nrf2 was constructed via a lentivirus expression system, and an activation model of NF-κB was achieved using phorbol 12-myristate-13-acetate (PMA). Cell viability was determined using a Cell Counting kit-8 assay. Relative measurement of relative protein and mRNA expression used western blotting and qRT-PCR, respectively. Intracellular reactive oxygen species (ROS) levels were detected using dichlorodihydrofluorescein diacetate, and malondialdehyde (MDA) and superoxide dismutase (SOD) levels were determined via commercial kits. The protein expression and distribution in the cells were visualized by immunofluorescence analysis. Enzyme-linked immunosorbent assay was used to detect the levels of inflammatory cytokines, including TNF-α, IL-6 and IL-1β.
We found that HO treatment significantly decreased cell viability and the level of SOD, increased the levels of ROS and MDA, and upregulated the expression of inflammatory cytokines, including TNF-α, IL-6 and IL-1β, in H9c2 cells. The expression levels of Nrf2, HO-1 and NQO-1 were significantly downregulated in the HO, while the phosphorylation of NF-κBp65 was promoted by HO. However, pretreatment with MOTS-c significantly reversed HO-induced damage in H9c2 cells. Moreover, both inhibition of the Nrf2/ARE pathway and activation of the NF-κB pathway significantly decreased the effects of MOTS-c, suggesting that MOTS-c might play a role in alleviating oxidative damage via the Nrf2/ARE and NF-κB pathways.
Our investigation indicated that MOTS-c could protect against HO-induced inflammation and oxidative stress in H9c2 cells by inhibiting NF-κB and activating the Nrf2/ARE pathways.
氧化应激和炎症反应促进心血管疾病的进展。本研究旨在探讨线粒体衍生肽 MOTS-c 是否可以通过激活核因子红细胞 2 相关因子 2（Nrf2）/抗氧化反应元件（ARE）和抑制 NF-κB 途径来减轻 H9c2 细胞中 HO 诱导的氧化应激和炎症状态。
获得大鼠 H9c2 心肌细胞，用 10、20 或 50μM MOTS-c 预处理 24 小时，然后用 100μM HO 处理 1 小时诱导氧化应激。通过慢病毒表达系统构建 sh-Nrf2 抑制模型，并用佛波醇 12-肉豆蔻酸 13-乙酸（PMA）激活 NF-κB 模型。使用细胞计数试剂盒-8 测定细胞活力。分别使用 Western blot 和 qRT-PCR 测定相对蛋白和 mRNA 表达的相对测量值。使用二氯二氢荧光素二乙酸检测细胞内活性氧（ROS）水平，并用商业试剂盒测定丙二醛（MDA）和超氧化物歧化酶（SOD）水平。通过免疫荧光分析观察细胞内蛋白质的表达和分布。酶联免疫吸附试验检测 TNF-α、IL-6 和 IL-1β 等炎症细胞因子的水平。
我们发现，HO 处理显著降低了 H9c2 细胞的活力和 SOD 水平，增加了 ROS 和 MDA 的水平，并上调了 TNF-α、IL-6 和 IL-1β 等炎症细胞因子的表达。HO 还显著下调了 H9c2 细胞中 Nrf2、HO-1 和 NQO-1 的表达水平，同时促进了 NF-κBp65 的磷酸化。然而，MOTS-c 预处理可显著逆转 H9c2 细胞中的 HO 诱导损伤。此外，抑制 Nrf2/ARE 途径和激活 NF-κB 途径均显著降低了 MOTS-c 的作用，表明 MOTS-c 可能通过 Nrf2/ARE 和 NF-κB 途径发挥作用，减轻氧化损伤。
本研究表明，MOTS-c 可通过抑制 NF-κB 和激活 Nrf2/ARE 途径来减轻 H9c2 细胞中 HO 诱导的炎症和氧化应激。
