The Mitochondrial-Derived Peptide MOTS-c Attenuates Oxidative Stress Injury and the Inflammatory Response of H9c2 Cells Through the Nrf2/ARE and NF-κB Pathways.

AIM

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.