Tropical Journal of Pharmaceutical Research

Original Research Article | OPEN ACCESS

Leflunomide inhibits inflammation and apoptosis of H9c2 cells induced by hydrogen peroxide

Jing Xie , Yeyu Qin, Cheng Yu

Department of Pharmacy, Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), Haikou 570311, China;

For correspondence:- Jing Xie Email: Van_joric@163.com Tel:+8689868642548

Accepted: 9 August 2021 Published: 30 September 2021

Citation: Xie J, Qin Y, Yu C. Leflunomide inhibits inflammation and apoptosis of H9c2 cells induced by hydrogen peroxide. Trop J Pharm Res 2021; 20(9):1887-1893 doi: 10.4314/tjpr.v20i9.15

© 2021 The authors.
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Abstract

Purpose: To investigate the effects of leflunomide (Lef) on inflammatory response and apoptosis after myocardial infarction, and to explore its molecular mechanisms of action.

Methods: H₂O₂ and H9c2 cells were used to establish myocardial cell injury model in vitro. H9c2 cells were divided into 3 groups: control group, H₂O₂ group, H₂O₂ + Lef group. The CCK-8 assay was used to determine the optimal concentration of H₂O₂ and Lef, while the expressions of TNF-α, IL-6, IL-1β, Bcl-2, Bax, Bad, TLR4, IκB-α, P65 and p-P65 were evaluated by Western blot. PCI was utilized to detect the expression of TNF-α, IL-6, IL-1β, Bcl-2, Bax and Bad mRNA. The levels of TNF-α, IL-6 and IL-1β in supernatant were assessed by ELISA, while apoptosis of the three groups was evaluated by TUNEL staining and flow cytometry.

Results: Compared with H₂O₂ group, TNF-α, IL-6, IL-1β, Bax and Bad expressions in H₂O₂+Lef group were significantly reduced (p < 0.05), but Bcl-2 expression significantly increased. The levels of TNF-α and IL-6 and IL-1β in supernatant of H₂O₂ + Lef group were also decreased compared to those in the H₂O₂ group (p < 0.05). In addition, TUNEL-positive cells and apoptotic rates were significantly reduced after treatment with Lef. Moreover, Lef inhibited expression of TLR4 and p-P65, but activated expression of IκB-α, indicating that Lef inhibited TLR4/NF-κB pathway (p < 0.05).

Conclusion: The results show that Lef inhibits H₂O₂-induced H9c2 cell apoptosis and inflammatory responses by inhibiting TLR4/NF-κB pathway. These findings may provide new targets for the treatment of myocardial infarction.

Keywords: Myocardial infarction, Leflunomide, Apoptosis, Inflammation, TLR4/NF-κB pathway