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

Yeyu Qin,Jing Xie,Cheng Yu

doi:10.4314/tjpr.v20i9.15

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.

Highlights

At present, ischemic heart disease (IHD) has become one of the diseases with the highest morbidity and mortality rates in humans [1]
In order to explore the optimal concentration of H2O2 for cell damage, we treated H9c2 cells with different concentrations (0, 50, 100, 150 and 200 μM) of H2O2
To observe the inflammation of the three groups of cells, we examined the expression of tumor necrosis factor (TNF)-α and IL-6 and IL-1β (Figure 2A)

Summary

INTRODUCTION

Ischemic heart disease (IHD) has become one of the diseases with the highest morbidity and mortality rates in humans [1]. The cell lysate was added to each group of cell culture plates, washed with phosphate buffered saline (PBS) and allowed to stand on ice for 5 minutes. After 24 h of cell culture, the supernatant was discarded, washed with PBS 1 to 2 times, and added per well with 90 μL of the sample dissolved in the serumfree medium (Life Technology, Wuhan, China) for 24 h. Total RNA from 3 groups of cells was extracted using TRIzol reagent (MCE, Nanjing, China). The supernatants of the three groups of cells were taken and the contents of TNF-α and IL-6 and IL-1β were detected by TNF-α and IL-6 and IL-1β ELISA kits (Dojindo, Kumamoto, Japan), respectively. Three groups of cells in a 6-well plate were collected, centrifuged and washed with PBS a total of 3 times.

RESULTS

DISCUSSION

Conflict of interest