Forsythiaside A inhibits hydrogen peroxide-induced inflammation, oxidative stress, and apoptosis of cardiomyocytes

Runqin Li,Dengfeng Ma,Wenxiu Li,Xiaoxuan Zheng,Zhihua Fu

doi:10.4314/tjpr.v20i10.3

Abstract

Purpose: To investigate the effect of forsythiaside A on heart failure.Methods: An in vitro cell model of myocardial injury was established by incubating H9c2 primary cardiomyocytes with hydrogen peroxide (H2O2). Apoptosis was measured by flow cytometry. Expression of inflammatory factors, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzymelinkedimmunosorbent assay (ELISA). Oxidative stress was evaluated by measuring malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels by ELISA.Results: Incubation with H2O2 increased H9c2 cell apoptosis (p < 0.001). Treatment with forsythiaside A reduced Bax expression and enhanced Bcl-2 expression which suppressed apoptosis of H2O2- induced H9c2 cells. Forsythiaside A also attenuated the H2O2-induced increase in TNF-α and IL-6expressions in H9c2 cells (p < 0.001). The H2O2-induced increase in MDA and decrease in SOD and GSH-Px in H9c2 cells were reversed by treatment with forsythiaside A. IκBα protein expression was downregulated, whereas p65 phosphorylation (p-p65), p-IκBα, nuclear factor erythropoietin-2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) were upregulated in H2O2-induced H9c2 cells. Forsythiaside A increased IκBα, Nrf2, and HO-1 expression and decreased p-p65 and p-IκBα expression in H2O2-induced H9c2 cells.Conclusion: Forsythiaside A exerts anti-inflammatory, anti-oxidant, and anti-apoptotic effects against H2O2-induced H9c2 cells through inactivation of NF-κB pathway and activation of Nrf2/HO-1 pathway. These results support the potential clinical application of forsythiaside A for the treatment of heart failure.

Highlights

Heart failure occurs when the heart is unable to pump blood adequately to maintain the body’s needs [1]
To establish an in vitro model of heart failure, H9c2 cells were incubated with H2O2 and analyzed by flow cytometry, which showed that apoptosis of H9c2 cells increased upon incubation with H2O2 (Figure 1 A)
Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses showed that Bax expression increased and Bcl-2 expression decreased upon incubation with H2O2 (Figure 1B)

Summary

INTRODUCTION

Heart failure occurs when the heart is unable to pump blood adequately to maintain the body’s needs [1]. Oxidative stress induces inflammation, which contributes to heart failure [4]. Forsythiaside A effectively inhibited hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in PC12 cells [7]. H2O2-induced myocardial injury in H9c2 primary cardiomyocytes was established as an in vitro cell model of heart failure. The effects of forsythiaside A on inflammation, oxidative stress, and apoptosis of H2O2-induced H9c2 cells were investigated. H9c2 cells were harvested and resuspended in the 1X binding buffer from the Annexin V/FITC kit (Beyotime, Shanghai, China). The membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000; Kangcheng Inc., Shanghai, China) and detection was performed using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA) and a bio-image analysis system (Bio-Rad, Baltimore, MD, USA). Data demonstrate the anti-oxidant effect of forsythiaside A in H2O2-induced H9c2 cells. A p value < 0.05 was considered statistically significant

RESULTS

DISCUSSION

CONCLUSION

Conflict of Interest