Ling-Gui-Zhu-Gan Decoction Protects H9c2 Cells against H2O2-Induced Oxidative Injury via Regulation of the Nrf2/Keap1/HO-1 Signaling Pathway.

Xiang Wang,Peng Zhou,Jinling Huang,Mengting Zhai,Tongjuan Tang,Liang Wang,Ruirui Ge,Mohammad Fattahi

doi:10.1155/2020/8860603

Abstract

Objectives Ling-Gui-Zhu-Gan decoction (LGZGD) is a potentially effective treatment for heart failure, and it showed significant anti-inflammatory potential in our previous studies. However, its ability to ameliorate heart failure through regulation of oxidative stress response is still unknown. This study was aimed to investigate the protective effect of LGZGD-containing serum on H2O2-induced oxidative injury in H9c2 cells and explore the underlying mechanism. Methods Eighteen rats were randomly divided into two groups: the blank control group and LGZGD group. The LGZGD group rats were administrated with 8.4 g/kg/d LGZGD for seven consecutive days through gavage, while the blank control group rats were given an equal volume of saline. The serum was extracted from all the rats. To investigate the efficacy and the underlying mechanism of LGZGD, we categorized the H9c2 cells into groups: the control group, model group, normal serum control (NSC) group, LGZGD group, LGZGD + all-trans-retinoic acid (ATRA) group, and ATRA group. Malonedialdehyde (MDA) and superoxide dismutase (SOD) were used as markers for oxidative stress. Dichlorodihydrofluorescin diacetate (DCFH-DA) staining was used to measure the levels of reactive oxygen species (ROS). The apoptosis rate was detected using flow cytometry. The expression levels of pro-caspase-3, cleaved-caspase-3, Bcl-2, Bax, Keap1, Nrf2, and HO-1 were measured using western blotting. The mRNA levels of Keap1, Nrf2, and HO-1 were measured using RT-qPCR. Results The LGZGD attenuated injury to H9c2 cells and reduced the apoptosis rate. It was also found to upregulate the SOD activity and suppress the formation of MDA and ROS. The expression levels of pro-caspase-3 and Bcl-2 were significantly increased, while those of cleaved-caspase-3 and Bax were decreased in the LGZGD group compared with the model group. As compared with the model group, the LGZGD group demonstrated decreased Keap1 protein expression and significantly increased Nrf2 nuclear expression and Nrf2-mediated transcriptional activity. ATRA was found to reverse the LGZGD-mediated antioxidative and antiapoptotic effect on injured H9c2 cells induced by H2O2. Conclusion Our results demonstrated that LGZGD attenuated the H2O2-induced injury to H9c2 cells by inhibiting oxidative stress and apoptosis via the Nrf2/Keap1/HO-1 pathway. These observations suggest that LGZGD might prevent and treat heart failure through regulation of the oxidative stress response.

Highlights

Heart failure (HF), the end stage of various cardiovascular diseases, is a clinical syndrome typically characterized by structural or/and functional cardiac abnormalities [1]
Effect of Ling-Gui-Zhu-Gan decoction (LGZGD) on the Expression Levels of reactive oxygen species (ROS), MDA, and SOD in H2O2-Treated H9c2 Cells. e expression levels of ROS and MDA significantly increased, while those of SOD sharply decreased in the model group compared with the control group (P < 0.01)
Treatment with LGZGD showed the opposite results. e apoptosis rate was significantly higher in the model group than in the control group (P < 0.01)

Summary

Introduction

Heart failure (HF), the end stage of various cardiovascular diseases, is a clinical syndrome typically characterized by structural or/and functional cardiac abnormalities [1]. E risk of survival of the patients who are given standardized treatment under the current guidelines is high. Experimental data and clinical evidence indicate that oxidative stress is an important risk factor that is involved in pathogenesis and development of HF [5, 6]. The balance between the production of reactive oxygen species (ROS) and endogenous antioxidants defense system is disturbed, which can lead to physiological conditions such as myocardial cellular damage, cardiomyocyte apoptosis, myocardial hypertrophy, systolic dysfunction, and arrhythmia [7]. The NF-E2-related factor 2(Nrf2-) Kelch-like ECH-associated protein 1 (Keap1) pathway is a well-characterized pathway involved in cellular defense against various types of oxidative damage [9, 10]. Studies have shown that modulating the Nrf2/Keap signaling pathway significantly attenuates HF [11]

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