Adiponectin Attenuates Lipopolysaccharide-induced Apoptosis by Regulating the Cx43/PI3K/AKT Pathway

Luqian Liu,Ketao Ma,Junqiang Si,Xinzhi Li,Ling Chen,Rui Yang,Meijuan Yan,Xuqing Qin,Li Li

doi:10.3389/fphar.2021.644225

Abstract

Cardiomyocyte apoptosis is a crucial factor leading to myocardial dysfunction. Adiponectin (APN) has a cardiomyocyte-protective impact. Studies have shown that the connexin43 (Cx43) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathways play an important role in the heart, but whether APN plays a protective role by regulating these pathways is unclear. Our study aimed to confirm whether APN protects against lipopolysaccharide (LPS)-induced cardiomyocyte apoptosis and to explore whether it plays an important role through regulating the Cx43 and PI3K/AKT signaling pathways. In addition, our research aimed to explore the relationship between the Cx43 and PI3K/AKT signaling pathways. In vitro experiments: Before H9c2 cells were treated with LPS for 24 h, they were pre-treated with APN for 2 h. The cytotoxic effect of APN on H9c2 cells was evaluated by a CCK-8 assay. The protein levels of Bax, Bcl2, cleaved caspase-3, cleaved caspase-9, Cx43, PI3K, p-PI3K, AKT and p-AKT were evaluated by Western blot analysis, and the apoptosis rate was evaluated by flow cytometry. APN attenuated the cytotoxicity induced by LPS. LPS upregulated Bax, cleaved caspase-3 and cleaved caspase-9 and downregulated Bcl2 in H9c2 cells; however, these effects were attenuated by APN. In addition, LPS upregulated Cx43 expression, and APN downregulated Cx43 expression and activated the PI3K/AKT signaling pathway. LPS induced apoptosis and inhibited PI3K/AKT signaling pathway in H9c2 cells, and these effects were attenuated by Gap26 (a Cx43 inhibitor). Moreover, the preservation of APN expression was reversed by LY294002 (a PI3K/AKT signaling pathway inhibitor). In vivo experiments: In C57BL/6J mice, a sepsis model was established by intraperitoneal injection of LPS, and APN was injected into enterocoelia. The protein levels of Bax, Bcl2, cleaved caspase-3, and Cx43 were evaluated by Western blot analysis, and immunohistochemistry was used to detect Cx43 expression and localization in myocardial tissue. LPS upregulated Bax and cleaved caspase-3 and downregulated Bcl2 in sepsis; however, these effects were attenuated by APN. In addition, the expression of Cx43 was upregulated in septic myocardial tissue, and APN downregulated Cx43 expression in septic myocardial tissue. In conclusion, both in vitro and in vivo, the data demonstrated that APN can protect against LPS-induced apoptosis during sepsis by modifying the Cx43 and PI3K/AKT signaling pathways.

Highlights

Numerous studies have shown that sepsis is a multiple organ dysfunction caused by the body’s immune response to microbial infections
The H9c2 cells were pre-treated with three concentrations of APN (0.5, 1 and 2 μg/ml) for 2 h and were treated with LPS to observe the effect of APN on cardiomyocyte toxicity induced by LPS
Cells exposed to LPS for 24 h exhibited disordered cell alignment and extensive cell shrinkage; the state of the cells in the co-treatment group was significantly improved. These results suggest that APN attenuates the cytotoxicity of H9c2 cells induced by LPS

Summary

Introduction

Numerous studies have shown that sepsis is a multiple organ dysfunction caused by the body’s immune response to microbial infections. Sepsis is often accompanied by organ damage and organ failure. Sepsis-induced myocardial dysfunction (SIMD) is a common complication in patients with sepsis. Compared with patients without cardiovascular dysfunction, sepsis patients with myocardial dysfunction have an increased mortality rate (by 3-fold), and these patients usually show myocarditis, abnormal contractility, increased interstitial collagen and mitochondrial damage (Blanco et al, 2008; Alvarez et al, 2016). Due to the complexity of myocardial injury in sepsis, its pathophysiological mechanism is not yet fully understood. Lipopolysaccharide (LPS), a major endotoxin derived from Gram-negative bacteria, has been widely used to induce sepsis myocardial injury models in vivo and in vitro (Luo et al, 2020). LPS induces cardiac dysfunction and the production of inflammatory factors (Fallach et al, 2010)

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