Quercetin Alleviates the Inflammatory Response and Oxidative Stress of Myoblasts after Ischemia/Reperfusion by Inhibiting NOX-2

Abstract

Objective: Limb ischemia-reperfusion injury (LIRI) may lead to tissue necrosis and loss of function, even life-threatening. Our previous study found that Tao-Hong-Si-Wu decoction (THSWD) had some efficacy in treating of LIRI. Quercetin, the major component of THSWD, was selected further to uncover the molecular mechanism underlying its treatment of LIRI. Methods: In this study, myoblasts were isolated from rat gastrocnemius muscle tissue, and an in vitro LIRI model was established. The cell counting kit-8 (CCK-8) and colony formation assay were used to evaluate the impact of quercetin on LIRI-induced myoblast viability and proliferation. Lactate dehydrogenase (LDH) activity was measured to detect myoblast injury in the LIRI model. The apoptosis of myoblasts was evaluated by Hoechst staining and flow cytometry. In addition, molecular docking analysis was performed to predict the interaction between quercetin and NADPH oxidase 2 (NOX-2). Subsequently, we investigated the molecular mechanism of quercetin in LIRI-induced myoblasts by overexpressing NOX-2. Results: The myogenic marker Desmin was highly expressed in isolated myoblasts. In the LIRI model, myoblast viability and proliferation were decreased, and cell injury and apoptosis levels were increased. In addition, NOX-2 was highly expressed in the LIRI model. At the same time, LIRI induction promoted the up-regulation of oxidative stress and inflammatory response. Quercetin significantly reversed the effects of LIRI treatment on myoblasts in a concentration-dependent manner. Molecular docking suggested an interaction between quercetin and NOX-2. Further overexpression of NOX-2 inhibited the effect of quercetin on LIRI-induced myoblasts. Conclusion: Quercetin could reduce inflammatory response and oxidative stress by inhibiting NOX-2, thus playing a therapeutic role in treating LIRI.