SanggenonC protects against cardiomyocyte hypoxia injury by increasing autophagy.

Abstract

Sanggenon C is isolated from Morus alba, a plant that has been used for anti-inflammatory purposes in Oriental medicine. Little is known about the effect of Sanggenon C on cardiomyocyte hypoxia injury. This study, using H9c2 rat cardiomyoblasts, was designed to determine the effects of Sanggenon C on cardiomyocyte hypoxia injury. Inflammatory cytokine levels were measured by reverse transcription-polymerase chain reaction, reactive oxygen species were measured by 2′,7′-dichlorofluorescin diacetate fluorescent probe, autophagy was detected using the LC3II/I ratio and cell apoptosis was detected by TUNEL staining. The molecular mechanisms underlying Sanggenon C-induced cyto-protection were also determined by western blotting, especially the possible involvement of autophagy and AMP-activated protein kinase (AMPK). Results indicated that samples pretreated with different concentrations of Sanggenon C (1, 10 and 100 µM) reduced the expression levels of pro-inflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-1 and IL-6, under hypoxia. The beneficial effects of Sanggenon C were also associated with reduced levels of reactive oxygen species generation and increased levels of antioxidant nitric oxide and superoxide dismutase. Sanggenon C enhanced hypoxia-induced autophagy as evidenced by the increased expression levels of autophagy-associated proteins Beclin and autophagy related 5 as well as the decreased the accumulation of p62, and increased the LC3II/I ratio. Sanggenon C also reduced hypoxia-induced apoptosis as detected by TUNEL staining and the expression of Bcl-2 proteins. The beneficial effects of Sanggenon C were associated with enhanced activation level of AMPKα and suppressed hypoxia-induced mechanistic target of rapamycin (mTOR) and forkhead box O3a (FOXO3a) phosphorylation. The AMPK inhibitor Compound C (CpC) was used, and the anti-apoptotic and pro-autophagy effects of Sanggenon C in response to hypoxia were abolished by CpC. In conclusion, the current study demonstrated that Sanggenon C possessed direct cytoprotective effects against hypoxia injury in cardiac cells via signaling mechanisms involving the activation of AMPK and concomitant inhibition of mTOR and FOXO3a.