Diosmetin alleviated cerebral ischemia/reperfusion injury in vivo and in vitro by inhibiting oxidative stress via the SIRT1/Nrf2 signaling pathway

Abstract

Diosmetin, a bioflavonoid that is abundant in dietary sources, could attenuate cerebral ischemia/reperfusion injury by inhibiting oxidative stress via the SIRT1/Nrf2 signaling pathway both in vivo and in vitro.

Abstract

Cerebral ischemia/reperfusion (I/R) injury is caused by blood flow recovery after an ischemic stroke, and effective treatments targeting I/R injury are still insufficient. Oxidative stress is known to play a pivotal role in the pathogenesis of cerebral I/R injury. Previous studies have revealed that diosmetin could protect against oxidative stress in cerebral I/R injury, but the underlying mechanisms have not been fully revealed. The present study was undertaken to investigate the effects and mechanisms of action of diosmetin on cerebral I/R injury. In vivo, rats were orally gavaged with diosmetin for seven days, and middle cerebral artery occlusion (MCAO) was established to simulate cerebral I/R injury. The neurological deficit score, cerebral infarct volume, and cortical pathological lesions were measured. In vitro, PC12 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R). To clarify the mechanism, the SIRT1 inhibitor EX527 and the small interfering RNA (siRNA) of SIRT1 were used to downregulate the SIRT1 protein level, respectively. The contents of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) were determined with commercial kits. The protein expressions of SIRT1, total Nrf2 (T-Nrf2), nucleus Nrf2 (N-Nrf2), NQO1 and HO-1 were measured by western blotting. The results showed that diosmetin pretreatment improved neurological outcomes, decreased the cerebral infarct volume and pathological lesions, and inhibited oxidative stress in cerebral I/R rats. In PC12 cells, diosmetin increased cell viability, reduced lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) level, and inhibited oxidative stress. Besides, diosmetin increased the protein expressions of SIRT1, T-Nrf2, N-Nrf2, NQO1 and HO-1 both in vivo and in vitro. However, administration of EX527 or silencing the SIRT1 gene with its siRNA eliminated the beneficial effects of diosmetin. Meanwhile, inhibition of SIRT1 decreased the levels of Nrf2 and the protein expressions of its downstream antioxidants NQO1 and HO-1. In conclusion, our data suggested that diosmetin could attenuate cerebral I/R injury by inhibiting oxidative stress via the SIRT1/Nrf2 signaling pathway.