Sestrin2 Slience Exacerbates Cerebral Ischemia/Reperfusion Injury Through Inhibiting AMPK/PGC-1α Pathway

Background:Sestrin-2(Sesn2) is a conserved antioxidant protein that is activated upon oxidative stress and protects cells against reactive oxygen injury.Studies have shown that Sesn2 exerts neuroprotective properties in some neurodegenerative diseases. However, the role of Sesn2 in cerebral ischemia and reperfusion injury disease is unclear. The AMP-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α(AMPK/PGC-1α) pathway plays an important role in regulating mitochondrial biogenesis, which helps prevent cerebral ischemia/reperfusion(I/R) injury.Objecitve:To determine whether Sesn2 affects I/R damage by regulating mitochondrial biogenesis through the AMPK/ PGC-1α signaling pathway.Methods:( 1) Transient cerebral ischemia and reperfusion was induced by middle cerebral artery occlusion(MCAO) using modified suture occlusiontechnique in rats. The rats were perfused transcardially and decapitated at6h、12h、24h、48h、72h and the proteins were extracted from the ischemic cortex. Real-time qPCR and Western blot assay were used to screen the highest expression time point of Sesn2.(2)Three Sesn2 si RNAs(Sesn2-777, Sesn2-1407, Sesn2-1554) and one control siRNA were built. siRNAs were injected intracerebroventricularly 24 h prior to MCAO. Real-time qPCR and Western blot assays were used to test the knockdown efficiency and filter out the most effective interference fragment.(3)After Sesn2 knockdown, neurological deficits, infarct volume,and histological assessment were measured to detect the cerebral injury.SOD activity and MDA content were used to assess the oxidative stress of the brain after cerebral I/R.( 4) The expression of AMPK and downstream factors, including mitochondrial biogenesis proteins, PGC1-α, NRF-1, TFAM, and mitochondrial related ROS-detoxifying proteins, SOD2 and UCP2, and mitochondrial-dependent apoptosis proteins, cytochrome c and AIF were examined after Sesn2 knockdown.(5)The MCAO model with AMPK activated after Sesn2 knockdown was built. AICAR, an AMPK activator was injected 30 min before MCAO model performed. Real-time qPCR and Western blot assays were used to detect the expression of Sesn2, AMPK, p-AMPK, PGC1-αand NRF-1,TFAM, SOD2, UCP2, cytochrome c and AIF.(6)Under Sesn2 silencing conditions, after AICAR was administered,neurological deficits, infarct volume, and SOD activity and MDA content were tested.Results:(1)The expression of Sesn2 began to increase at 6h, peaked at 24 h,and then decreased at 48 h and 72 h after cerebral I/R.( 2) Compared with the MCAO group, the inhibitory effect of Sesn2-777 was the strongest one, with interference efficiency over 60%.No significant difference was found between MCAO group and control siRNA group.( 3) Neurological deficits were exacerbated, infarct volume was enlarged, and oxidative stress and neuronal damage were aggravated in the Sesn2 siRNA group in comparison with those in MCAO group.(4)Expression levels of Sesn2, p-AMPK, PGC-1α, NRF-1, TFAM,SOD2, UCP2, cytochrome c and AIF were significantly increased following cerebral I/R. However, except cytochrome c and AIF were dramatically up-regulated, other proteins were decreased after Sesn2 knockdown. In contrast, activation of AMPK with AICAR reversed the effects of Sesn2 siRNA.(5)Compared with the Sesn2 siRNA group, Neurological deficits were improved, infarct volume was reduced, and oxidative stress wasweakened after AICAR administered.Conclusions:(1)The cerebral I/R injury was exacerbated after Sesn2 silenced,furthermore represented that Sesn2 exerted neuroprotective effects during cerebral I/R injury.(2)These results suggest that Sesn2 silencing may down-regulate mitochondrial biogenesis, reduce mitochondrial biological activity, and finally aggravate cerebral I/R injury through inhibiting the AMPK/PGC-1αpathway.