Pinocembrin Protects Brain From Ischemia/Reperfusion By Attenuating Endoplasmic Stress Induced Injury

Ischemic stroke is harmful to human health with high morbidity, high morbidity and high mortality. Finding a safe and effective treatment is of great significance. Recently, neuroprotective drugs have caused more and more attention of people for the protection against ischemia and ischemia-reperfusion injury of the brain.Pinocembrin (5,7-dihydroxyflavanone), a natural flavonoid in propolis and many plants, Our previous studies showed that it had potent neuroprotective effects by improving mitochondrial function, decreasing oxidative damage, reducing neuronal apoptosis and inhibiting inflammatory responses in middle cerebral artery occlusion rat models. These results indicate that pinocembrin may be a multiple-targets drug.Cerebral ischemia/reperfusion injury triggers a series of injury cascade, resulting in protease activation and gene expression changes, eventually leading to cell necrosis or apoptosis. Recently, accumulating evidences also show that ER stress induced apoptosis plays an important role in the mechanisms underlying ischemia/reperfusion neuronal damage.This study aimed to elucidate whether pinocembrin suppressing ER stress-induced apoptosis is a potential mechanism of its attenuation on brain ischemia/reperfusion damage.Part I The Relationship between Pinocembrin and Cerebral ischemia reperfusionThis section provides an analysis of relationship among cerebral ischemia-reperfusion injury, endoplasmic reticulum stress, and pinocembrin, which is the background and purpose of the study.Part II Therapy effects of Pinocembrin on on experimental focal cerebral brain ischemia reperfusionIt is found that pinocembrin has good therapeutic effects on the rats both of acute focal cerebral ischemia 2 h followed reperfusion 6 h and 24 h in a dose dependent manner under 1 mg/kg,3 mg/kg,10 mg/kg. Specific performances in:(1) Improving acute focal cerebral ischemia reperfusion rats neurobehavioral damage; (2) Reducing the acute focal cerebral ischemia reperfusion in rats of cerebral infarction volume; (3) Reducing cerebral edema percentage in the rats of acute focal cerebral ischemia reperfusion; (4) Improving the morphology of brain cortex, striatum and hippocampus of acute focal cerebral ischemia reperfusion; (5) Reducing serum NSE and S-100β level of the acute focal cerebral ischemia reperfusion rats; (6) Reducing serum inflammatory factors of TNFα and IL-1β level of the acute focal cerebral ischemia reperfusion rats. In addition, the results also showed that the injury of brain with focal cerebral ischemia 2 h following reperfusion 6 h was more serious than 24 h reperfusion.Part Ⅲ Pinocembrin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress induced apoptosisResults of this section suggest that pinocembrin has the dose-dependent (1 mg/kg,3 mg/kg,10 mg/kg) action as follows:(1) Reducing cell apoptosis of penumbra in focal cerebral ischemia/reperfusion rats; (2) Increasing the expression of GRP78 protein, and improve the level of GRP78mRNA of penumbra; (3) Reducing the expression of CHOP/GADD153 and caspase-12, which are related with endoplasmic reticulum stress-induced apoptosis; (4) Pinocembrin reduces the expression of CHOP/GADD153 by regulating PERK-eIF27α-ATF4 signal pathway, and it can decrease eIF2a phosphorylation cope with the reduction of ATF4 expression; (5)Up-regulating Hsp70 expression; (6) Increasing Xbp-1 mRNA level.The study provides experimental evidences that pinocembrin protect against cerebral ischemia/reperfusion injury, and one of the mechanisms underlying the neuroprotection is related with attenuation of ER stress-induced apoptosis.Part Ⅲ The protective effect of pinocembrin on SH-SY5Y cells under endoplasmic reticulum stress conditions or oxygen-glucose deprivation/recovery conditonsResults of this section suggest that under endoplasmic stress conditions or oxygen-glucose deprivation/recovery conditons, pinocembrin has the dose-dependent effects as follows during 0.01 μM to 1 μM concentration range:(1) Increasing nerve cells vitality and reducing the nerve cell apoptosis; (2) Increasing the expression of GRP78 and Hsp70; (3) Reducing expression of CHOP/GADD153 and caspase-12; (4) Reducing the intracellular calcium ion concentration; (5) Regulating the PERK-eIF2α-ATF4-CHOP/GADD153 signaling pathway to reduce the expression of CHOP/GADD153 by suppressing eIF2a phosphorylation and the expression of ATF4; (6) At the cellular level, the maximize efficiency of pinocembrin is underlied the concentrations between 1μM to 10 μM.In conclusion, pinocembrin could inhibit endoplasmic reticulum stress induced apoptosis by down-regulating eIF2a phosphorylation, and the expression of ATF4, CHOP/GADD153 after cerebral ischemia/reperfusion in rats, which maybe one of the mechanisms of its attenuation of ischemical reperfusion injury.