6’-O-galloylpaeoniflorin Attenuates Inflammation And Oxidative Stress Induced Injury In Cerebral Ischemia/Reperfusion Model Via PI3K/Akt/Nrf2 Pathway

Cerebral vascular disease is refers to the cerebral vascular disease caused by brain dysfunction.Nerve function cause defect syndrome due to the local brain blood circulation obstacle.The incidence 、 mortality and morbidity of cerebral vascular disease is high.Cerebral vascular and heart disease,malignant tumor constitute the three leading cause of death of human beings.Urban morbidity and mortality of stroke is 219/100 thousand and 116/100 thousand respectively.Cerebral ischemia reperfusion injury is a complex pathological process.The mechanism of each period is interaction,.According to the results of the present study found that more involved in the mechanism of cerebral ischemia-reperfusion injury and connected to each other,and influence each other,form a "mesh" structure,the pathogenesis of the article "main line" is unclear.Therefore,Questing a major cause of cerebral ischemia-reperfusion injury incidence is still the focus of the research in this field,in order to provide a certain basis for the prevention and control.6’-O-galloylpaeoniflorin as ranunculaceae plants peony or radix paeoniae rubra the main effective components,has clear the effect of heat cool blood and the scattered stasis pain.6’-O-galloylpaeoniflorin belong to the monoterpene glycoside compounds,its molecular formula for C23H28O11,molecular weight of 480.45.In recent years,scholars at home and abroad have more in-depth research on the pharmacological effects of 6’-O-galloylpaeoniflorin.6’-O-galloylpaeoniflorinin have many kinds of biological effect on inhibition of intracellular calcium overload,fight free radical damage,reduce blood viscosity,platelet aggregation,dilate blood vessels,improve microcirculation,oxidation resistance and resistance to nerve toxicity and so on.but6’-O-galloylpaeoniflorin by PI3K/Akt/Nrf2 pathway to reduce ischemia reperfusion of oxidative stress injury research have not been reported.This study was divided intothree parts to explore the protective effects of 6’-O-galloylpaeoniflorin on Cerebral ischemia reperfusion injury and the underlying mechanism in vitro and in vivo.Part Ⅰ: 6’-O-galloylpaeoniflorin attenuate cerebral ischemia reperfusion injury induced oxidative stress and inflammation in PC12 cellsIn this part of study,we observed the effects of 6’-O-galloylpaeoniflorin treatment on oxidative stress,neuroinflammation and cell viablity in PC12 cells exposed to cerebral ischemia reperfusion injury in vitro.The results showed that cerebral ischemia reperfusion injury could induce the production of reactive oxygen species(ROS)in PC12 cells,which was confirmed by DCFH-DA.And6’-O-galloylpaeoniflorin can reduce the ROS content in PC12 cells and down-regulate the mitochondrial membrane potential damage caused by cerebral ischemia reperfusion injury.Moreover,the 6’-O-galloylpaeoniflorin can up-regulate SOD activity and down-regulate the level of MDA.The results indicated that the6’-O-galloylpaeoniflorin had the effect of reducing the oxidative stress in PC12 cells induced by cerebral ischemia reperfusion injury.The expression of TNF-α and IL-1βin PC12 cells was significantly increased by the treatment of erebral ischemia reperfusion injury by q RT-PCR and ELISA.The expression and release of inflammatory factors in PC12 cells induced by erebral ischemia reperfusion injury were significantly decreased by 6’-O-galloylpaeoniflorin in a dose-dependent manner,indicating that 6’-O-galloylpaeoniflorin could alleviate the inflammatory response induced by erebral ischemia reperfusion injury in vitro.At the end of this study,CCK-8 and LDH activity and apoptosis detection were used to detect cerebral ischemia reperfusion injury cell injury and apoptosis,and the results showed that6’-O-galloylpaeoniflorin could protect PC12 cells from cerebral ischemia reperfusion injury induced neurotoxicity in a dose-dependent manner.In summary,the results of this study show that 6’-O-galloylpaeoniflorin can protect cerebral ischemia reperfusion injury PC12 cells by reducing cerebral ischemia reperfusion injury cell oxidative stress and neuroinflammatory response.However,the mechanism by which the 6’-O-galloylpaeoniflorin played a neuroprotective effect is not yet fully understood.In the second part of this study,the mechanism underlying antioxidant and anti-inflammatory effects of 6’-O-galloylpaeoniflorin was further explored.Part Ⅱ 6’-O-galloylpaeoniflorin modulates oxidative stress andinflammation in PC12 cellsIn this part of the study,we further explored on the mechanism underlying antioxidant and anti-inflammatory effects of 6’-O-galloylpaeoniflorin.It was found that both low-dose and high-dose 6’-O-galloylpaeoniflorin had no significant effect on the expression of Nrf2 in PC12 cells.However,the expression of Nrf2 in the nucleus was significantly increased after stimulation with 6’-O-galloylpaeoniflorin in a dose-dependent manner.Knockdown of Nrf2 in PC12 cells via RNA interference significantly weakened the antioxidant and anti-inflammatory ability of 6’-Ogalloylpaeoniflorin,as well as the neuroprotective effect of 6’-O-galloylpaeoniflorin.A PI3K-specific inhibitor inhibited the effect of 6’-O-galloylpaeoniflorin on Nrf2 translocation.Furthermore,A PI3K-specific inhibitor also inhibited the effect of6’-O-galloylpaeoniflorin on membrane potential,MDA level and the inflammatory cytokines TNF-α and IL-1β induced by cerebral ischemia reperfusion injury,and attenuated the protective effect of 6’-O-galloylpaeoniflorin on cell viability and apoptosis.In summary,the results show that 6’-O-galloylpaeoniflorin can promote the activation of Nrf2 through Akt signaling pathway,leading to antioxidant and anti-inflammatory ability to protect PC12 cells exposed to cerebral ischemia reperfusion injury.However,in vitro studies do not adequately address the therapeutic effect of 6’-O-galloylpaeoniflorin on cerebral ischemia disease.Therefore,in the next part of the study,we will further observe and explore the effect of 6’-Ogalloylpaeoniflorin on oxidative stress,inflammatory response and memory learning ability in MCAO mice.Part Ⅲ: Therapeutic effects of 6’-O-galloylpaeoniflorin on MCAO mice and the underlying mechanismBased on its anti-oxidant and estrogenic properties,we tested the effects of chronic 6’-O-galloylpaeoniflorin administration at three different doses on various behavioural and neuropathological markers in transgenic cerebral ischemia reperfusion injury mice.Endpoints of the study were motor activity,locomotion,learning,memory,IL-1β,TNF-α,malondialdehyde(MDA),and glutathione(GSH)levels.Untreated transgenic MCAO mice showed impaired learning and memory.The administration of 6’-O-galloylpaeoniflorin rectified in a dose-dependent manner the learning and memory impairments,as well as the various pathological markers in via PI3K/Akt/Nrf2 pathwaybrain and peripheral tissues of the cerebral ischemia reperfusion injury mice.In addition,these beneficial effects of 6’-O-galloylpaeoniflorin in transgenic cerebral ischemia reperfusion injury animals were significantly abolished by co-administration of a selective Akt-specific inhibitor.We conclude that 6’-O-galloylpaeoniflorin rescues many aspects of brain function in MCAO model mice via activation of the PI3 K pathway,and may have therapeutic potential for the treatment of cerebral ischemia reperfusion injury.