The Role Of Resveratrol In Cerebral Ischemia-reperfusion Injury In Rats And Its Mechanism

Stroke is the second leading cause of death in the world.It is the most serious and deadly disease of the nervous system.It seriously affects the quality of life of patients and brings a huge economic burden on families and society."Cerebral stroke" is also known as "stroke" or "cerebral vascular accident"(CVA).It is an acute cerebrovascular disease commonly seen in sudden rupture of the basilar artery or local vascular embolism leading to a sharp decrease in local blood supply to the brain.Cerebral stroke includes ischemic and hemorrhagic stroke.The incidence of ischemic stroke is the highest,accounting for 60% to 70% of the total stroke,and ischemic stroke is mainly caused by middle cerebral artery occlusion(MCAO)[1].The onset age is more than 40 years old,with male majority.Resveratrol(RSV)is a symmetrical diphenylethylene compound that has significant pharmacological effects in mammals.RSV can inhibit the proliferation of tumor cells and promote its apoptosis through various ways.In addition,RSV has a certain anti-oxidation effect,and improves the cognitive ability of Alzheimer disease(AD)mice by regulating acetylcholinesterase [2].Fukao et al.found that RSV pretreatment can reduce myocardial ischemia-reperfusion injury,dilate coronary arteries,and prevent atherosclerosis in rats [3].Lanzillotta et al.found that RSV pretreatment reduced oxygen and glucose deprivation(OGD)-induced apoptosis of primary cortical neurons in mice [4].Recently,RSV was reported to promote the expression of Vascular Endothelial Growth Factor B(VEGF-B)leading to reduce myocardial ischemia-reperfusion injury in mice.A further study showed that VEGF-B is widely expressed in brain tissue [5].While,it is not clear whether RSV participates in cerebral ischemia-reperfusion induced endogenous neuroprotection by regulating VEGF-B expression in rats.Objective To explore the effect and mechanism of resveratrol on cerebral ischemia-reperfusion injury in rats,and the effect of changes in the expression of Vascular Endothelial Growth Factor B(VEGF-B)on the neurological effect of resveratrol.Methods(1)Clean grade healthy adult male SD rats,260-270 g,were randomly divided into five groups according to random number table(n=6): control group(C group),ischemia-reperfusion group(I/R group),low-dose resveratrol pretreatment group(RSV25 group),medium dose resveratrol pretreatment group(RSV50 group),high-dose resveratrol pretreatment group(RSV100 group).C group was the same as I/R group but did not embolize the middle cerebral artery;I/R group reperfusion after MCAO 2 h;RSV25 group,RSV50 group and RSV100 group were received intraperitoneal injections of RSV 25 mg/kg,50 mg/kg,100 mg/kg,respectively,every day for one week before MCAO,and then reperfusion after MCAO 2 h.At 6 h after reperfusion,the brains were decapitated and the ischemic penumbra brain tissue was extracted.The expression of caspase-3 and anti-apoptotic factor Bcl-2 was detected by western blot.At 12 h after reperfusion,the brains were decapitated and the ischemic penumbra brain tissue was extracted.Real-time fluorescence quantitative PCR(RT-PCR)was used to detect inflammatory cytokines TNF-? and IL-1?.At 24 h and 72 h after reperfusion,the neurological function of rats after MCAO was evaluated using a standard neurological deficit score scale,brains were then decapitated and stained with 2% TTC,then used Image J software for image analysis to calculate the size of the infarct volume,and used the correction formula to reduce the impact of cerebral edema after ischemia on the experimental results.(2)Clean grade healthy adult male SD rats,260-270 g,were randomly divided into six groups according to random number table(n=6): control group(C group),ischemia-reperfusion group(I/R group),Vehicle group(V group),VEGF-B-siRNA negative control group(siRNA-c group),VEGF-B-siRNA group(siRNA group),RSV100+VEGF-B-siRNA group(R-V group).C group was the same as I/R group but did not embolize the middle cerebral artery;I/R group reperfusion after MCAO 2 h;V group,siRNA-c group and siRNA group were injected with 8 ?L transfection reagent(Vehicle)jet PEI,8 ?L(4 ?g)VEGF-B-siRNA-c and 8 ?L(4 ?g)VEGF-B-siRNA,respectively,at the 24 h,48 h and 72 h before MCAO;R-V group,100 mg/kg RSV was intraperitoneally injected every day for one week before MCAO,and then 8 ?L(4 ?g)of VEGF-B-siRNA was injected into the lateral ventricle of the rats at 24 h,48 h and 72 h before MCAO.At 6 h after reperfusion,the brain was decapitated and stained with 2% TTC,then extracted ischemic penumbra brain tissue,and the expression of cleaved caspase-3,Bcl-2,Phospho-CREB(p-CREB)and Phospho-Akt(p-Akt)was detected by western blot;At 12 h after reperfusion,the brain was decapitated and stained with 2% TTC,then extracted ischemic penumbra brain tissue,and the expression of TNF-? and IL-1? was detected by RT-PCR;At 24 h and 72 h after reperfusion,the neurological function of rats after MCAO was evaluated using a standard neurological deficit score scale,brains were then decapitated and stained with 2% TTC,then used Image J software for image analysis to calculate the size of the infarct volume,and used the correction formula to reduce the impact of cerebral edema after ischemia on the experimental results.Results(1)Compared with group C,the expression of cleaved caspase-3,TNF-? and IL-1? was increased in I/R group,RSV25 group,RSV50 group and RSV100 group,and the expression of Bcl-2 was decreased,the volume of cerebral infarction was increased,and the neurological deficit score was decreased;Compared with I/R group,the expression of cleaved caspase-3,TNF-? and IL-1? was decreased in RSV25 group,and the neurological deficit score was increased at 72 h;Compared with I/R group,the expression of cleaved caspase-3,TNF-? and IL-1? was decreased,the expression of Bcl-2 was increased,the volume of cerebral infarction was decreased,and the neurological deficit score was increased in RSV50 group;Compared with RSV50 group,the expression of cleaved caspase-3,TNF-? and IL-1? was decreased,the expression of Bcl-2 was increased,the volume of cerebral infarction was decreased,and the neurological deficit score was increased in RSV100 group.(2)Compared with group C,the expression of cleaved caspase-3,TNF-?,IL-1?,p-Akt,and p-CREB was increased in I/R group,V group,siRNA-c group,siRNA group and R-V group,and the expression of Bcl-2 was decreased,the volume of cerebral infarction was increased and the neurological deficit score was decreased at 24 h and 72 h;Compared with siRNA-c group,the expression of cleaved caspase-3,TNF-? and IL-1? was increased in siRNA group and R-V group,the expression of Bcl-2,p-Akt and p-CREB was decreased,the volume of cerebral infarction was increased,and the neurological deficit score was decreased.Group siRNA and group R-V,the difference was not statistically significant in the expression of cleaved caspase-3,TNF-?,IL-1?,Bcl-2,p-CREB and p-Akt,and the difference was not statistically significant in cerebral infarction volume and neurological deficit score;Group I/R,group V and group siRNA-c,the difference was not statistically significant in the expression of cleaved caspase-3,TNF-?,IL-1? and Bcl-2,and the difference was not statistically significant in cerebral infarction volume and neurological deficit score.Conclusion(1)Resveratrol pretreatment reduces cerebral ischemia-reperfusion injury in rats,while inhibiting VEGF-B expression reduces the neuroprotective effect of resveratrol.(2)Akt signaling pathway participates in the regulation of neuroprotective effect of resveratrol.(3)Resveratrol may antagonize cerebral ischemia-reperfusion injury in rats by promoting VEGF-B expression.The mechanism may be related to the Akt signaling pathway.