EAAT2Regulate NDRG2Involved In Cerebral Ischemic Tolerance Induced By Electroacupancture

Stroke is the second most common cause of death and results in a largenumber of people with disability worldwide. Therefore, it is a huge and urgentmedical need to develop novel and rational strategies aimed at preventing strokeas well as reducing impairments caused by ischemia/reperfusion (I/R) injury.EAis a novel therapy based on traditional acupuncture combined with modernelectrotherapy. Due to the beneficial effects of acupuncture to different brainand heart diseases, EA has been used in treatment as an improvement ontraditional acupuncture.In recent years, numerous studies have shown that EAalso induced pretreatment effect, inducing ischemic tolerance as well. Manystudies have shown that protective mechanisms of EA preconditioning mayinvolve a series of regulatory molecular pathways including activityenhancement of antioxidant, regulation of the endocannabinoid system,inhibition of apoptosis, etc. Astrocytes is the first cell type in the central nervoussystem to encounter insult after brain ischemia, and induced initiation ofischemic cascade．NDRG2is expressed in astrocytes, and may involve in the modulation of glial cell function in the central nervous system. However, therole of NDRG2in the EA induced cerebral ischemic tolerance is unknown. Thisstudy was designed to investigate the role of NDRG2in neuroprotective effectof EA, and to explore its possible up and downstream regulation mechanism.Experiment1Purpose: N-myc downstream regulated gene2(NDRG2) was reported to bewidely expressed in the nervous system. However, the expression and potentialrole of NDRG2in focal cerebral ischemia brain remain unclear. Herein, weinvestigated whether or not the NDRG2was involved in the ischemic toleranceinduced by EA preconditioning in rats.Methods: Male SD rats were radomly divided into three groups as follows:Sham, MCAO and EA groups. After24h period of reperfusion, brain sampleswere harvested to determine the expression of NDRG2in the brain by reversetranscriptase-polymerase chain reaction (RT-PCR), Western Blot analysis andimmunohistochemical staining. Cellular apoptosis was assessed by TUNELstaining.Results: By using double immunofluorescence, NDRG2signals in astrocyteswas suppressed in EA group at24h after reperfusion, and NDRG2proteinexpression was weak in the nucleus and strong in the cytoplasm in the EA group,but strong in nucleus in the MCAO group. Triple immunofluorescent stainingfor TUNEL, NDRG2, and DAPI showed that most of NDRG2signalsco-localized with apoptotic cells. Moreover, the number of apoptotic cellsdecreased with attenuation of NDRG2-positive signals in EA group comparedwith MCAO group. Conclusion: NDRG2is involved in anti-apoptosis induced byelectroacupuncture preconditioning after focal cerebral ischemia in rats。Experiment2Purpose: To investigated if EAAT2regulate NDRG2involved in cerebralischemic tolerance induced by electroacupancture.Methods: Male SD rats were radomly divided into three groups as follows:Sham, MCAO, EA, CXT and DHK groups. After24h period of reperfusion,the animals were examined for neurological scores and then killed to measuredinfarct volumes, brain samples were harvested to determine the expression ofNDRG2in the brain by Western Blot analysis and immunohistochemicalstaining.Results: The neurological scores were higher in EA and CXT groups than thatin MCAO group, no statistical difference was found between DHK group andMCAO group.The infarct volumes in EA and CXT groups were significantlysmaller than that in MCAO group, no statistical difference was found betweenDHK and MCAO group. EAAT2signals was suppressed in DHK group andactivated in EA and CXT groups.The expression of NDRG2protein expressionwas weak in the EA and CXT groups, but strong in the DHK group.Conclusion: Electroacupancture preconditioning suppressed NDRG2viaupregulated the expression of EAAT2involved in cerebral ischemic tolerance.Experiment3Purpose: To investigated the role of NDRG2regulate STAT3in protectiveeffect induced by2-AG in primary astrocytes after OGD.Methods: After transfecting the NDRG2pSRL-SIH1-H1-GFP vector, OGD4h, reoxygenation was subjected to primary astrocytes in24h. The cell growth wasmonitored by MTT and LDH assay, NDRG2and STAT3protein level wasdetected by Western Blot and immunocytochemistry. Then ransfecting theNDRG2pSRL-CDH1-GFP and add2-AG. After OGD was subjected to primaryastrocytes, the cell growth was monitored by MTT and LDH assay, NDRG2andSTAT3protein were detected by Western Blot and immunocytochemistry.Results: The cell viability was inhibited after OGD, no statistical difference wasfound between pSRL-SIH1-H1-GFP and Sham groups. The level of NDRG2,pSTAT3in ODG group were higher than those in sham group, and comparedto OGD group, the upregulation of NDRG2, pSTAT3in pSRL-SIH1-H1-GFPgroup were supressed. The cell viability in2-AG group was higher thanthat inOGD group, and ransfecting the NDRG2pSRL-CDH1-GFP vector reversed theprotective effects of2-AG. no statistical difference was found betweenpSRL-SIH1-H1-GFP and OGD groups. The level of NDRG2, pSTAT3in2-AGgroup were loeer than those in OGD group, no statistical difference was foundbetween pSRL-SIH1-H1-GFP and OGD groups.Conclusion:2-AG suppressed phosphorylation of STAT3by decreasedexpression of NDRG2induced neuroprotection.