The Role And Mechanism Of TRPM7 In Cerebral Ischemia-reperfusion Injury

Objective:To investigate the mechanism of TRPM7 in cerebral ischemiareperfusion injury by studying the function of membrane protein TRPM7 and its ion channel inhibitors in cerebral ischemia-reperfusion injury.Methods:1)RT-PCR and Western blotting were used to detect the expression of TRPM7 mRNA and protein in rat cerebral cortex and cortical neurons.Immunofluorescence staining was applied to detect the localization of TRPM7 protein;2)We established an oxygen glucose deprivation and reperfusion(OGD/R)model of primary cortical neurons in vitro,and detected the expression of TRPM7 by Western blotting and immunofluorescence staining.Then in the primary cortical neuron OGD/R model,the function of TRPM7 was regulated by carvacrol or 2-APB,which are the ion channel inhibitors of TRPM7.TUNEL method was used to detect neuronal apoptosis rate;3)We established a model of middle cerebral artery occlusion and reperfusion(MCAO/R).Twenty-four adult rats were randomly divided into sham group(n=3)and MCAO/R group,then the MCAO/R group were further randomly divided into 7 groups(n=3/group),which were 0h?4h?8h?12h?24h?48h?72h group.Western blotting and IHC were used to detect the expression of TRPM7 in the ischemic prefrontal cortex(infarct core area and surrounding area);4)Eighty-nine SD adult rats were randomly divided into Normal group(n=13),Sham group(n=13),I/R group(n=20),I/R+DMSO group(n=5),I/R+CAR group(n=20)and I/R+2-APB group(n=18).They were treated with intraperitoneal injection of normal saline,0.5% DMSO,CAR(4 mg/kg)and 2-APB(50 mg/kg).The apoptosis factors were detected by qRTPCR 24 h after surgery.Neurobehavioral function was assessed on 1 day,3 days,and 7 days after surgery with modified Garcia scoring criteria,and the end of the treatment,the rats were sacrificed and their brains were processed.The histological changes of cerebral infarction were assessed by TTC staining.Results:1)TRPM7 is mainly expressed in neurons and astrocytes,barely expressed in microglia and oligodendrocytes;2)In the OGD/R model of cortical neurons,the expression of TRPM7 in OGD/R24 h group was higher than that in Control group(P<0.05),and CAR(500?M)or 2-APB(100?M)significantly reduced the apoptotic rate of neurons by TUNEL method(P<0.05);3)the expression of TRPM7 in the ischemic prefrontal cortex increased significantly with the reperfusion time in 72h(P<0.05),and peaked at 24-48 h after reperfusion(P<0.05);4)In the MCAO/R model,the neurobehavioral scores of the I/R+CAR group and the I/R+2-APB group were significantly improved(P<0.05)and the volume of cerebral infarction was significantly reduced compared with the I/R+DMSO group(P <0.05).The expression of antiapoptotic factor BCL-2 was up-regulated and the expression of pro-apoptotic factor Bax was down-regulated in the pro-cerebral cortex of I/R+CAR group and I/R+2-APB group(P<0.05).Conclusion: 1)TRPM7 is consistently expressed in the anterior cortex,mainly in the cell membrane of neurons and astrocytes;2)The expression of TRPM7 increased in a time-dependent manner during the acute phase of cerebral ischemia-reperfusion injury,and it involved in the process of acute cerebral ischemia-reperfusion injury;3)carvacrol or 2-APB,which are ion channel inhibitors of TRPM7,improved cerebral ischemia-reperfusion injury in rats,and its molecular mechanism is through the regulation of the anti-apoptotic factor BCL-2 and pro-apoptotic factor Bax in the apoptotic signaling pathway.