Study On The Role And Mechanism Of TRPM2/CREB Signaling Pathway In Neuronal Injury After Cerebral Ischemia/Reperfusion

BackgroundCerebrovascular disease,as one of the major causes of human death,has been of great concern in recent years.With the progress of scientific research and medical technology,the incidence and mortality rate of cerebrovascular accidents in many developed countries have been on a decreasing trend,but the overall incidence and mortality rate of cerebrovascular disease in many developing countries,including China,are still on a slightly increasing trend.Cerebral ischemic stroke(CIS)is one of the most common and most prevalent of the many classifications of cerebrovascular disease.The main cause of CIS is the reduction or interruption of cerebral blood flow due to the blockage of cerebral vessels caused by various factors,which leads to ischemia and hypoxia in the brain.Although timely intravenous thrombolysis and embolization treatment can recanalize the occluded vessels,it is often accompanied by reperfusion,which further aggravates the metabolic disorders and even structural damage of brain cells,leading to brain tissue damage and neurological dysfunction,i.e.cerebral ischemia reperfusion injury(CIRI).There are no targeted therapeutic options for CIRI,therefore,there is an urgent need to further investigate the mechanism of neuronal injury caused by ischemia/reperfusion and to find new therapeutic targets.Ischemia/reperfusion-induced neuronal injury is a complex pathological process involving multiple factors,mainly including excitatory glutamate toxicity,calcium overload,increased oxygen free radicals and nitric oxide,and brain cell edema.Among them,excitatory glutamate toxicity is considered to be an important mechanism of ischemic neuronal injury.However,for a long time,successive generations of glutamate receptor antagonists have not shown the expected neuroprotective effects when applied in human stroke and neurodegenerative diseases.Therefore,it is important to investigate the mechanism of non-glutamate-dependent pathway-mediated cerebral ischemia/reperfusion neuronal injury.Transient receptor potential melastatin 2(TRPM2),a member of the transient receptor potential channels(TRP)family,is activated by stimuli such as ischemia and hypoxia,leading to increased Ca2+ inward flow,increased production of reactive oxygen species(ROS)and activation of multiple apoptotic pathways.It can be activated by stimuli such as ischemia and hypoxia,leading to increased Ca2+ inward flow and increased production of reactive oxygen species(ROS),activating various apoptotic pathways and leading to cell death.However,the time course of TRPM2-induced neuronal damage is not clear,and the regulatory mechanisms involved in these processes remain to be explored.It has been shown that high expression of transient receptor potential channels 6(TRPC6),a member of the TRP family,significantly attenuates ischemic neuronal injury and that TRPC6 is involved in this process through the activation of Calcium/calmoduLin-dependent protein kinase Ⅳ/cAMP-response element binding protein(CaMKIV/CREB)signaling pathway.TRPM2 has significant homology with amino acid sequence of TRPC6,therefore,it is likely that TRPM2 has a similar signaling pathway to TRPC6,the mechanism of which needs to be further explored.In this study,we propose to establish in vivo and ex vivo models of cerebral ischemia/reperfusion,construct TRPM2 overexpression/knockdown models and CaMKIV/CREB low expression models,as a basis to investigate the role and possible mechanisms of TRPM2 in neuronal injury caused by ischemia/reperfusion.Objective1.To clarify the role of TRPM2 on neuronal injury due to ischemia/reperfusion and the time course of action.2.To observe the role of TRPM2 on CaMKIV/CREB expression in ischemia/reperfusion neuronal injury.3.To elucidate the role of CaMKIV/CREB signaling pathway in TRPM2-mediated cerebral ischemia/reperfusion neuronal injury.Methods1.The human neuroblastoma cell line(SH-SY5Y)was cultured in vitro to establish a cellular oxygen-glucose deprivation/reoxygenation(OGD/R)model.The cell morphology,viability,apoptosis ratio and TRPM2 expression at different hypoxia and reoxygenation time points were examined to clarify the time course of TRPM2 effect on neuronal injury caused by ischemia/reperfusion.2.To construct plasmids to transfect SH-SY5Y cells to overexpress TRPM2 and CTZ to inhibit TRPM2 expression,respectively,and to establish OGD/R cell models.qRT-PCR and Western Blot were used to detect the expression of Bcl-2,Bax,Cleaved Caspase-3,c-Fos and JunB.TRPM2-/-and TRPM2+/-C57BL/6 mice were prepared to construct transient middle cerebral artery embolism(tMCAO)models,and the degree of neurological deficits in mice was assessed using the Longa method neurobehavioral score and modified neurological damage severity scale,respectively,and TTC staining was used to detect brain infarct volume and TUNEL staining to detect apoptosis.The expression of Bcl-2,Bax,Cleaved Caspase-3,c-Fos and JunB were detected by immunofluorescence,qRT-PCR and Western Blot.3.Sham group,tMCAO+WT,tMCAO+TRPM2+/-(HE)and tMCAO+TRPM2-/(HO)C57BL/6 mouse transient middle cerebral artery/occlusion(tMCAO)models were prepared,and total RNA was extracted from the cerebral cortex of mice in the infarct area,transcriptomic sequencing was performed to screen for differential genes.4.To construct TRPM2 high/low expression OGD/R cell models,SH-SY5Y cells were transfected with plasmids to overexpress TRPM2 and CTZ to inhibit TRPM2 expression,respectively.TRPM2-/-and TRPM2+/-C57BL/6 mouse tMCAO models were prepared,and the expression of CaMKIV,pCaMKIV,CREB and pCREB in different groups of cells and tissues were detected by Western Blot.5.OGD/R cell model with low expression of CaMKIV/CREB was constructed by using CaMKIV specific inhibitor KN-62 and CREB specific inhibitor KG-501 in vitro.AAV-U6-shRNA(CaMKIV)-CMV-GFPand AAV-U6-shRNA(CREB)-CMV-GFP were injected into mouse tail vein in vivo to construct tMCAO model of CaMKIV/CREB low expression mice.The degree of neurological deficits in mice was assessed using the Longa method neurobehavioral score and the modified neurological damage severity scale respectively,and the brain infarct volume was detected by TTC staining.The expression of Bcl-2,Bax,Cleaved Caspase-3,c-Fos,JunB and TRPM2 were detected by qRT-PCR and Western Blot.Results1.SH-SY5Y cell viability showed a gradual decrease with the prolongation of OGD time,with the most obvious decrease at OGD6h,while the apoptosis level showed the opposite trend.The cellular TRPM2 expression increased with the prolongation of OGD time and reached the peak at OGD6h.After OGD6h,the cell viability gradually decreased with the increase of reoxygenation time and reached the lowest point at OGD6h/R3h,and then the cell viability gradually increased.The apoptosis level and TRPM2 expression showed the opposite trend with cell viability.(p<0.05).2.The expression of apoptosis-related factors such as Bax,Cleaved-Caspase3,cFos,JunB and pCaMKIV/CaMKIV,pCREB/CREB in the OGD/R+TRPM2 and tMCAO+WT groups were significantly higher than those in the OGD/R+CTZ and tMCAO+HO groups.The trend of Bcl2 expression was the opposite.The neurological deficits,brain infarct volumes and neuronal apoptosis ratios were significantly lower in the tMCAO+HO group than in the tMCAO+WT group.(p<0.05)3.The expression of CaMKIV and CREB was inhibited by AAV-U6shRNA(CaMKIV)-CMV-GFP,AAV-U6-shRNA(CREB)-CMV-GFP and CaMKIV inhibitor KN62,CREB inhibitor KG-501 in vivo and in vitro respectively.The expression of apoptosis-related factors was significantly decreased,the neurological deficit and the volume of cerebral infarction was significantly reduced(p<0.05).However,there was no significant effect on TRPM2 expression(p>0.05).Conclusion1.TRPM2 is involved in OGD/R-mediated SH-SY5Y cell injury with a peak action time course of OGD6h/R3h.2.TRPM2 plays a role in promoting apoptosis in delayed cerebral ischemia/reperfusion injury.3.CaMKIV/CREB signaling pathway is one of the regulatory mechanisms involved in cerebral ischemia/reperfusion neuronal injury by TRPM2,and inhibition of CaMKIV/CREB activity significantly ameliorates cerebral ischemia/reperfusion injury.