Study On Function And Mechanism Of Capacity Calcium Entry In Cerebral Ischemia-reperfusion Injury

?Background?Cerebral ischemic stroke,is one of the most critically emergents of central nervous system,which is mainly presented with focal neurological deficits.Cerebrovascular congenital abnormalities or some of acquired factors(hypertension,hyperlipidemia,diabetes,etc.)cause cerebrovascular cavity stenosis,occlusion or embolism,resulting in the decline or even disruption of local or whole cerebral blood flow and then the occurrence of cerebral ischemic stroke.With high rate of morbidity,disability and mortality,cerebral ischemic stroke has been a serious threat to our health and life quality.Nowadays,some of reperfusion therapies,like acute thrombolysis,interventional thrombosis/stenting,or cerebrovascular bypass,are extensively applied in clinic.Although some of patients have gained satisfactory outcomes,ischemia-reperfusion injury has also been observed in clinic and compromised the therapeutic effects.Accumulating investigations have indicated that a variety of injury mechanisms,such as calcium overload,excitotoxicity,oxidative stress and endoplasmic reticulum stress,have been intensively involved in the cerebral ischemia-reperfusion injury.Although considerable investigations have been aimming to elucidate the injury mechanisms of cerebral ischemia-reperfusion injury and to find potential intervention targets,the current research situation is not optimistic.Capacity calcium entry(CCE)is an important regulatory mechanism in maintaining the intracellular calcium dynamic.By monitoring the calcium concentration in the endoplasmic reticulum,CCE mediates the influx of calcium,replenishing intracellular calcium or calcium store.The CCE complexes compose of stromal interaction molecule(STIM),calcium release activated calcium channel(CRACs)and some of regulatory proteins.CCE is triggered by some distinct stress conditions,like oxidative stress,calcium store depletion and intracellular acidification.Some investigations have also found that CCE is intensively involved in the occurrence and development of some acute and chronic central nervous system disease.As mentioned above,both oxidative stress and calcium overload are important injury mechinisms of cerebral ischemia-reperfusion injury.Whether CCE has some roles in the occurrence of cerebral ischemia-reperfusion injury,is not yet known and needs to be clarified.?Purposes?This study has been designed to clarify a full picture of(1)the expression changes and role of CCE in cerebral ischemia-reperfusion injury,(2)the neuroprotection mechanism of CCE regulation,(3)some potential molecules regulating CCE.?Methods?1.Oxygen-glucose deprivation & reperfusion(OGD/Rep.)model was used to mimic cerebral ischemia-reperfusion injury in vitro.Glutamate-induced oxidative stress model in HT-22 cells was been applied to studied the interaction between Homer1 a and CCE.2.The mRNA and protein levels of CCE constituents,apoptosis and autophagy were detected by using real-time quantitative PCR and Western Blot(WB)assay,respectively.3.The neuronal injury was evaluated by detecting the release of Lactate dehydrogenase(LDH).The neuronal apoptosis was detected by using terminal dUTP nick end labeling(TUNEL)assay.4.Fluorescent protein labeled plasmids and immunofluorescence staining were used to visualize the spatial localization of CCE constituents.5.The levels of STIM1,STIM2 and Homer1 a protein were regulated by using lentivirus-mediated short hairpin RNA(shRNA)or CRISPR/Cas9 technique.6.The levels of autophagy and autophagy flux were visualized by transfection of LC3-GFP and mRFP-GFP-LC3 fluorescent protein plasmids.7.Some of inhibitors or agonists of CCE,calmodulin-dependent protein kinase II(CaMKII)or autophagy were used to regulate the activity of CCE,CaMKII or autophagy,respectively.8.Calsium indicators were used to monitor the CCE-mediated calcium influx and the change of cytoplasmic calcium concentration.9.Co-immunoprecipitation(co-IP)was used to observe the protein interactions.?Result?Section one: After treatment of OGD/Rep.,the changes of CCE constituents,including STIM1,STIM2,Orai1,Orai2 and TRPC1,were not significant in mRNA and protein levels;However,STIM1 puncta were increased significantly after OGD/Rep.treatment;Inhibition of CCE significantly reduced OGD/Rep induced neuronal calcium overload;Both down-regulation or knockout of STIM1 expression and using of CCE inhibitors significantly alleviated OGD/Rep.-induced neuronal damage,inhibited neuronal apoptosis,and promoted neuronal survival.These results indicated that CCE is involved in OGD/Rep.-mediated neuronal damage and inhibition of CCE have some of neuroprotective effects against cerebral ischemia-reperfusion injury.Section two: After treatment of OGD/Rep.,the autophagy activity was increased,but an impaired autophagic degradation was also observed;Activation of autophagy alleviated OGD/Rep.-mediated neuronal damage;Inhibition of CCE could activate neuronal autophagy,and improve impaired autophagic degradation;After treatment of OGD/Rep.,the phosphorylation level of CaMKII was increased significantly,and inhibition of CaMKII caused the decline of mTOR phosphorylation level and improved the activation of autophagy.After inhibition of CCE,the phosphorylation level of CaMKII and mTOR decreased significantly.There were no significant additive effects of CCE inhibition and CaMKII inhibition were observed.These results indicated that inhibition of CCE might not only promote autophagy through inhibiting the activtiy of CaMKII-mTOR,but also improve the impaired autophagic degradation.Section three: The protein levels of STIM1 and Orai1 did not change significantly,but the STIM1 puncta were significantly increased after glutamate treatment;Both downregulation or knockout of STIM1 expression and using of CCE inhibitors reduced oxidation stress-induced injury;Upregulation of Homer1 a expression significantly inhibited CCE-mediated calcium influx,intracellular calcium overload and mitochondrial oxidative stress injury;The protein interactions among STIM1,Homer1 a,and Orai1 were detected by co-IP assay;Upregulation of Homer1 a expression significantly inhibit glutamate-induced STIM1-Orai1 complex formation.The above results showed that inhibition of CCE could reduce neuronal oxidative stress injury,and Homer1 a could negatively regulate CCE through inhibiting the STIM1-Orai1 complex formation.?Conclusion?This study has indicated as follows:(1)CCE was involved in the injury mechanism of cerebral ischemia-reperfusion injury;(2)Inhibition of CCE played neuroprotective effects in cerebral ischemia-reperfusion injury and oxidative stress-mediated injury;(3)The neuroprotection of CCE inhibition might be exerted by the promotion of autophagy,the improvement of impaired autophagic influx and the inhibition of apoptosis;(4)Homer1a might be an endogenous negative regulating protein of CCE.The present study has further revealed the injury mechanism of cerebral ischemia-reperfusion injury,and also found an endogenous regulatory molecule of this injury mechanism,providing some new intervention targets of cerebral ischemic stroke.