Effects Of Inhibition Of CaMKⅡα On Oxidative Stress In Cerebral Ischemic Reperfusion Rats And Related Mechanism Research

Stroke is a major emergency disease with high morbidity,mortality and recurrence rate.The number of stroke patients in China is the highest in the world,and ischemic stroke accounts for about 70-80%.The cardinal therapeutic goal of ischemic stroke is to restore blood supply in time,open the blocked blood vessels,and achieve reperfusion of ischemic brain tissue.Sudden recovery of blood supply might lead to more serious injuries,namely cerebral ischemia-reperfusion(I/R)injury.During the cerebral ischemia reperfusion phase,many pathophysiological changes occur,such as,calcium overload,free radical damage.During calcium overload,the Ca2+concentration in neurons increases,and then Ca2+in combination with calmodulin(CaM)can activate the Ca2+dependent enzyme(such as CaMK II,nitric oxide synthase).Among them,the Ca2+/calmodulin dependent protein kinase Ⅱ(CaMK Ⅱ)is an important member of the calcium/calmodulin activated protein kinase family,and it is a serine/threonine protein kinase.CaMK Ⅱ α is an important subtype of CaMK Ⅱ,highly expressed in the neurons,and plays an important role in regulation of neuronal death and survival.After the occurrence of ischemic stroke,brain tissue ischemia and hypoxia,ATP synthesis disorder,increased intracellular calcium ion concentration,mitochondrial depolarization,and insufficient antioxidant protection mechanism in cells lead to the accumulation of a large number of reactive oxygen radicals,which are toxic to cells and attack the body,namely oxidative stress.Previous study showed that during ischemia reperfusion,in addition to calcium overload,ROS may also directly activate CaMK II,which makes its substrate phosphorylation and its phosphorylation and involved in oxidative stress injury.After CaMK Ⅱ is activated,phosphorylation type CaMK Ⅱ(p-CaMK Ⅱ)increases.In order to restore basic inactivation,protein phosphatase is needed for dephosphorylation,and protein phosphatase itself also is controled by oxidative stress.Oxidative stress can participate in the activation of CaMK Ⅱ by several ways,and then participate in the process of cell damage in the form of its phosphorylation(p-CaMK Ⅱ).In the process of oxidative stress,reduced glutathione(GSH)is the main antioxidant molecule in the body.It plays a very important antioxidant role in preventing and reducing cell damage caused by reactive oxygen species such as free radicals and peroxides.The reduction of GSH mainly depends on the level of reduced nicotinamide adenine dinucleotide phosphoric acid(NADPH),Glucose 6-phosphate dehydrogenase(G6PD)is the rate-limiting enzyme in the pentose phosphate pathway,which can mediate the production of NADPH and play an antioxidant role by maintaining the reduction state of GSH.During ischemia-reperfusion,reactive oxygen species is accumulated,and lead to increased consumption of GSH and NADPH and aggravation of oxidative stress,which can trigger compensatory increase of G6PD expression,which plays an important role in maintaining the balance of intracellular REDOX state.Oxidative stress is involved in activating CaMK Ⅱ phosphorylation,with p-CaMK Ⅱ forms involved in cellular damage,studies have shown that inhibition of CaMK Ⅱ can relieve oxidative damage through the balance between oxidant and antioxidant,G6PD expression level has an important effect in oxidative stress,and oxidative stress has intimate connection with them.so we hypothesized that CaMK Ⅱ α and G6PD may exist interaction in the process of oxidative stress in cerebral ischemia reperfusion,and there is no relevant research reports till now,which provides a direction for us to explore CaMK Ⅱ inhibitors in the role of cerebral ischemia-reperfusion mechanism.Based on the above theoretical basis,in this study,firstly we establish the rat model of middle cerebral artery occlusion(MCAO)which realize the cerebral ischemia reperfusion injury through the line switch method,and then in different time after ischemia-reperfusion,detect the change of REDOX indicators,the change of CaMK Ⅱ α,p-CaMK Ⅱ α,G6PD levels,and the expression location of p-CaMK Ⅱ αand G6PD.Secondly,neuronal ischemia reperfusion-oxygen glucose deprivation(OGD)model is set up,and CaMK Ⅱα siRNA transfect hippocampal neuronal cells of mice in vitro to suppress the expression of CaMK Ⅱα,and detect the effect on ischemia reperfusion of neurons and the expression of G6PD after CaMK Ⅱαinhibited.Then we injected CaMK Ⅱα siRNA transfection complex through lateral intraventricular,MCAO model is constructed,we assess the nerve function defect score and cerebral infarction volume,detect the gene silence of CaMK Ⅱα and REDOX indicators and G6PD levels after inhibition of CaMK Ⅱα,and then analysis the effect of CaMK Ⅱα inhibition on oxidative stress in cerebral ischemia reperfusion and the expression of G6PD in this process.Finally,we inject G6PD siRNA transfection complex through lateral intraventricular,MCAO model is set up,we detect the change of REDOX indicators and the level of CaMK Ⅱα to verify the effect of G6PD on oxidative stress during cerebral ischemia reperfusion and explore the relation between G6PD and CaMK Ⅱα.The aim of this study is to explore the effect of CaMK Ⅱα inhibition on oxidative stress in ischemia-reperfusion rats and possible mechanism,and provide the experimental and theoretical reference for the application of the CaMK Ⅱ inhibitors in the clinical treatment of ischemic stroke.This project includes the following four parts:1.The change of REDOX index and the expression of CaMK Ⅱα and G6PD in the middle cerebral artery occlusion model of rats;2.The effects of transfection CaMK Ⅱα siRNA in vitro on ischemia reperfusion neurons and the expression of CaMK Ⅱα and G6PD.3.The effects of CaMK Ⅱα inhibition in vivo on oxidative stress and the change of G6PD expression in cerebral ischemia reperfusion;4.The effects of G6PD inhibition in vivo on oxidative stress and CaMK Ⅱαexpression in cerebral ischemia reperfusion.The first chapter:The change of REDOX index and the expression of CaMK Ⅱα and G6PD in the middle cerebral artery occlusion model of ratsMethod:1.The MCAO model of rats was established,and then 35 rats were grouped into the sham operation group(n=5),the MCAO group 3 hours after MCAO(3h;n=5),MCAO group after 6 hours(6h;n=5),MCAO after 12 hours(12h;n=5),MCAO after 24 hours(24h;n=10)and MCAO after 48 hours(48h;n=5)according to different time points after ischemia reperfusion.2.The REDOX indicators represented by ROS content,GSH/GSSG ratio and NADPH/NADP+ratio were detected by the corresponding test kit in the infarcted cerebral cortex of each group at different time points after ischemia-reperfusion.3.By immunofluorescence staining to display the localization of p-CaMK Ⅱαand G6PD after cerebral ischemia reperfusion.Western Blot method was used to detect the level of CaMK Ⅱα,p-CaMK Ⅱα and G6PD.Finally,we analyzed and compared the change trend of REDOX indicators and the dynamic change of CaMKⅡ α,p-CaMK Ⅱα and G6PD after cerebral ischemia reperfusion.Result:1.Compared with the sham operation group,the ROS content in each group after MCAO operation was significantly increased(p<0.05),and it was progressively increased with the prolongation of the time of ischemia-reperfusion.The ratio of GSH/GSSG and NADPH/NADP+decreased significantly(p<0.05),and showed a progressive decline over time.The imbalance of oxidation and anti-oxidation was progressively aggravated within 48 hours after cerebral ischemia-reperfusion.2.P-CaMK Ⅱα and G6PD were mainly expressed in the neurons and astrocytes in the cortices of the rats after MCAO.3.Compared with the sham operation group,the level of CaMK Ⅱα expression had no obviously difference(p>0.05),the level of p-CaMK Ⅱα and p-CaMK Ⅱα/CaMK Ⅱα were increased(p<0.05),the expression of G6PD increased(p<0.05),and gradually increased over time in each group after MCAO operation.After cerebral ischemia reperfusion,oxidative stress activated CaMK Ⅱα and the phosphorylation increases,at the same time the expression of G6PD was triggered to compensatorily increase.The second chapter:The effects of transfection CaMK Ⅱα siRNA in vitro on ischemia reperfusion neurons and the expression of CaMKⅡα and G6PDMethod:1.Hippocampal neurons of HT22 mice were cultured in vitro,and the purity of Hippocampal neurons was determined by immunofluorescence cytochemistry on the 5th day of cell culture.2.SiRNA was designed and synthesized,and transfection concentration gradients were designed during the transfection of neurons in vitro:10nM,50nM and 100nM.Real-time PCR was performed 24h after transfection to determine the final transfection concentration.3.CaMK Ⅱα siRNA transfection in vitro was conducted after cell cultured on day4(corresponding to Scramble siRNA as a negative control),ischemia reperfusionoxygen glucose deprivation(OGD)model was builded after cell cultured on day5.Four groups were divided:control group,OGD group,OGD+negative control group,OGD+CaMK Ⅱα siRNA group.The OGD model exchanged liquid for oxygen as a starting point,12h after reoxygenation,the necrosis of neurons were detected,and 0h,12h,24h,48h after reoxygenation,Western blot was used to detect the level of CaMK Ⅱα,p-CaMK Ⅱα and G6PD,real-time PCR was used to detect the level of CaMK Ⅱα mRNA and G6PD mRNA.Result:1.Immunofluorescence cytochemistry showed high purity of hippocampal neurons of HT22 mice cultured in vitro;2.The transfection efficiency raised with the increase of concentration of transfection during hippocampal neurons transfected with CaMK Ⅱα siRNA in vitro.There was no statistically significant difference between 50 nM and 100 nM,and 50 nM was selected for final transfection concentration.3.12h after reoxygenation,the rate of neuronal necrosis in OGD group was significantly higher than that in control group(p<0.05),and the model was successfully established.Compared with OGD+negative control group,the rate of neuronal necrosis in OGD+CaMK Ⅱα siRNA group was significantly lower(p<0.05),there was no significant difference between the OGD+negative control group and OGD group(p>0.05).Inhibition of CaMK Ⅱα had a protective effect on neurons in ischemia-reperfusion injury.4.0h,12h,24h,48h after reoxygenation,compared to OGD group,OGD+negative control group and the control group,the expression level of CaMK Ⅱα had no significantly difference(p>0.05),but the phosphorylation of CaMK Ⅱα increased significantly(p>0.05).Compared with OGD+negative control group,in OGD+CaMKⅡα siRNA group,0h,12h,24h after reoxygenation,the expression and the phosphorylation of CaMK Ⅱα decreased obviously(p<0.05),48h after reoxygenation,the expression and the phosphorylation of CaMK Ⅱα had no significant difference(p>0.05).CaMK Ⅱα siRNA transfection in vitro can inhibit the expression of CaMKⅡα and reduce its phosphorylation in the short term.5.12h,24h and 48h after reoxygenation,the expression level of G6PD in the OGD group was significantly higher than that in the control group(p<0.05),and it increased gradually with the time prolongation.Compared with OGD+negative control group,in OGD+CaMK Ⅱα siRNA group,0h,12h,24h after reoxygenation,the expression level of G6PD increased significantly(p<0.05),48h after reoxygenation,the expression level of G6PD had no significantly difference(p>0.05).The expression of G6PD increased after ischemia-reperfusion,and the inhibition of CaMK Ⅱα can further increase G6PD the expression in the short term.The third chapter:The effects of CaMK Ⅱα inhibition in vivo on oxidative stress and the change of G6PD expression in cerebral ischemia reperfusionMethod:1.Firstly we injected CaMK Ⅱα siRNA transfection complex(Scramble siRNA as a comparison)through lateral intraventricular,and then established the MCAO model.The 40 rats were randomly divided into sham operation group(n=10),MCAO group(n=10)and MCAO+Scramble siRNA group(Scr;n=10),MCAO+CaMK ⅡαsiRNA group(si-CaMK Ⅱα;n=10).All the rats were evaluated for neurological deficit score before sacrificed.The infarct volume was calculated by TTC staining after the rats were killed.2.We used Western Blot to detect the expression level of CaMK Ⅱα and p-CaMKⅡα,and real-time PCR to detect the level of CaMK Ⅱα mRNA,and observe the effect of CaMK Ⅱα gene silencing.3.The REDOX indicators were determined by the corresponding test kit.Western blot and real-time PCR were used to detect the expression of G6PD.We analyzed and compared the neurological deficit score,the infarct volume,REDOX indicators and the change of G6PD in each group.Result:1.Compared with the Scr group,the expression level of CaMK Ⅱα was significantly decreased(p<0.05)in si-CaMK Ⅱα group.CaMK Ⅱα siRNA transfection complex injection through lateral intraventricular can inhibit CaMK Ⅱαexpression.2.Compared with MCAO group and Scr group,the phosphorylation of CaMKⅡα decreased significantly(p<0.05),the neurological deficit score was obviously lower(p<0.05),the cerebral infarction volume significantly was reduced(p<0.05),ROS level was significantly decreased(p<0.05),GSH/GSSG ratio and NADPH/NADP+ratio were significantly increased(p<0.05),the expression level of G6PD was significantly increased(p<0.05)in si-CaMK Ⅱα group.Inhibition of CaMK Ⅱα can raise the expression level of G6PD,improve the imbalance between oxidation and antioxidant after ischemia reperfusion,reduce the phosphorylation of CaMK Ⅱα,improve the nerve function defect,reduce infarct volume,and play a neuroprotective role.The fourth chapter:The effects of G6PD inhibition in vivo on oxidative stress and CaMK Ⅱα expression in cerebral ischemia reperfusionMethod:1.In order to verify the impact of G6PD on oxidative stress and explore the relationship of G6PD and CaMK Ⅱα,we adopt the method of CaMK Ⅱα inhibition to inhibit G6PD expression.Firstly,the G6PD siRNA transfection complex(the corresponding Scramble siRNA was used as control)was injected into the lateral ventricle,and then the MCAO model was established.Twenty rats were randomly divided into three groups:sham operation group(n=5),MCAO group(n=5),MCAO+Scramble siRNA group(Scr;n=5),MCAO+G6PD siRNA group(SI-G6PD;n=5).2.We used Western Blot to detect the expression level of G6PD,and real-time PCR to detect the level of G6PD mRNA,and observed the effect of G6PD gene silencing.3.The REDOX indicators were determined by the corresponding test kit.Western blot was used to detect the level of CaMK Ⅱα and p-CaMK Ⅱα and real-time PCR was used to detect the level of CaMK Ⅱα mRNA.We analyzed and compared the REDOX indicators and the change of CaMK Ⅱα and the phosphorylation of CaMKⅡα in each group.Result:1.Compared with the Scr group,the expression level of G6PD significantly decreased(p<0.05)in si-CaMK Ⅱα group.G6PD siRNA transfection complex injection through lateral intraventricular can inhibit G6PD expression.2.Compared with MCAO group and Scr group,ROS was significantly increased(p<0.05)),GSH/GSSG ratio and NADPH/NADP+ratio were significantly decreased(p<0.05),the expression level of CaMK Ⅱα had no significant difference(p>0.05),but the phosphorylation of CaMK Ⅱα was significantly increased(p<0.05)in si-G6PD group.That means inhibition of G6PD expression can intensify the imbalance between oxidation and antioxidant,and increase the phosphorylation of CaMK Ⅱα.Conclusion:1.Within 48 hours after cerebral ischemia reperfusion,oxidative stress gradually aggravates,and activation of CaMK Ⅱα increases,at the same time the expression of G6PD was triggered to compensatorily increase by oxidative stress.2.After ischemia reperfusion,CaMK Ⅱα expression of neurons has no obvious change,but the phosphorylation increases.Transfection of siRNA designed characteristically for mice CaMK Ⅱα gene in vitro,could inhibit CaMK Ⅱαexpression and decrease the phosphorylation in the short term,which has a protective effect on ischemia-reperfusion injury of neurons.3.The expression of G6PD increases after ischemia reperfusion,and gradually increases with the extension of time trend.Inhibition of CaMK Ⅱα can obviously increase the expression of G6PD,and its rising level is significantly higher than that of G6PD expression after ischemia reperfusion.4.G6PD involved in oxidative stress,inhibition of G6PD expression can aggravate the oxidative stress after cerebral ischemia reperfusion,increase the phosphorylation of CaMK Ⅱα,and aggravate nerve damage.While inhibition of CaMK Ⅱα expression can relieve oxidative stress by raising G6PD expression,reduce the phosphorylation CaMK Ⅱα,alleviate nerve damage,and has obvious nerve protective effect.