The Effect Of SIK2 On Cerebral Ischemia-Reperfusion Injury And Its Mechanism

Objective: Role of SIK2 in cerebral ischemia-reperfusion injury,Molecular mechanism of SIK2 involved in regulation of vascular regeneration after cerebral ischemia-reperfusion injury.Methods: To study the feasibility and effect of PeriCam PSI blood flow imaging perfusion system guiding the establishment of ischemia-reperfusion injury model in rats.The experimental rats were divided into normal group,ischemia 2 h group,reperfusion 3 h group,6 h group,12 h group,24 h group and 48 h group.Detection of Sik2 expression by Q-PCR and Western-blot.The construction protein overexpression animal by Intracerebral stereotaxic injection of adenovIrus were divided into follow groups: Control,Control+hSIK2,MCAO,MCAO+hSIK2,Detection of Cerebral Infarction in rats by ttc staining.Observation of brain edema and blood-brain barrier injury in rats by EB staining.Observation of collateral circulation vascular regeneration by immunofluorescence labeled cd31 in rats.Detection of SIK2,CREB,pCREB,VEGF expression in brain tissue of rats by western-blotting.Results:1.Establishment and Identification of Rat Model of Cerebral Ischemia-reperfusion injury assisted by PeriCam PSI blood flow imaging perfusion system.The blood perfusion in the brain and the distribution of blood vessels were clearly observed in the sham operation group,and the data of rats in the sham operation group were normal.In 2h ischemic group,the arterial flow was interrupted in the right cerebral artery,and the blood flow in the middle arterial blood supply was significantly decreased.After the recovery of 24 h,the artery in the right side of the brain was restored to blood flow,but the blood flow in the partial supply areadecreased,unable to recover to normal level.The TTC staining results indicated that there were obvious infarcts in the right brain tissue of the model group,while the sham operation group was normal.The results of pathological staining showed that the structure of brain tissue in the sham operation group was normal,and the morphological rules of nerve cells were not change.The ischemia brain tissue side of model group rats: we could found cortex and ischemia half dark stripe,nerve cell degeneration,necrosis and glial fiber disintegration,liquefaction,and it is light color,screen mesh in ischemic central area.2.The change trend of sik2 expression level during cerebral ischemia-reperfusion injury.Compared with Control group,the results of Q-PCR showed that the expression of sik2 began to decrease in rats with 2 h ischemia,but there was no statistical significance(P>0.05).The expression of SIK2 decreased significantly after reperfusion 3h(P<0.05).After 6 hours of reperfusion,it reached the lowest level(P<0.01),then increased gradually,and gradually approached the normal level after24 hours of reperfusion.At the same time,Western blot detection showed that the expression of SIK2 began to decrease at 2h after ischemia when compared with the normal group,(P<0.05).With the recanalization of cerebral embolism vessels,the expression of SIK2 decreased,and reached the lowest level after 6 hours of reperfusion(P<0.01),and then increased gradually.After 24 hours of reperfusion,it gradually approached the normal level.The results of two kinds of technical methods are basically the same.3.Construction and Identification of SIK2 overexpression Recombinant adenovirus transfected Animal Model.Western blot results show:The expression of SIK2 in brain tissue of Ad-hSIK2 group was significantly higher than that of Control group(P<0.01),However,the expression of SIK2 in brain tissue of Ad-GFP rats was not significantly different from that of control group(P>0.05).4.The effect of SIK2 expression on cerebral ischemia-reperfusion injury.The results of TTC staining in brain tissue showed: Brain tissue staining was red in Control and Control+hSIK2 groups,and no infarct was found.In MCAO group and MCAO+hSIK2 group,there were obvious white infarct foci in brain tissue,The percentage of cerebral infarction volume in MCAO+hSIK2 group(32.2 ±2.3)% was significantly higher than that in MCAO group(28.0 ±3.1)%,there is a statistical difference between the two group(P<0.05).The results of EB staining showed,compared with control rats,blood-brain barrier permeability was significantly increased in MCAO group and MCAO+hSIK2 group(P<0.05).The permeability of blood-brain barrier in MCAO+hSIK2 group was higher than that in MCAO group,and there was statistical difference between them(P<0.05).Immunofluorescence results show: compared with control group,vascular proliferation in ischemic area in MCAO group was significantly increased(p < 0.05),but there was no significant change in Control+hSIK2 group(P>0.05).Compared with MCAO group,vascular proliferation in ischemic area in MCAO+hSIK2 group was lower than that in MCAO group(P<0.05).5.Molecular Mechanism of SIK2 regulating collateral vessel Regeneration in Cerebral Ischemia-reperfusion injury.Correlation between SIK2,CREBb,pCREB and VEGF in rat brain tissue was detected by western-blotting.The results show that compared with Control group,the expression of SIK2 in brain tissue of MCAO group decreased significantly(P<0.01).However,the expression of pCREB,and VEGF was significantly increased.And there was no significant change in the expression level of CREB(P>0.05).When the expression of SIK2 in control+hSIK2 group increased significantly(P<0.05),the expression of CREB,pCREB and VEGF did not change significantly(P>0.05).Compared with MCAO group,the expression of sik2 in brain tissue of MCAO+hSIK2 group was higher than that of MCAO group(P<0.05),while the expression of pCREB,and VEGF showed a downward trend(P<0.05).And there was no significant change in the expression level of CREB.Conclusion:The MCAO rat model constructed by Pericam PSI blood flow imagerhas good stability and reliability.SIK2 is involved in the occurrence and development of cerebral ischemia-reperfusion injury.With the progression of disease,the expression level of SIK2 has a significant change trend.Overexpression of SIK2 in rat brain can aggravate cerebral ischemia reperfusion injury.During cerebral ischemia-reperfusion injury,SIK2 can regulate the expression of downstream target gene VEGF by CREB.The expression of SIK2 was negatively correlated with the expression of pCREB and VEGF,which affected collateral vessel regeneration.