| Objective:In this experiment,a rat middle cerebral artery embolization model(MCAO model)was established by suture method,Then,ischemia-reperfusion,ischemic postconditioning are performed.The expressions of LC3-II,Beclinl and autophagy-related genes Atg5 and Atg7 in the cerebral ischemic penumbra were detected,and the expression of IncRNA LOC102550394,lncRNA LOC100363521,lncRNA Cistr-act,IncRNA cb707485,IncRNA RGD1564482 in the cerebral ischemic penumbra was detected.To study the possible interaction between autophagy and IncRNA in brain protection mechanisms after cerebral ischemia.Methods:1.A rat middle cerebral artery embolization model(MCAO model)was established by suture method,The group was subjected to ischemia-reperfusion,ischemic postconditioning are performed.Subsequently,the rat modified mNSS neurological function score and fresh whole brain specimen TTC staining were used to measure the infarct volume.2.The protein expression levels of LC3-? and Beclinl in the cerebral ischemic penumbra were detected by Western Blot.The gene expression levels of LC3-? and Beclinl were detected by Q-PCR.Immunohistochemistry was used to detect the expression levels of Atg5 and Atg7 in the cerebral ischemic penumbra.To study the neuroprotective effects of different infarct duration,ischemia-reperfusion and ischemic postconditioning treatment on the regulation of autophagy in rats with cerebral ischemia-reperfusion injury.3.Q-PCR was used to detect the expression level of IncRNA LOC100363521,IncRNA LOC100363521,IncRNA Cistr-act,IncRNA cb707485,lncRNA RGD1564482 in the cerebral ischemic penumbra.To investigate the expression of these five IncRNAs in brain tissue after different infarct duration,ischemia-reperfusion and ischemic postconditioning,To find out the possible interaction between autophagy and IncRNA,and further explore the mechanism of neuroprotection after ischemic postconditioning.Results:1.The MCAO model of male Sprague-Dawley rats was successfully established:the rats in the enrolled group were embolized to the contralateral limb hemiplegia.The enrollment condition was 7-12 points of the modified neurological deficit score(mNSS score)in rats,and it was confirmed by TTC staining that the brain tissue of the right middle cerebral artery in the rat was white and the normal brain tisue was red.The mortality of the MCAO model rats was high(31.25%),and the mortality of the rats in the 6h group was greater than 90%.2.The mNSS scores showed that compared with the I/R2h group,the rats in the IPostC2h group had lower neurological deficit scores and improved symptoms(P<0.05,the difference was statistically significant).Compared with I/R4.5h group,the,ats in the IPostC4.5h group had lower neurological deficit scores and improved symptoms(P<0.05,the difference was statistically significant).Compared with I/R2h group,the rats in I/R4.5h group had higher neurological deficit scores and worsened symptoms(P<0.05,the difference was statistically significant).Compared with the IPostC2h group,the rats in the IPostC4.5h group had higher neurological deficit scores and worsened symptoms(P<0.05,the difference was statistically significant).3.The results of TTC staining showed that the infarct volume of brain tissue in the IPostC2h group was smaller than that in the I/R2h group(P<0.05,the difference was statistically significant).The infarct volume of brain tissue in the IPostC4.5h group was smaller than that in the I/R4.5h group(P<0.05,the difference was statistically significant).The infarct volume of brain tissue in I/R4.5h group was greater than that in I/R2h group(P<0.05,the difference was statistically significant).The infarct volume of brain tissue in the IPostC4.5h group was greater than that in the IPostC2h group(P<0.05,the difference was statistically significant).4.The results of Western Blot showed that the protein expression levels of LC3-II and Beclinl in the cerebral ischemic penumbra of the I/R2h group were lower than those in the IPostC2h group,and the difference was statistically significant(P<0.05).The protein expression levels of LC3-II and Beclinl in the I/R4.sh group were lower than those in the IPostC4.5h group,and the difference was statistically significant(P<0.05).The protein expression levels of LC3-II and Beclinl in the I/R4.5h group were higher than those in the I/R2h,group,and the difference was statistically significant(P<0.05).The protein expression levels of LC3-II and Beclinl in the IPostC4.5h group were higher than those in the IPostC2h group,and the difference was statistically significant(P<0.05).5.The results of Q-PCR showed that the expression levels of LC3-II mRNA and Beclinl mRNA in the cerebral ischemic penumbra of the I/R2h group were lower than those in the IPostC2h group,and the difference was statistically significant(P<0.05).The expression levels of LC3-II mRNA and Beclinl mRNA in the I/R4.5h group were lower than those in the IPostC4.5h group,and the difference was statistically significant(P<0.05).The expression levels of LC3-II mRNA and Beclinl mRNA in the I/R4.5h group were higher than those in the I/R2h group,and the difference was statistically significant(P<0.05).The expression levels of LC3-II mRNA and Beclinl mRNA in the IPostC4.5h group were higher than those in the IPostC2h group,and the difference was statistically significant(P<0.05).Five IncRNAs screened were detected,but IncRNA LOC102550394 was not detected.The expression levels of lncRNA LOC100363521,lncRNA Cistr-act,IncRNA cb707485,IncRNA RGD1564482 were higher in the IPostC2h group than those in the I/R2h group-However,the expressions of IncRNA in the first three groups were statistically significant(P<0.05),the expression of IncRNA cb707485 was not statistically significant(P>0.05).The expression levels of IncRNA LOC100363521,IncRNA Cistr-act,IncRNA cb707485,lncRNA RGD1564482 were higher in the IPostC4.5h group than those in the I/R4.5h group.However,the expression difference of IncRNA LOC100363521 was statistically significant(P<0.05),the expression of the remaining three IncRNAs was not statistically significant(P>0.05).The expression level of IncRNA LOC100363521 was lower in the IPostC4.5h group than that in the IPostC2h group,and the difference was statistically significant(P<0.05).6.The results of immunohistochemistry showed that the expression levels of autophagy-related genes Atg5 and Atg7 in the cerebral ischemic penumbra of the IPostC2h group were lower than those in the I/R2h group,and the difference was statistically significant(P<0.05).The expression levels of Atg5 and Atg7 in the IPostC4.5h group were lower than those in the I/R4.5h group,and the difference was statistically significant(P<0.05).The expression levels of Atg5 and Atg7 in the I/R4.5h)group were higher than those in I/R2h group,and the difference was statistically significant(P<0.05).The expression levels of Atg5 and Atg7 in the IPostC4.5h group were higher than those in the IPostC2h group,and the difference was statistically significant(P<0.05).Conclusions:1.Ischemic postconditioning has protective effects on focal cerebral ischemia and hypoxia in rats.When blood flow is restored and reperfused,it can effectively reduce reperfusion injury,reduce the volume of cerebral infarction,and improve the symptoms of neurological deficit in rats after cerebral infarction2.With the prolongation of ischemia time,the infarct volume of rats and the mNSS scores increased significantly,the symptoms of neurological deficits also increased significantly,and the mortality of rats increased.3.Ischemic postconditioning provides a protective effect in ischemia-reperfusion injury of the brain by down-regulating the expression of autophagy-related genes Atg5 and Atg7,and reducing the level of autophagy(Confirmed by detecting the expression levels of LC3-? and Beclin1).4.Ischemic postconditioning may play a neuroprotective role by up-regulating the expression of lncRNA LOC100363521,lncRNA Cistr-act,lncRNA cb707485,lncRNA RGD1564482 to reduce the level of autophagy,but the specific molecular pathway remains to be further studied.5.With the prolongation of cerebral infarction time,the above protective effects of ischemic postconditioning are relatively weak.That is to say IncRNA regulates the level of autophagy in cells and exerts neuroprotective effects in the early stage of cerebral infarction. |
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