| Objective: To observe the expression of PAK1 and PAK3 in brain tissue of rats with cerebral ischemia-reperfusion(IR)injury at different time points.Methods: Adult male SD rats were randomly divided into normal group,sham operation group and model group(6 h,24 h,72 h,1 w,2 w after ischemia-reperfusion).The rat model of cerebral ischemia-reperfusion was established by middle cerebral artery occlusion(MCAO),and the degree of neurological deficit was evaluated by Zea-Longa score.The behavioral changes of rats were observed by balance beam test,the volume of cerebral infarction was measured by TTC staining,the pathological changes after cerebral ischemia-reperfusion injury were observed by HE staining,and the expression of PAK1 and PAK3 in each group was detected by Western blot.The expression sites of the two proteins were observed by immunohistochemistry.Results:1.Behavioral results: The Zea-Longa scores of normal group,sham operation group,IR 6h group,IR 24 h group,IR 72 h group,IR 1w group and IR 2w group were respectively(0)score?(0)score?(2.40±0.52)score?(2.30±0.48)score?(1.80±0.42)score?(1.10±0.57)score?(0.60±0.52)score;and the balance beam score were respectively(1.00±0.00)score?(1.00±0.00)score?(4.40±0.52)score?(4.20±0.42)score?(2.30±0.48)score?(1.80±0.42)score?(1.50±0.53)score?Zea-Longa score and balance beam score increased in different degrees in each subgroup of cerebral ischemia-reperfusion model,and the rats in each group were scored at the corresponding time point after operation.The results showed that the degree of neurological deficit decreased gradually with the prolongation of reperfusion time.Compared with 24 h ischemia-reperfusion group,the neurological deficit score decreased gradually at 72 h,1 w and 2 w(P<0.05).2.TTC staining results: The results of the TTC staining showed that infarct volume of normal group,sham operation group,IR 6h group,IR 24 h group,IR 72 h group,IR1 w group and IR 2w group were respectively(0)%?(0)%?(46.00±0.96)%?(47.03±1.62)%?(46.44±1.45)%?(47.23±2.18)%?(46.17±1.48)%?There were no infarcted foci in normal group and sham operation group,and infarcted foci were found in each ischemia-reperfusion model group,but there was no significant difference in the percentage of cerebral infarction volume in each group(P>0.05).3.HE staining results:After ischemia-reperfusion,the number of neurons in brain tissue decreased,the structure was disordered,the nucleus pyknosis,the size was different,and vacuoles could be seen around the cells.4.Western blot results:PAK1 and PAK3 were expressed at the basic level in the brain of rats in normal group and sham operation group.Compared with normal group and sham operation group,the expression of PAK1 protein decreased at 6 h,24 h and 72 h after ischemia-reperfusion,and began to increase at 1 w and 2 w after reperfusion,and there was no significant difference between the two groups and normal group and sham operation group(P>0.05).The expression of PAK3 was higher than that of normal group and sham operation group at 6 h after cerebral ischemia-reperfusion(P<0.05).With the prolongation of reperfusion time,the expression of PAK3 decreased gradually and decreased to the lowest level at 72 h after reperfusion,which was compared with that of normal group and sham operation group.The expression of PAK3 began to increase again at 1 week after reperfusion,which was significantly higher than that in normal group and sham operation group(P<0.05),and returned to baseline level 2 weeks after reperfusion.Compared with the normal group and sham operation group,There was no significant difference(P>0.05).5.Immunohistochemical results:Immunohistochemical results showed that PAK1 and PAK3 proteins were widely expressed in the brain,PAK1 protein was mainly expressed in the cerebral cortex,hippocampus CA1,DG region,and significantly expressed in the nucleus and cytoplasm.However,PAK3 was mainly expressed in hippocampal DG region,cerebral cortex and hippocampal CA1 region,and mainly expressed in cytoplasm.6.Correlation analysis: The gray ratio of PAK1/ ?-actin was negatively correlated with Zea-Longa score and balance beam score at each time point after cerebral ischemia-reperfusion in rats(rs=-0.559,rs=-0.539,P<0.05).However,there was no significant correlation between PAK3/ ?-actin gray ratio and behavioral score(rs=-0.117,rs=-0.063,P>0.05).Conclusion:After cerebral ischemia-reperfusion,PAK1 and PAK3 in the infarcted brain tissue of rats show dynamic changes,PAK1 decreases in the early stage of reperfusion,PAK1 increases in the late stage of reperfusion,while PAK3 increases at first and then decreases in the early stage of reperfusion,and then increases again in the late stage of reperfusion.The expression of PAK1 is negatively correlated with the behavior of rats at each time point after ischemia-reperfusion,suggesting that PAK1 may be involved in the repair process of cerebral ischemia-reperfusion injury. |
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