| Objective To observe the changes of interleukin-1 receptor-associated kinase 1 (IRAKI) expression in cerebral cortex after cerebral ischemia reperfusion injury in rats and to explore its mechanism.Method 1. In vivo experiment: Male SD rats were randomly assigned to normal control group, sham group and ischemia group. According to the different reperfusion time, the ischemia group was divided into 6h,12h, 1d,3d,7d groups. Modified Pulsinelli four vessel occlusion method was used to built whole brain ischemia reperfusion model. Western blot, immunohistochemistry, immunofluorescence methods were used to measure IRAKI and Cx43 in cortex. Gap26 was injected intraperitoneally 1h before the model was made, and the expression of IRAKI was determined by Blot Western method.2. In vitro experiment:Cobalt chloride induced astrocyte neuron of ischemia model was established. According to the stimulation at different time points for 1h,3h,6h,12h,24h groups. Western blot method was used to determination of IRAK1 and Cx43 expression. SiRNA was used to remove IRAK1 in culture cells Cx43 expression were measured by Western blot.Result In vivo experiment:(1) Compared with the sham group, IRAK1 in cortex began to increase at 6h in rat cerebral ischemia after reperfusion injury (P< 0.05), and reach to peak at 3d (P< 0.05), there was statistically significant difference, then the expression decreased at 7d(P< 0.05). Cx43 protein expressed low in sham group. Compared with the sham operation group,Cx43 began to increase at 6h in rat cerebral ischemia and reperfusion (P> 0.05), reach to peak at 3d(P< 0.05), then decreased at 7d (P> 0.05),channel protein Cx43 and IRAKI expression coupling changes. (2) Immunohistochemical method was used to detect the IRAKI protein expression in cerebral cortex, it was found that IRAK1 protein express low in the cerebral cortex in the sham group. Compared with the sham group, Compared with sham group, the expression of IRAKI protein was significantly increased in the ischemic reperfusion injury, and the expression of protein was significantly different (P< 0.05) (3) Immunofluorescence was used to detect the IRAKI protein expression and localization,it was found that in sham group and ischemia reperfusion group, GFAP express (+) while IRAKI express (+), Neun express (+) while IRAKI express (-), IBA1 express (+) while IRAKI express (-).Compared with sham operation group, the expression of IRAKI protein was significantly increased in the cortex after global cerebral ischemia reperfusion injury, and the difference was significant (P< 0.05). (4) Using Gap26 to inhibit the expression of Cx43 protein, it was found that IRAKI expression also decreased compared with nonintervention group,, the difference was significant (P< 0.05).In vitro experiment:(1)IRAK1 protein expressed low in control group.Compared with the normal group, in CoCl2 stimulated astrocytes, IRAK1 began to increased significantly at 3h (P< 0.05), and reach to peaked at 6h (P< 0.05), which has statistical significance and expression gradually reduced,12 hours have been reduced to the normal level. At the same time, the expression of Cx43 was detected, and the expression of Cx43 was increased at 3h(P< 0.05), which has statistical difference,and reach to peak at 6h(P< 0.05), which was statistically significant compared with the normal group.(2) In the chemical ischemia model, the expression of Cx43 was decreased (P<0.05), which was statistical signigicant after inhibiting the expression of IRAK1 compared with that in the non suppressed group.Conclusion l.The expression of IRAK1 and Cx43 were increased in rats after global cerebral ischemia and reperfusion, and the trend of the two groups showed a change of coupling pattern.2.IRAK1 was mainly localized in astrocytes, and channel protein Cx43 was the most expressed in astrocytes.3.Using astrocytes to establish a chemical ischemia model, it was found that the inhibition of IRAK1 expression could inhibit the expression of Cx43, while using of specific channel blockers Gap26 to inhibit Cx43 expression could also inhibit the expression of IRAKI.4. The increased expression of IRAK1 in rats with cerebral ischemia reperfusion injury may be related to the pathological changes of Cx43. |
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