Role And Mechanism Of Gap Junctions Formed By Cx43Protein In Rat Cerebral Ischemic Postconditioning

Objectives:1. To realize the influence of gap junctions regulated by kinase in cerebral ischemicpostconditioning on cerebral ischemia-reperfusion injury.2. To investigate the role of IP3, Caspase-9, Caspase-3and the ratio of Bax/Bcl-2inexpanded I/R injury by gap junctions.Methods:Thread occlusion method was used to make MCAO model. Neurological scores of ratwere evaluated by Longa’ score. Infarct volume of brain tissue was measured by TTCstaining. Histopathology of cerebral tissue was detected by HE staining. The expressionsof Cx43、Cx32、PKC、ERK1/2、p-PKC、Bax/Bcl-2、Caspase-3and Caspase-9proteinwere detected by Western Blotting. The expressions of Cx43mRNA in cerebral tissuewere detected by RT-PCR. The expressions of IP3in cerebral tissue were detected byElisa.Results:1. Neurological deficit scores showed that there was no neurologic change in sham group.In I/R group, neurologic deficit scores decreased significantly compared to the Shamgroup. Meanwhile, in IPO group, neurologic deficit scores increased extremely compared to I/R group. Similarly, in IPO+CBX group neurologic deficit scores increased obviouslycompared to IPO group.2. TTC staining showed that no infarct volume could be found in sham group. In I/Rgroup, infarct volume increased extremely compared to the Sham group. Meanwhile, inIPO group, infarct volume decreased significantly compared to the Sham group. Similarly,in IPO+CBX group infarct volume decreased obviously compared to IPO group.3. HE staining showed that no brain tissue pathological changes in sham group. In I/Rgroup, histopathological changes increased extremely compared to the Sham group.Meanwhile, in IPO group, histopathological changes decreased significantly compared tothe Sham group. Similarly, in IPO+CBX group histopathological changes decreasedobviously compared to IPO group. The results suggest that IPO has the protective effecton cerebral I/R injury and the protective effect of IPO could increased extremely byinhabition of GJ.4. Western blotting showed that in I/R group Cx43protein increased significantlycompared to the sham group. Meanwhile, in IPO group Cx43protein decreased extremelycompared to I/R group. Similarly, in IPO+CBX group Cx43protein decreasedsignificantly compared to IPO group. The expression levels of Cx32were no differenceand Cx26were lowly by western blotting.5. RT-PCR showed that the expression levels of Cx43mRNA were no different. Theresults suggest that IPO could decrease the expression levels of Cx43mRNA after Reversetranscription.6. Western blotting showed that in I/R group PKC protein decreased significantly compared to the sham group. Meanwhile, in IPO group PKC protein increased extremelycompared to I/R group. Similarly, in IPO+CBX group PKC protein increasedsignificantly compared to IPO group. In I/R group p-PKC protein increased significantlycompared to the sham group. Meanwhile, in IPO group p-PKC protein decreasedextremely compared to I/R group. The expression levels of ERK1/2were no difference.The results suggest that gap junction inhibition in the ischemic postconditioning isprobably related to the increased PKC protein expression.7. Western blotting showed that in I/R group ratio of Bax/Bcl-2protein increasedsignificantly compared to the sham group. Meanwhile, in IPO group ratio of Bax/Bcl-2protein decreased extremely compared to I/R group. Similarly, in IPO+CBX group ratioof Bax/Bcl-2protein decreased significantly compared to IPO group. In I/R groupCaspase-9protein increased significantly compared to the sham group. Meanwhile, inIPO group Caspase-9protein decreased extremely compared to I/R group. Similarly, inIPO+CBX group Caspase-9protein decreased significantly compared to IPO group. InI/R group Caspase-3protein increased significantly compared to the sham group.Meanwhile, in IPO group Caspase-3protein decreased extremely compared to I/R group.Similarly, in IPO+CBX group Caspase-3protein decreased significantly compared to IPOgroup. The results suggest that decreased apoptosis in IPO through GJ founctioninhibition is possibly related to the ratio of Bax/Bcl-2, Caspase-9and Caspase-3.8. Elisa showed that in I/R group IP3increased significantly compared to the sham group.Meanwhile, in IPO group IP3decreased extremely compared to I/R group. Similarly, inIPO+CBX group IP3decreased significantly compared to IPO group. The results suggest that decreased apoptosis in IPO through GJ founction inhibition is possibly related to thereduced IP3.Conclusions:1. Gap junction of Cx43inhibition in the ischemic postconditioning is probably related tothe PKC.2. Decreased apoptosis in IPO through GJ founction inhibition is possibly related to thereduced the ratio of Bax/Bcl-2, Caspase-9, Caspase-3and IP3.