Mechnism Of Cx36 And Pannexin-1 In Protective Effect Of NADPH Oxidase Inhibitior-apocynin Against Cerebral Ischemia Reperfusion Injury Of Rats

Objectives: 1. To determine the mechnism of Cx36 and Pannexin-1 in protective effect of NADPH oxidase inhibitior-apocynin against cerebral ischemia reperfusion injury of ratsMethods: 1. The middle cerebral artery occlusion(MACO) method was used to make focal cerebral ischemia reperfusion injury model. 60 male SD rats were randomly divided into four groups, including sham group, ischemia-reperfusion group(I/R group), ischemia-reperfusion + apocynin group(I/R+Apo group) and ischemia-reperfusion +apocynin+GF109203X group(I/R+Apo+GF group). Neurological scores were observed by Longa 5 methods, the brain infarct volume and the presence of apoptotic cells were detected by TTC and TUNEL staining. The expressions of Cx36, Pannexin-1, PKC, Bax, Bcl-2 were detected by Western Blotting. The expressions of ROS, SOD, MDA and GSH in brain tissue of rats were detected by Chemiluminescence, WST-1, TBA and micro ELISA method. 2. The primary cultured neurons went with hpoxia/reoxygenation injury model. Neurons of the study were divided into normal control group, hypoxia-reoxygenation group, hypoxia-reoxygenation+apocynin group(H/R+Apo group) and hypoxia-reoxygenation+apocynin+GF109203X group(H/R+Apo+GF group). Neuronal viability and the cell apoptosis were detected by Methyl thiazolyltetrazolium(MTT) assay and Hochest 33258; The function of gap junction were evaluated by fluorescent tracer and the expressions of Cx36, Pannexin-1, PKC, Bax, Bcl-2 and were detected by Western Blotting.Results: 1. The neurological scores, the percentage of infarct volume and the cell apoptosis in the brain tissues of rats were detected and the results show that compared with sham group, the percentage of infarct volume, neurological scores and the TUNEL-positive cells rate of rats were increased significantly in I/R group. Compared with I/R group, the percentage of infarct volume, neurological scores and the TUNEL-positive cells rate were decreased significantly in I/R+Apo group. Compared with I/R+Apo group, the percentage of infarct volume, neurological scores and the TUNEL-positive cells rate were increased significantly in I/R+Apo+GF group. 2. The protein expression of Cx36, Pannexin-1, PKC, Bax and Bcl-2 in the brain tissues of rats was detected and the results show that compared with the sham group, the protein expression of Cx36, Pannexin-1 and PKC were reduced and the Bax/Bcl-2 ratio was increased significantly in I/R group. Compared with I/R group, the Cx36, Pannexin-1 and PKC protein expression were increased and the Bax/Bcl-2 ratio was reduced significantly in I/R+Apo group. Compared with I/R+Apo group, the Cx36, Pannexin-1 and PKC protein expression were reduced and the Bax/Bcl-2 ratio was increased significantly in I/R+Apo+GF group. 3. The content of ROS, SOD, MDA and GSH in brain tissue of rats was detected and the results show that compared with control group, the content of ROS, MDA in brain tissue of rats increased significantly, while the content of SOD and GSH decreased in the I/R group. Compare with I/R group, the SOD and GSH levels were increased and the ROS, MDA content were reduced significantly in I/R+Apo group. Compared with I/R+Apo group, the ROS, MDA content were increased significantly, while the content of SOD and GSH were decreased in I/R+Apo+GF group.4. The neuronal viability and the cell apoptosis were detected and the results show that compared with control group, the neuronal viability was reduced and the cell apoptosis was increased significantly in H/R group. Compared with H/R group, the neuronal viability was increased and the cell apoptosis was reduced significantly in H/R+Apo group. Compared with H/R+Apo group, the neuronal viability was significantly reduced and the cell apoptosis was increased in H/R+Apo+GF group. 5. The function of gap junction was detected and the results show that compared with control group, the function of gap junction reduced significantly in H/R group. Compared with H/R group, the function of gap junction was increased significantly in H/R+Apo group. Compared H/R+Apo group, the function of gap junction was reduced significantly in H/R+Apo+GF group. 6. The protein expression of Cx36, Pannexin-1, PKC, Bax and Bcl-2 in the neurons was detected and the results show that compared with the control group, the protein expression of Cx36, Pannexin-1 and PKC were reduced and the Bax/Bcl-2 ratio was increased significantly in H/R group. Compared with H/R group, the Cx36, Pannexin-1 and PKC protein expression were increased and the Bax/Bcl-2 ratio was reduced significantly in H/R+Apo group. Compared H/R+Apo group, the Cx36, Pannexin-1 and PKC protein expression were reduced and the Bax/Bcl-2 ratioin was increased significantly in H/R+Apo+GF group.Conclusions: 1. The protective effect of NADPH oxidase inhibitior-apocynin on the cerebral I/R injury may be related to increasing the Cx36, Pannexin-1 protein expression. 2. Effect of NADPH oxidase inhibitior-apocynin on the Cx36, Pannexin-1 protein expression may be related to increasing the PKC kinase. 3. Inhibition of NADPH oxidase reduces cell apoptosis after cerebral I/R injury may be related to reducing the Bax/Bcl-2 ratio.