Effect And Possible Mechanism Of Gap Junction In Propofol Against Cerebral Ischemic/Reperfusion Injury

Objective:1. To investigate the effect and mechanism of gap junction in propofol against focal cerebral ischemia reperfusion injury in rats.Methods:1. A thread occlusion method was used to make focal cerebral ischemia reperfusion injury model. To screen the concentration of propofol against cerebral ischemic/reperfusion（I/R） injury, fifty adult male SD rats were randomly divided into sham group, I/R group, Propofol low-dose(I/R+P₂₅, 25mg/kg) group, medium-dose(I/R+P₅₀, 50mg/kg), high-dose(I/R+P₁₀₀, 100mg/kg) group. To research the mechanism of propofol against I/R injury, fifty adult male SD rats were randomly divided into sham group, I/R group, I/R+P₁₀₀ group, I/R+CBX group and I/R+P₁₀₀+CBX group. The neurological behavior was evaluated by Longa,scores. The cerebral infarction volume was measured by TTC staining. The expression of Cx43, PKC, Bax and Bcl-2 in brain tissue of rats was detected by Western Blot.2. The hypoxia/reoxygenation（H/R） injury model was obtained with primary cultured astrocytes. To screen the concentration of propofol against H/R injury, the study of astrocytes were divided into control group, hypoxia/reoxygenation（H/R） group, propofol low-dose(H/R+P₂₅, 25mg/kg) group, medium-dose(H/R+P₅₀,50mg/kg), high-dose(H/R+P₁₀₀,100mg/kg) group. To research the mechanism of propofol against H/R injury, the study of astrocytes were divided into control group, H/R group, H/R+P₁₀₀ group, H/R+P₁₀₀+RA group, H/R+P₁₀₀+CBX and H/R+P₁₀₀+GF group. The cell viability and apoptosis were detected by Methyl thiazolyl-tetrazolium（MTT） assay and Hochest 33258. The fluorescent tracer was used to evaluate the function of gap junction and the Western Blot was used to detect the expression of Cx43, PKC, Bax and Bcl-2 of astrocytes.Results:1. The results of the neurological behavior and TTC staining showed that compared with I/R group, the rats pretreated with moderate and high dose of propofol significantly reduced the neurological behavior scores and the cerebral infarction volume percentage in varying degrees except for I/R+P₂₅ group, and the better effect of propofol pretreatment is I/R+P₁₀₀ group. And CBX increased the protective effect of propofol against ischemia/reperfusion injury obviously.2. The results of expression of Cx43, PKC, Bax, Bcl-2 showed that compared with sham group, the protein expression of Cx43 and the Bax/Bcl-2 ratio were increased and the protein expression of PKC was reduced in I/R group. Compared with I/R group, administration of propofol high-dose significantly reduced the protein expression of Cx43 and the Bax/Bcl-2 ratio and increased the protein expression of PKC. And CBX improved the effect of propofol on changing the expression of protein.3. The results of the purity of the astrocytes showed that the purity of the cells was 96.3%±1.2%, and the cell cultures could be used in the next study.4. The results of cell viability showed that, compared with H/R group, administration of propofol significantly increased the cell viability except for propofol low-dose group and the better effect of propofol pretreatment is high-dose group. And, CBX increased the cell viability following propofol treatment. But, compared with H/R+P₁₀₀ group, the cell viability were decreased by RA and GF.5. The results of the function of gap junction in astrocytes showed that, compared with the control group, the function of gap junction（GJIC）in H/R group was increased. Compared with H/R group, administration of high-dose propofol significantly decreased the GJIC. And, CBX improved the effect of propofol on inhibition of GJIC. Then, compared with propofol high-dose group, the GJIC of cells were increased in H/R+P₁₀₀+RA group and H/R+P₁₀₀+GF group.6. The results of protein expression of Cx43 and PKC in astrocytes showed that compared with control group,the protein expression of Cx43 was increased and the protein expression of PKC was reduced in H/R group. Compared with H/R group, administration of propofol high-dose significantly reduced the protein expression of Cx43 and increased the protein expression of PKC. And those effects of propofol could be improved by CBX. Compared with H/R+P₁₀₀ group, the protein expression of Cx43 was increased and the protein expression of PKC was reduced in varying degrees in H/R+P₁₀₀+RA group and H/R+P₁₀₀+GF group.7. The results of cells apoptosis showed that, compared with control group,the cells apoptosis was increased in H/R group. Compared with H/R group, administration of propofol high-dose significantly reduced the cells apoptosis. And those effects of propofol could be improved by CBX. Compared with H/R+P₁₀₀ group, the cells apoptosis was increased in varying degrees in H/R+P₁₀₀+RA group and H/R+P₁₀₀+GF group.8. The results of protein expression of apoptosis-related protein Bax and Bcl-2 showed that, compared with control group, the Bax/Bcl-2 ratio was increased in H/R group. Compared with H/R group, administration of propofol high-dose significantly reduced the Bax/Bcl-2 ratio. And those effects of propofol could be improved by CBX. Compared with H/R+P₁₀₀ group, the ratio of Bax/Bcl-2 was increased in varying degrees in H/R+P₁₀₀+RA group and H/R+P₁₀₀+GF group.Conclusion:1. Propofol has protective effects on I/R injury and the better effect of propofol pretreatment is high-dose（100mg/kg） group.2. Inhibition of the gap junction via PKC signaling pathway may be involved in the protective effect of propofol against cerebral I/R injury.3. Propofol reduces the cell apoptosis of astrocytes after cerebral icchemia reperfusion injury, which may be associated with reducing the Bax/Bcl-2 ratio.