Role Of Mitochondrial Respiratory Chain Inhibition On Propofol-induced Central Neurotoxicity In Hippocampus In Developing Rats

Part 1 Effect of Propofol on Mitochondrial Function of Primary Hippocampal NeuronsObjective:To investigate the effect of propofol on mitochondrial function of primary hippocampal neurons.Methods:Primary hippocampal neurons were cultured for 7 days in vitro were randomly divided into control group（C group）,Intralipid group（I group）,propofol 1μM group（P₁ group）,Propofol 10μM group(P₁₀ group)and propofol 100μM group(P₁₀₀ group)by pretreated with different concentration of propofol.Fresh maintenance medium,Intralipid,propofol 1μM,propofol 10μM and propofol 100μM were replaced with medium of C group,I group,P₁group,P₁₀ group and P₁₀₀ group,respectively.Primary hippocampal neurons were randomly divided into control group（C group）,Intralipid（I group）,and propofol 100μM for 3 h,6 h and 9 h（P₃,P₆ and P₉ group）by pretreated with different time of propofol.Fresh maintenance medium,Intralipid,propofol 100μM or 3 h,6 h and 9 h were replaced with medium of C group,I group,P₃ group,P₆ group and P₉ group,respectively.The ATP,ROS and Mitochondrial membrane potential were measured by ATP,ROS and JC-1 assay kit.The expression of Cleaved-Caspase-9,Cytochrome C,Bax,Bcl-2 and Apaf-1were determined by Western blotting.Results:（1）Concentration-dependent:Compared with C group,the content of ATP was decreased,the content of ROS was increased,the Mitochondrial membrane potential was decreased,the expression of Bcl-2 protein was decreased,and the expression of Cleaved-Caspase-9,Cytochrome C,Bax and Apaf-1 protein were increased in P₁₀ group and P₁₀₀ group（P<0.05）.Compared with P₁₀ group,the content of ATP was decreased,the content of ROS was decreased,the Mitochondrial membrane potential was decreased,the expression of Bcl-2 protein was decreased,and the expression of Cleaved-Caspase-9,Cytochrome C,Bax and Apaf-1 protein were increased in P₁₀₀ group（P<0.05）.（2）Time-dependent group:Compared with C group,the content of ATP was decreased,the content of ROS was increased,the Mitochondrial membrane potential was decreased,the expression of Bcl-2 protein was decreased,and the expression of Cleaved-Caspase-9,Cytochrome C,Bax and Apaf-1 protein were increased in P₃ group,P₆ group and P₉ group（P<0.05）.Compared with P₃ group,the content of ATP was decreased,the content of ROS was increased,the Mitochondrial membrane potential was decreased,the expression of Bcl-2protein was decreased,and the expression of Cleaved-Caspase-9,Cytochrome C,Bax and Apaf-1 protein were increased in P₆ group and P₉ group（P<0.05）.Compared with P₆ group,the content of ATP was decreased,the content of ROS was increased,the Mitochondrial membrane potential was decreased,the expression of Bcl-2 protein was decreased,and the expression of Cleaved-Caspase-9,Cytochrome C,Bax and Apaf-1 protein were increased in P₉ group（P<0.05）.Conclusions:Propofol has a damaging effect to mitochondria with a time dependent and concentration dependent manner in hippocampal of neonatal rats.Part 2 Role of Mitochondrial Respiratory Chain of Propofol-Induced Central Neurotoxicity in Hippocampus in Developing RatsObjective: To investigate the effect of mitochondrial respiratory chain of propofol-induced central neurotoxicity in hippocampus in developing rats.Methods: Primary hippocampal neurons were cultured for 7 days in vitro were randomly divided into control group（C group）,Intralipid group（I group）,0.25% dimethyl sulfoxide group（D group）,propofol 100 μM group（P group）,Rotenone 0.1 μM group（R group）,Coenzyme Q10 10 μM group（Q group）and propofol 100 μM + Coenzyme Q10 10 μM group（PQ group）.Fresh maintenance medium,Intralipid,0.25% dimethyl sulfoxide,100 μM propofol,0.1 μM Rotenone and 10 μM Coenzyme Q10 were replaced with medium of C group,I group,D group,P group,R group and Q group,respectively.The cells in Group PQ were incubated with 10 μM Coenzyme Q10 before 100 μM propofol exposure.The ATP,ROS and Mitochondrial membrane potential were measured using ATP,ROS and JC-1 assay kit.The expression of Cleaved-Caspase-9,Cytochrome C,Bax,Bcl-2 and Apaf-1were determined by Western blotting.Results: Compared with C group,the content of ATP was decreased,the content of ROS was increased,the Mitochondrial membrane potential was decreased,the expression of Bcl-2 protein was decreased,and the expression of Cleaved-Caspase-9,Cytochrome C,Bax and Apaf-1 protein were increased in P group and R group（P <0.05）.Compared with P group,the content of ATP was increased,the content of ROS was decreased,the Mitochondrial membrane potential was increased,the expression of Bcl-2 protein was increased,and the expression of Cleaved-Caspase-9,Cytochrome C,Bax and Apaf-1 protein were decreased in PQ group（P <0.05）.Conclusions: Propofol has a damaging effect by inhibiting the mitochondrial respiratory chain,and induced neuroapoptosis in hippocampal of neonatal rats.