Inhibitory Effect And Mechanism Of Insulin On Hydrogen Peroxide-induced Apoptosis In PC12 Cells

Objective:To establish hydrogen peroxide-induced PC 12 cell injury model,to discuss whether insulin has the function to inhibit apopsis,and to explore its mechanism.Method:(1)The establishment of hydrogen peroxide-induced apoptic model:culture PC 12 cells with various concentrations of hydrogen peroxide for 24 h,then cell viability was measured by MTT in order to determine the optimal damage concentration.(2)Protection experiment:After determining the concentration of damage,PC 12 cells were pretreated with different concentrations of insulin for 30min,then cultured with hydrogen peroxide for 24 hours.Cell viability by MTT and fluorescent staining by Hoechst 33342 determined whether insulin had the protection from hydrogen peroxide injury,at the same time to determine the optimal concentration of the drug.(3)Intracellular reactive oxygen species level,mitochondrial membrane potential and its ultrastructure were observed.(4)The change of Nrf2 in the cytoplasm and the nucleus were observed by immunohistochemistry and western blot experiments,meanwhile intracellular content of malondialdehyde(MDA),the activity of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and peroxidase(CAT)were detected by kits.Result:(1)It was revealed that hydrogen peroxide declined PC 12 cells viability in a concentration-dependent way.We chose the concentration of 600?mol/L hydrogen peroxide as a damage model.(2)600?mol/L hydrogen peroxide significantly induced apoptosis in PC12 cells,increased the rate of apoptosis and the level of intracellular ROS,declined mitochondrial transmembrane potential.Obvious mitochondrial damage was observed by electron microscopy.Antioxidant enzyme activities including SOD,GSH-Px,CAT decreased,lipid peroxidation was occured through the detection of MDA content.100nmol/L insulin can significantly inhibit hydrogen peroxide-induced PC 12 cells apoptosis,reduce the rate of apoptosis of PC 12 cells,and significantly inhibit hydrogen peroxide-induced decline of mitochondrial transmembrane potential and reduce the level of intracellular ROS and promote nuclear translocation of Nrf2,and enhanced SOD,GSH-Px,CAT activity,reduce the content of MDA.These protected the structural integrity of mitochondria and promoted cell viability.Conclusion:Insulin inhibited hydrogen peroxide-induced PC 12 injury.Insulin can improve cell viability,reduce the rate of apoptosis.Insulin strengthened the antioxidant enzyme activity by upregulating the expression of nuclear Nrf2,inhibited mitochondrial damage.These released insulin played a role in antagonizing hydrogen peroxide-induced apoptosis in PC12 cells,and its mechanism of action may protect mitochondria by Nrf2 signaling pathway activation.With the depth of insulin basic research,insulin will open up new avenues for the treatment of neurodegenerative disease.