Potective Effects Of Trilobatin And Its Potential Mechanism On Hydrogen Peroxide-Induced Oxidative Injury In PC12 Cells

Objective:To investigate the neuroprotective effect of trilobatin（TLB）and its underlying mechanisms were investigated in vitro using hydrogen peroxide（H₂O₂）-induced oxidative stress model in a neuron-like PC12 cell.Methods:The oxidative stress injury model was induced by H₂O₂ 48 h in PC12 cells.N-acetyl-lcysteine（NAC）,a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:Control group,H₂O₂ group,Control+TLB 60μM group,H₂O₂+TLB 15μM group,H₂O₂+TLB 30μM group,H₂O₂+TLB 60μM group,H₂O₂+NAC 20μM group,Control+AMPK inhibitor group,Control+TLB 60μM+AMPK inhibitor group,H₂O₂+AMPK inhibitor group,H₂O₂+TLB 60μM+AMPK inhibitor group,Nrf2 transfection group,Nrf2 transfection+TLB 60μM group,H₂O₂+Nrf2 transfection group,H₂O₂+TLB 60μM+Nrf2 transfection group,Nrf2 transfection negative control group.Cell viability and LDH leakage rate were respectively measured by MTT assay and lactate dehydrogenase（LDH）ELISA.Used Hoechst 33342 staining and flow cytometry were used to detect apoptosis,DCFH-DA,Mito-SOX Red and Rhodamine 123 were used to detect intracellular ROS,ROS in mitochondria and mitochondrial membrane potential（MMP）.Activity of Caspase 3/7,NAD⁺/NADH ratio and activity of mitochondrial enzyme and ATPase were detected by assay kit.In addition,the level of Sirt3 and mtDNA gene were detected by RT-PCR.Furthermore,the expressions of Cyto c,Sirt3,FoxO3a,SOD2,ac-SOD2,GPx-1,IDH2and UCP2 were detected by Western blot.Results:Compared with control group,H₂O₂ decreased the PC12 cell viability and increased leakage rate of LDH as well as the apoptosis rate,increased intracellular ROS and ROS in mitochondria,decreased MMP,decreased Bax/Bcl-2 in mitochondria,which was increased intracellular at the same time.In addition,H₂O₂ not only increased the expressions of Cyto c,the activity of Caspase-3/7 and the content of MDA,but also reduced the activities of GPx,IDH2,SOD2,ATP and the level of p-AMPK and the expressions of ERRα,Sirt3,IDH2,FoxO3a,ac-SOD2,UCP2,GPx-1.However,TLB significantly increased cell viability,decreased LDH leakage rate,apoptosis,intracellular ROS and ROS in mitochondria,as well as restored MMP.Notably,TLB increased intracellular Bax/Bcl-2 ratio and decreased Bax/Bcl-2 ratio in mitochondria the expressions of Cyto c and the content of MDA.In addition,TLB rised the activities of GPx,IDH2,SOD2,ATP,the level of p-AMPK and the expressions of ERRα,Sirt3,IDH2,FoxO3a,ac-SOD2,UCP2,GPx-1.Interestingly,Compound C,an AMPK inhibitor,or Nrf2 siRNA,which can knockdown Nrf2,almost abolished the beneficial effects of TLB on H₂O₂-induced neuronal cell injury.Conclusion:In conclusion,our findings demonstrate that TLB protects against oxidative injury in neuronal PC12 cells through regulating mtROS homeostasis,increasing activities of mitochondrial enzyme and regulating the AMPK/Nrf2/Sirt3signaling pathway.