ICSⅡ Protects Against PC12 Cell Damage Induced By Oxygen-glucose Deprivation And Reoxygenation And Explore Its Mechanism

Objective: To investigate ICS Ⅱ protects against PC12 cell damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.Methods: The oxidative stress injury model was induced by OGD 2 h/R 24 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,Control+ICS Ⅱ 50 μM group,OGD/R group,OGD/R+ICS Ⅱ 12.5 μM group,OGD/R+ICS Ⅱ 25 μM group,OGD/R+ICS Ⅱ 50 μM group,OGD/R+NAC 100 μM group.Cell viability and LDH leakage rate were measured by MTT assay and lactate dehydrogenase(LDH)ELISA kit,respectively.Moreover,reactive oxygen species reactive oxygen species(ROS)ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of Nrf2,Keap1,HO-1,NQO-1,SIRT3,IDH2,Bax,Bcl-2 and Caspase-3 were detected by Western blot analysis.Results: The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICS Ⅱ 50 μM alone(P < 0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blot results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and active-caspase-3 level,while the decrease in the expression of nucleus-Nrf2 and HO-1,NQO-1,SIRT3,IDH2,Bcl-2(P < 0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P < 0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasm-Nrf2,Keap1,Bax and the level of active-Caspase-3,whereas,it increased the expressions of nucleus-Nrf2,HO-1,NQO-1,SIRT3,IDH2 and Bcl-2(P<0.05).Conclusion: ICS Ⅱ reduced OGD/R-induced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.