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摘要
OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/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,control+ICS Ⅱ50 μmol·L-1,OGD/R,OGD/R+ICSⅡ 12.5 μmol·L-1,OGD/R + ICS Ⅱ 25 μmol·L-1,OGD/R + ICS Ⅱ50 μmol·L-1,and OGD/R+NAC 100 μmol·L-1 groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,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 nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO-1,NQO-1,silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting 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 ICSⅡ 50 μmol·L-1 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 blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO-1,NQO-1,SIRT3,IDH2 and 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 cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO-1,NQO-1,SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.
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