The Effect And Underlying Mechanism Of Timosaponin B-Ⅱ On RGC-5Necroptosis Induced By Hydrogen Peroxide

Objective:To study the protective effect and possible molecular mechanism of Timosaponin B-II, one of the single chemical extraction from rhizoma anemarrhenae, on the necroptosis of retinal ganglion cell line (RGC-5) induced by hydrogen peroxide.Methods:Cultured RGC-5cells were randomly divided into six groups:normal control (A group), H2O2treatment (B group),1μmol/L Timosaponin B-Ⅱ+H2O2treatment (C group),10μmol/L Timosaponin B-Ⅱ+H2O2treatment (D group),100μmol/L Timosaponin B-Ⅱ+H2O2treatment (E group),1000μmol/L Timosaponin B-Ⅱ+H2O2treatment (F group) groups. Cells of group C, D, E and F were pretreated with different dose of Timosaponin B-Ⅱ for24hours. Then, all groups except group A were treated with300μmol/L H2O2for24hours. The morphologic changes were detected by Inverted microscope. Necroptosis of RGC-5induced by H2O2treatment were labeled by double staining of Annexin V and PI and then detected by flow cytomctry. Cell viability was determined by a modified version of the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Amounts of malondialdehyde (MDA) in RGC-5were determined using MDA kits. The level of Tumor necrosis factor(TNF-alpha) was detected by ELISA assay.Results:60%of RGC-5was lost24hours after treatment of300μmol/L H2O2. flow cytometry detected lots of RGC-5labeled by Annexin V and PI after treatment of300μmol/L H2O2, suggesting necroptosis of RGC-5. pretreatment of1,10or100μmol/L of Timosaponin B-Ⅱ for24hours reduced RGC-5loss induced by H2O2. Especially,100μmol/L of Timosaponin B-II significantly decreased necroptosis of RGC-5induced by H2O2(p<0.05). Its protection on RGC-5was dose-dependent. In addition, Timosaponin B-Ⅱ significantly decreased the amount of MDA and TNF-αConclusion:Timosaponin B-Ⅱ could protect necroptosis of RGC-5induced by H2O2in a dose-dependent manner...