title: The effect and underlying mechanism of Timosaponin B-II on RGC-5 necroptosis induced by hydrogen peroxide.
abstract: BACKGROUND
  Necroptosis is an important mode of cell death, which is due to oxidant stress accumulation. Our previous study indicated that oxidant stresses could be reduced by Timosaponin B-II (TBII), a kind of Chinese herb RhizomaAnemarrhenae monomer extraction. We wonder the possible effect of Timosaponin B-II, whether it can protect cells from necroptosis via reducing the oxidant stress, in RGC-5 following hydrogen peroxide (H2O2) insult.
  
  
  METHODS
  RGC-5 cells were grown in DMEM, the model group was exposed in H2O2 with the concentration of 300 μM, and the experimental group was pre-treated with Timosaponin B-II at different concentrations (1 μM, 10 μM, 100 μM and 1000 μM) for 24 hrs. MTT assay was carried out to measure the cytotoxicity of H2O2, MDA concentration assay was executed to evaluate the degree of oxidative stress, TNF-α ELISA Assay was used to measure the concentration of TNF-α, finally, the degree of necrosis were analyzed using flow cytometry.
  
  
  RESULTS
  We first constructed the cell injury model of necroptosis in RGC-5 upon H2O2 exposure. Morphological observation and MTT assay were used to evaluate the degree of RGC-5 death. MDA assay were carried out to describe the degree of oxidant stress. Annexin V/PI staining was used to detect necroptotic cells pre-treated with or without Timosaponin B-II following H2O2 injury. TNF-α ELISA was carried out to detect the TNF-α accumulation in RGC-5. Upon using Timosaponin B-II with concentration of 100 μM, the percentage of cell viability was increased from 50% to 75%, and the necrosis of cells was reduced from 35% to 20% comparing with H2O2 injury group. Oxidant stress and TNF-α was reduced upon injury which decreased the ratio of RGC-5 necroptosis.
  
  
  CONCLUSIONS
  Our study found out that Timosaponin B-II might reduce necroptosis via inhibition of ROS and TNF-α accumulation in RGC-5 following H2O2 injury.
authors.forename: San-Hong
authors.initials: SH
authors.lastname: Jiang
authors.forename: Lei
authors.initials: L
authors.lastname: Shang
authors.forename: Li-Xiang
authors.initials: LX
authors.lastname: Xue
authors.forename: Wei
authors.initials: W
authors.lastname: Ding
authors.forename: Shuang
authors.initials: S
authors.lastname: Chen
authors.forename: Ruo-Fei
authors.initials: RF
authors.lastname: Ma
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha, Hunan 410013, China. huangjufang@csu.edu.cn.
authors.forename: Ju-Fang
authors.initials: JF
authors.lastname: Huang
authors.forename: Kun
authors.initials: K
authors.lastname: Xiong
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000009:adverse effects / Q000378:metabolism*; D019668:Liliaceae / Q000737:chemistry*; D051379:Mice; D009336:Necrosis; D018384:Oxidative Stress / Q000187:drug effects*; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D027343:Rhizome; D012503:Saponins / Q000494:pharmacology*; D013256:Steroids / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D004365:Drugs, Chinese Herbal; D012503:Saponins; D013256:Steroids; D014409:Tumor Necrosis Factor-alpha; C523027:timosaponin B-II; D006861:Hydrogen Peroxide