Protective effect of total flavone of puerarin against H2O2-induced oxidative damage of cultured PC12 cells
- VernacularTitle:葛根总黄酮对过氧化氢所致PC12细胞氧化损伤的防护
- Author:
Shuzhen SONG
;
Zhengyu FANG
;
Yaping TIAN
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2006;10(31):171-173
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Puerarin possesses various biological efficacies, such as the protective efficacy on hypertension, cardiac disease, diabetes mellitus and blood disease, and the extracts of puerarin can inhibit the proliferation of S180 sarcoma and Lewis lung cancer to some extent.OBJECTIVE: To investigate the modulation effects of total flavone of puerarin (TFP) on the growth of pheochromocytoma PC12 cells and the protective efficacy on the H2O2-induced cellular oxidative damage.DESIGN: Complete randomization design and control experiment.SETTING: Department of Biochemistry and Institute of Geriatrics, Chinese PLA General Hospital.MATERIALS: Puerarin was bought from Tongrentang drug store, and TFP was extracted and purified routinely by ethanol and ether acetate, then was evaluated with thin-layer chromatography. The content of puerarin in extracts was 31.79% in quantitative assay. Methyl thiazolyl tetrazolium (MTT), luminal and anti-oxidative activity reagent xanthine oxidase were all from Sigma Company. PC12 cells were given by Institute of Geriatrics,Chinese PLA General Hospital as a present.METHODS: The experiment was conducted at the Institute of Geriatrics, Chinese PLA General Hospital from January to July in·2001.① The cells were cultured in 20 g/L DMEM (pH 7.1-7.2), and randomized into two groups: TFP group and H2O2-injury+TFP group, and each group was divided into 5 mass concentrations (0, 1.0, 10, 100 mg/L and 1.0 g/L). There were 8 holes for parallel culture with 100 mL culture medium in each hole (containing 1 ×l09 L-1 cells). TFP group:TFP was added for 72-hour culture at 37 ℃; H2O2 injury+TFP group:TFP was firstly cultured for 48 hours at 37 ℃, then 500 mmol/L H2O2 was added and co-cultured for other 24 hours.②The activity of cultured PC12 cells was monitored by MTF assays, the content of nitrite was measured by Griess reagents, and the antioxidant activity of superoxidedismutase (SOD) was monitored by hypoxanthine/xanthine oxidase.H2O2-initiated PC12 cellular oxidative damage had been used as experimental model to study the protective efficacy of TFP, and expressed as inhibition ratio [(blank A value-detection A value)/blank A value × 100%]. The higher inhibition ratio indicated the strong ability of clearing O2-. ③ One-factor analysis of variance was used to compare the difference between data. MAIN OUTCOME MEASURES: The influence of TFP on the nitrite content, SOD activity and cell activity in PC12 cells.RESULTS: ①Effect of TFP on PC12 cell activity: 1-10 mg/L TFP hadno obvious effects on the growth of PC12 cells, and 100 mg/L TFP in creased the cell growth (P ＜ 0.05), whereas the TFP concentration was increased to 1.0 g/L, the activity of PC12 cells was inhibited obviously (P ＜ 0.01). TFP of 1-100 mg/L could protect the cultured cells from the oxidative damages of H2O2 concentration dependently (P ＜ 0.05).② Effect of TFP on clearing O2-: The ability of clearing O2 increased withthe mass concentrations of TFP in both groups with obvious dose-effect relation, except when 1 mg/L TFP was added in the H2O2 injury+TFP group. The SOD activity in PC12 cell culture liquid was obviously en hanced after adding 100 mg/L and 1 g/L TFP, compared with that without TFP addition (P ＜ 0.05-0.01). ③Modulation of TFP on nitrite: TFP of low concentration (1-100 mg/L) reduced the production of cellnitrite, whereas increased the nitrite production at the concentration of1.0 mg/mL.CONCLUSION: ①TFP can regulate the growth of PC12 cells, which canbe enhanced by low-concentration (1-100 mg/L) TFP whereas inhibited byhigh-concentration (1 g/L) TFP. However, the anti-oxidation of TFP is themost powerful. ②TFP can protect the PC12 cells obviously from the oxidative damages induced by H2O2 at low concentration.
