Protective Effect Of Resveratrol Against Oxidative Stress Induced By Formaldehyde On PC12 Cells

Objective: To observe the protective effect of Resveratrol against oxidative stress induced by formaldehyde on PC12 cells and the protective mechanism.Methods: Rat adrenal pheochromocytoma monoclonal lines (PC12) were cultured with 1640 in vitro and selected the cells in the exponential phase of growth to experiment. The cells were treated with different formaldehyde concentrations (10μmol/L, 20μmol/L, 40μmol/L, 80μmol/L, 160μmol/L) for 24h. To investigate the protective effect of Resveratrol (Res) on damages of PC12 induced by formaldehyde, the cells were divided into control group, 80μmol/L formaldehyde group, solvent group (2‰DMSO), aminoguanidine group (100μmol/L) and five Res groups (6.25μmol/L, 12.5μmol/L, 25μmol/L, 50μmol/L, 100μmol/L). Various concentrations Res were preincubated with cells for 2h, then detected indexes mentioned below. The viability of PC12 cells is detected by a colorimetric 3-[4, 5-dimethyl thiazol-2-yl]-2, 5 -diphenyltetrazolium bromide (MTT); The transudation content of lactate dehydrogenase (LDH) was observed by colorimetry reaction; The degree of PC12 cells injury was evaluated by measurement the content of malondialdehyde (MDA) in conditioned medium; the activity of superoxide dismutase (SOD) and the activity of glutathione (GSH-Px) in cells were measured by kits respectively. The level of nitric oxide(NO) and was determined by nitrate reductase method; The activity of NOS and iNOS was measured by biochemical approach; Collect each cell, Western blot,PCR was used to detect the effect of formaldehyde Cytochrome c oxidase subunitⅡprotein and mRNA expressed in PC12 cells; Apoptosis is measured by Hoechst 33258 fluorescence staining and Flow cytometry. Statistical analyses were performed with SPSS 13.0 statistical software. Data for each experimental group were subjected to analysis of variance (ANOVA). The data are expressed as the mean±standard deviation.Results: Formaldehyde could induce cell membrane injury. The level of LDH and MDA in FA groups were significantly increased in dose-dependent manner, compared with control (P<0.05). The activities of SOD and the level of GSH-Px in FA groups were decreased significantly, compared with control (P<0.05). With the increase of the concentration of formaldehy, there are more NO in cell culture media and the activity ability of NOS and iNOS in cell will increase as well. With the increasing of the's concentration of formaldehy, the expression of Cytochrome c oxidase subunitⅡprotein and mRNA was decreased step by step. When preincubated Res with cells for 2h, LDH, MDA, NO, NOS and iNOS were reduced; SOD, GSH-Px were raised; the expression of Cytochrome c oxidase subunitⅡprotein and mRNA was enhanced, compared with 80μmol/L FA group(P<0.05). Res can alleviate formaldehyde induced oxidative damages in dose-dependent manner by Hoechst 33258 fluorescence staining and Flow cytometry, apoptosis rate to decrease.Conclusion:1. Resveratrol protective for PC12 cells induced by formaldehyde oxidatie stress injuries. Resveratrol at a dose of 6.25~100 may be in dose-dependent manner.2. Formaldehyde can decrease release of Cyt C through ROS/oxidative stress,NOS/NO/free radicals ways, induced mitochondria-associated damage, resulting in PC12 cell apoptosis.3. Resveratrol treat can enhance the expression of COⅡprotein and decrease the release of Cyt C from mitochondria, thereby resveratrol can attenuate cell apoptosis and protect cell.