Study On Protection Of (R)-α-Lipoic Acid Against Mitochondrial Membrane Potential Damage Induced By Hydroquinone In V79 Chinese Hamster Cells

PrefaceIn recent years, with the improvement of people's living standards, interior decoration and decoration are very common, so that people become concerned about indoor pollution problems. Compared with adults, children are in rapid growth period, and indoor pollution on children and teenagers became more serious health hazard. These years, children suffering from respiratory diseases in particular, the incidence of asthma is increasing year by year, and the reason for this may increase the degree of environmental pollution, indoor and outdoor air containing the dust, carbon dioxide, formaldehyde, benzene, ammonia and smoke cigarettes, etc. However, the mechanism of lung injury caused by these pollutants is not yet very clear. In this study, Hydroquinone (HQ) is used as the poison which widely exists in the paint, decoration materials and is the main metabolites of benzene, to study the mechanism of the toxicity of lung tissue, and to explore effective prevention and therapy.Hydroquinone which is the main metabolites of benzene widely exists in nature and it is also one main component of cigarette smoke and tar. However, the mechanism of damage by hydroquinone is unclear. It is known that mitochondrion was the target of cell injuries, especially the oxidative damage. The damage of mitochondrion is one of pathomechanisms which can induced many diseases. R-α-Lipoic Acid(LA) is one of antioxidants which is widely used in prevention and therapy of diabetes mellitus. In order to clarify the mechanism of injuries by hydroquinone, MTT and JC-1 are used to observe the effects of different HQ concentrations on cell viability and mitochondrial membrane potential of V79 Chinese Hamster cells. At the same time, we also explore different LA concentrations intervened in such damages. We try to offer theoretical basis for such damages about benzene or smoking from happening and protecting people's health.Objective1,To investigate the toxic effects of HQ with different concentrations on V79 cells.2,To explore mitochondrial membrane potential damage of V79 cells by different doses of HQ exposure.3,To study the intervention of the LA with suitable concentrations in such damage and protection mechanism.Materials and methods1,MaterialsV79 Chinese Hamster Cells, HQ, LA, DMEM, Fetal bovine serum, Trysin-EDTA, JC-1, MTT.2,Methods(1) Cell CultureThe V79 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin, and 100μg/mL streptomycin. Cell cultures were maintained at 37℃in a humidified atmosphere of 5% CO₂. The medium was changed every 1 or 2 days.The V79 cells were seeded at 10⁵ cells/ml in 96-well plate, 6-well plate and culture bottles. After being cultured for 24h, the V79 cells were maintained in DMEM supplemented with 1% fetal bovine serum. Then the cells were divided into different groups: (1)Control group. (2)HQ group: the concentrations of HQ was10, 20, 30, 40 50, 100, 150μmol/L. (3)LA group: the concentrations of LA was 50 and 100μmol/L. (4)LA+HQ group 1: the concentrations of LA was 50 and 100μmol/L; the concentrations of HQ was 50μmol/L. (5)LA+HQ group 2: the concentrations of LA was 50 and 100μmol/L; the concentrations of HQ was 100μmol/L. Then the cells were continue to be cultured for 24h.(2) MTT Assay for Cell Viability(3) JC-1 Assay for Mitochondrial Membrane Potential(4) Statistical analysisData was presented as the mean±SD of two or three separate experiments. Statistical significance was calculated with one-way ANOVA. P<0.05 was considered significant.Results1,Effects of HQ on cell viability in V79 cellsThe viability of the V79 cells was sharply decreased compared with the control when HQ was at 40μmol/L(P<0.05). It also has the dose-effect relation when HQ was 40 to 150μmol/L.2,Protective effect of LA on the HQ-induced decrease in cell viability in V79 cellsLA itself had no apparent effect on cell viability in the concentrations used (50 and 100μmol/L LA in V79 cells). The treatments of V79 cells with LA resulted in a significant protection against 50, 100μmol/L HQ induced toxicity when the concentrations of LA was 50 and 100μmol/L (P<0.05).3,Protective effect of LA on the HQ-induced decrease in Mitochondrial Membrane Potential in V79 cellsThe red-green JC-1 fluorescence ratio of V79 cells was 1.12. LA itself had no apparent effect on mitochondrial membrane potential in V79 cells at the concentration used (50μmol/L LA in V79 cells). Treatment with 50, 100μmol/L HQ decreased the red-green fluorescence ratio of JC-1 dye to 0.22 and 0.03 in V79 cells, respectively. Treatment with 50μmol/L LA resulted increased in the red-green JC-1 fluorescence ratio relative to 0.79 and 0.63 in 50, 100μmol/L HQ-treated cells, respectively.Conclusions1,The damage of mitochondrion in V79 cells maybe play a very important role of the mechanisms induced by HQ.2,A certain level of LA showed a protective effect on cyctoxicity and mitochondrial membrane potential damage induced by HQ in V79 cells.