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The Prooxidant Effect Of High Concentration Quercetin On HepG2 Cells And Its Potential Mechanism

Posted on:2017-04-17Degree:MasterType:Thesis
Country:ChinaCandidate:F T WenFull Text:PDF
GTID:2334330488982538Subject:Nutrition and Food Hygiene
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Oxidative stress induced by free radicals such as reactive oxygen is closely related to the development and progression of a variety of chronic disease of human body. As the representative of natural antioxidant function factor, quercetin can scavenge free radicals and increase the activity of antioxidant enzyme system function, which has positive significance on the regulation of the body's redox state.But with the further research, a growing number of studies have found that quercetin in high concentration, long time will increase the body ROS levels and decline antioxidant enzyme activity. So the study on mechanism of quercetin promotes cell oxidative stress is of great significance for more safe application of quercetin. This research use AAPH, 2,2'-Azobis( 2-amidinopropane) dihydrochloride, as oxidant to produce ROS stably and the HepG2 cells to set up oxidative stress model. To explore the influence and possible mechanism of effect on oxidative stress of cells of quercetin and their oxidation products at different concentrations, AAPH was mixed with quercetin in chemical reaction system to generate quercetin oxidation product.In this study, normal or AAPH treated Hep G2 cells were incubated with quercetin of different concentrations. Viable cells were harvested, and the cell viability, ROS level and redox related indicators were assayed. The results showed that, at low concentration(10 or less ?mol/L) and short period of time(24 h), quercetin can significantly improve the antioxidant enzyme activity, GSH content and GSH/GSSG ratio and cell survival rate, lower levels of cellular ROS and MDA content(P < 0.05), and have antioxidant effect on cells. But with the increase of the concentration and incubation time, cell survival rate quercetin dropped significantly, ROS levels MDA content significantly increased, cellular antioxidant index were significantly reduced(P < 0.05). And the ROS levels of AAPH treated cell group were of significantly higher than the normal cells. This may be related to AAPH accelerates the formation and accumulation of quercetin oxidation products in cells.To investigate whether quercetin oxidation products significantly promoted the cell oxidative stress more than quercetin, this study use AAPH and quercetin to generate quercetin-AAPH oxidation product under 37? in 12 h, the structure of the oxidation product was identificated and analysed by ultraviolet-visible wavelength scanning and HPLC-MS, and incubated HepG2 cells with the oxidation product, then determined the cell viability, ROS level, and redox related indicators. According to the results, the product of quercetin react with AAPH after 12 h had characteristic absorption at 294 nm, LC-MS analytical results show that the oxidation reaction system generate four main products,and the relative molecular mass of the products were 154, 170, 350 and 300. Compared with quercetin incubated cell group, 100 ?mol/L quercetin oxidation products significantly reduced the cell viability(P < 0.05), elevated ROS level and MDA level, reduce the T- AOC, CAT, GSH-px, SOD activity, GSH content and GSH/GSSG, the results show that high concentration quercetin oxidation product has a stronger role in promoting the free radicals generation of cell oxidative stress than quercetin itself, quercetin oxidation product may be the active form of quercetin promoting oxidation.To investigate the potential mechanism of oxidant products of quercetin in promoting oxidation, the antioxidant and prooxidant gene pathways expression were measured by qRT-PCR. It turned out that low concentration of quercetin or its oxidation products upregulated Nrf2, HO-1, NQO1, Prdx mRNA expression, while Keap-1, Nox-2 and Cox-2 mRNA level was downregulated, which was contrary to the effect of high concentration of quercetin at 100?mol/L. Besides, the products generated by quercetin showed the most severe oxidation effect on cells in our protocol, and individual processing of AAPH led to siginificant downregulation of Nrf2, HO-1 and NQO-1 mRNA expression compared with that of the control group.It indicates that quercetin mRNA expression of cell oxidation reduction related gene has significant differences under different incubation model, which it's probably related to the content of quercetin oxidation products in the cell under different incubation conditions.Above all, quercetin can protect cell from oxidation when in low concentration and short incubation time. But the cell viability rate will reduce and ROS level will elevate with the increase of concentration and incubation time, the protection of quercetin on cells transfers to prooxidant effect. Quercetin with high concentration may reduce the antioxidant activity of HepG2 cells and promote the oxidative stress by generating amount of oxidation products when scavenging free radical. The oxidation products accelerate cell oxidative stress development mainly by consuming GSH and down-regulating Nrf2 antioxidant signal pathway expression and autioxidant enzyme activity. Quercetin with high concentration has a certain prooxidant effect on cell, therefore, it needs more dose-response study of quercetin when it will be developed as a dietary supplement.
Keywords/Search Tags:quercetin, oxidation products, HepG2 cells, prooxidant effect, oxidative stress
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