Effects Of Quercetin On Some Metabolic Pathways In Oxidatively Stressed Rat Hepatocytes

ObjectiveThe rat hepatocytes were cultured in serum-free culture medium and oxidative stress was induced by addition of H₂O₂. The regulation of some metabolic pathways by quercetin was investigated in hepatocytes under oxidative stresse in order to explore the mechanisms associated with the antioxidant action of quercetin.Methods1. The rat hepatocytes were isolated by in situ perfusion and cultured in serum-free culture medium;2. H₂O₂ was used to induce oxidative stress;3. The absorption of quercetin by hepatocytes was determined by high performance liquid chromatography (HPLC);4. The content of malondialdehyde (MDA) in cell homogenate, and the activity of lactate dehydrogenase (LDH) in culture medium were assayed with commercial reagent kits, and total antioxidative capacity (T-AOC) in culture medium was assayed by FRAP method;5. Flow cytometry (FCM) was used to measure the apoptosis rate of cultured hepatocytes;6. Nuclear magnetic resonance (NMR) technique was employed to analyze the metabonomic changes of cultured hepatocytes;7. The amino acid profiles in culture medium were measured by Amino Acid Analyzer based on ion exchange principle;8. The content of homocysteine (Hcy) in culture medium was determined by Automatic Biochemical Analyzer based on enzymatic cycling assay. Results1. The rat hepatocytes were cultured successfully in serum-free medium;2. After the hepatocytes were exposed to H₂O₂ (2.2, 4.4 and 8.8 mmol/L respectively) for 6h, the content of MDA, the activity of glutathione peroxidase (GSH-Px) in cell homogenate, and the activity of LDH and T-AOC in culture medium increased significantly, especially in 8.8mmol/L H₂O₂ treated group compared to the control. Thus, 8.8mmol/L was selected as the optimal concentration of H₂O₂ to induce oxidative stress;3. The hepatocytes were cultured in the medium containing 10, 20 and 30μmol/L quercetin for 12, 24 and 36h respectively. The absorption of quercetin by hepatocytes was confirmed, as the quercetin peak was identified on all chromatograms. The highest absorption of quercetin was found among the hepatocytes being treated with 20μmol/L quercetin for 24h. No quercetin peak was identified from the cell extracts of control. There were some unknown peaks appeared on the chromatograms;4. The hepatocytes were pretreated with 10, 20 and 30μmmol/L quercetin for 24h. Then, H₂O₂ (8.8mmol/L) was added to induce oxidative stress. The results showed that the LDH release was lowered significantly (P<0.05) in all quercetin pretreated groups as compared to the H₂O₂ group. The content of MDA decreased remarkably in quercetin pretreated groups (P<0.05) in comparison with the H₂O₂ group, but no significant difference was found between any quercetin groups and control. The T-AOC of quercetin groups was higher than that of control and H₂O₂ group (P<0.05);5. Compared to the control, the apoptosis rate increased remarkably (P<0.05) in the H₂O₂ group. With the pretreatment of quercetin, the apoptosis rate decreased significantly and was close to that of control. No difference was found between 20 and 30μmol/L quercetin groups;6. The results of 1H NMR analysis followed by partial least-squares discriminant analysis (PLS-DA) showed that different metabolic patterns were identified for different groups. The differences were attributed mostly by the 1H NMR signals atδ1.34, 1.38, 1.94, 2.38, 2.42, 2.74, 2.94, 3.18, 3.26, 3.46, 3.5, 3.86, 3.94, in which,δ1.34, 1.94, 2.38, 2.42, 2.74, 3.26 corresponded to lactate (Lac), acetate, pyruvate, glutamine (Gln), dimethylamine (DMA), and taurine (Tau) respectively, and the rest corresponding toδ1.38,2.94,3.18,3.46,3.5,3.86,3.94 remained to be identified. Compared to the control, Lac and pyruvate decreased, acetate and DMA increased significantly, Gln increased moderately in the quercetin pretreated group. Lac, acetate and Tau increased remarkably, and pyruvate and histidine (His) (δ7.05,7.76) decreased notably in H₂O₂ group. Lac, acetate, DMA and Tau increased distinctly, and Gln increased moderately while His and pyruvate decreased prominently in quercetin+H₂O₂ group. Among them, Lac, acetate and Tau in quercetin+H₂O₂ group increased more remarkably than in H₂O₂ group. The contents of isoleucine (Ile) (δ0.94,1.02), leucine (Leu) (δ0.95,0.97), valine (Val) (δ1.00,1.04), ethanol (δ1.18-1.20,δ3.65-3.68), tryptophan (Trp) (δ7.05,7.76) and phenylalanine (Phe) (δ7.33-7.34,7.41-7.45) were not changed significantly;7. The results of amino acid analysis indicated that the contents of Ala, Met and His decreased significantly, while Tau increased remarkably in H₂O₂ group and quercetin+H₂O₂ group compared to that of control. The changes in quercetin+H₂O₂ appeared more distinct than in H₂O₂ group;8. The content of Hcy in H₂O₂ group was higher than in control and quercetin+H₂O₂ group (P<0.05).Conclusion1. H₂O₂ in certain concentration range could induce oxidative stress in primary cultured rat hepatocytes;2. The rat hepatocytes could absorb quercetin to some extent. The absorbed quercetin could protect the hepatocytes against oxidative stress by decreasing the content of MDA, reducing LDH release, and lowering apoptosis rate; 3. Based on metabonomic analysis, quercetin could accelerate glycolysis and promote fatty acids oxidation, so as to provide more energy for the hepatocytes against oxidative stress. Quercetin could also affect the metabolism of some nitric compounds;4. The metabolism of sulfur amino acids seemed to be promoted by quercetin as reflected by the changes in the content of Met, Tau. It is postulated that the Met transsulfuration pathway in oxidatively stressed hepatocytes could be accelerated by quercetin, which may help protect hepatocytes against oxidative stress;5. Quercetin could decrease the yield of Hcy, one of the intermediate products in Met metabolism by oxidatively stressesd hepatocytes. Therefore, quercetin is effective in diminishing the damage generated by Hcy in oxidative stress.