Effect and its underlying mechanism of antioxidant quercetin in the mice in vivo and NIH-3T3 cells in vitro
Cui-cui GONG; Nai-gang ZHENG; Jing-lan WU; Pei-xia HE; Yi-ling WANG
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Objective To compare the difference in quercetin against oxidative stress response between in mouse plasma and in H2O2-pre-/post-treated NIH-3T3 cells, and to explore the underlying mechanism for the quercetin antioxidant. Methods The cultured NIH-3T3 cells were randomly divided into 4 groups: quercetin (Q) pre-protective group (Qb) firstly treated with quercetin for 24h followed by incubation with H2O2 for 30 min; post- protective group (Qa) firstly treated with H2O2 for 30 min followed by incubation with quercetin for 24h; H2O2 group (H2O2) after exposure with H2O2 for 30 min, incubated with DMEM medium and the control group (C) only cultured with DMEM medium. The survival rate and apoptotic rate were detected respectively with MTT and TUNEL in NIH-3T3 cell suspension samples. The expression of cyclin D1, PTEN, NF-kB, HSP-70, Bcl-2, Bax and caspase-3 was examined with immunocytochemistry and immunoblotting. Besides, 20 Wistar rats were divided into control group and experimental group, the latter was given with quercetin in the doze of 0.13mmol/kg. The levels of T-AOC，SOD，GSH-Px，GSH，MDA, NOS and NO2-/NO3- were detected both in the cleaved NIH-3T3 cells in vitro and in the plasma in vivo from both experimental and control animals prior to and post-1 h, 2h and after 24 h. Results When the Qb group was compared with H2O2 or Qa group, the survival rate was higher and the apoptotic rate was lower. When the H2O2 group was compared with C group，the expression of cyclin D1、PTEN or Bcl-2 was down-regulated；while that of Bax、HSP-70、NF-kB or caspase-3 was up-regulated; the level of T-AOC，SOD，GSH-Px or GSH was decreased; that of NOS、NO2-/NO3- or MDA enhanced in the cleft NIH-3T3 cells. When the Qb group was compared with Qa or H2O2 group, the reversed H2O2 effects, especially elevated the decreased A-TOC and GSH-Px and depressed the enhanced MDA effects by H2O2, were more marked. When the plasma level of the anti-oxidative enzyme system prior to- compared with post-1h and 2h-treatment with Q, the level of T-AOC, SOD, GSH-Px and GSH, especially the former two, were higher; MDA, lower; NOS or NO2-/NO3- promoted. However, the above parameters basically became normal 24h after treatment with Q. Conclusions Quercetin could down-regulate the promoted expression of HSP70, NOS, NO2-/NO3- and NF-kB etc. in H2O2-treatment NIH-3T3 cells. Qb could reverse the H2O2 damage effects more markedly, including up-regulation of PTEN expression, inhibiting PI3k/Akt signal transduction, inactivating NF-kB expression, and depressing Bcl2/Bax ratio and apoptosis through modulating cyclin D1 expression level. Moreover, the quercetin could exert anti-oxidant protective effect through modulating the anti-oxidative enzyme system both in vivo and in vitro. However, based on the cell heterogeneity in none- or pre/post-H2O2-treatment state, a difference in quercetin antioxidant response exhibited.