探究金雀异黄酮(genistein, Gen)对叔丁基过氧化氢(tert-butyl hydroperoxide, TBHP)诱导的H9c2细胞衰老的保护作用及其机制。将H9c2细胞随机分为对照组、模型组(TBHP 200 μmol/L)及Gen低、中、高剂量组(0.04、0.2、1 μmol/L)。采用MTT比色法检测细胞存活率;β-半乳糖苷酶(senescence-associated β-galactosidase, SA-β-gal)染色法观察细胞衰老程度;比色法测定细胞总抗氧化能力(total antioxidant capacity, T-AOC)水平和超氧化物歧化酶(superoxide dismutase, SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)、过氧化氢酶(catalase, CAT)活性及丙二醛(malondialdehyde, MDA)含量;Western blotting法检测P53、P21、Kelch样环氧氯丙烷相关蛋白-1(Kelch-like epichlorohydrin-related protein-1, Keap1)、核因子E2相关因子(nuclear factor erythroid-2-related factor 2, Nrf2)、血红素氧合酶1(heme oxygenase 1, HO-1)蛋白表达以及细胞磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase, PI3K)、蛋白激酶B(protein kinase B, AKT)、细胞外调节蛋白激酶(extracellular regulatory protein kinase1/2, ERK1/2)、P38丝裂原活化蛋白激酶(P38 mitogen-activated protein kinase, P38)、c-Jun 氨基端激酶(c-jun amino-terminal kinase, JNK)蛋白及其磷酸化蛋白表达水平。结果表明,与模型组比较,Gen组H9c2细胞的细胞存活率显著升高,SA-β-gal染色阳性率显著降低;T-AOC、SOD、GSH-Px、CAT活性显著升高,MDA含量显著降低;同时Gen组Keap1表达下降,Nrf2、HO-1蛋白表达水平上升;P53、P21蛋白表达降低,PI3K、AKT磷酸化蛋白表达水平升高,ERK1/2、P38、JNK磷酸化蛋白表达降低。该研究证明Gen可延缓H9c2细胞衰老,提高H9c2细胞的抗氧化能力,其机制可能与其通过PI3K/AKT、MAPKs信号通路上调Nrf2表达有关。
To investigate the protective effects of genistein (Gen) on tert-butyl hydroperoxide (TBHP)-induced H9c2 cell aging and its mechanism, H9c2 cells were randomly divided into control group, model group (200 μmol/L TBHP) and low, medium and high dose Gen groups (0.04, 0.2 and 1 μmol/L of Gen, respectively). The survival rate was determined by the MTT method. Cell senescence was observed by β-galactosidase (SA-β-gal) staining. The total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activities, and malondialdehyde (MDA) content were determined by the colorimetric assay. The expressions of P53, P21, Kelch-like epichlorohydrin-related protein-1(Keap1), nuclear factor E2-related factor (Nrf2) and heme oxygenase 1(HO-1) proteins, and the phosphorylation of phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT), extracellular regulatory protein kinase1/2 (ERK1/2), P38 mitogen-activated protein kinase (P38) and c-jun amino-terminal kinase (JNK) were detected with the western blotting method. Compared with the model group, the survival rate was increased, the positive staining of SA-β-gal was decreased; T-AOC, SOD, GSH-Px and CAT activities were increased, and MDA content was decreased in Gen groups. Meanwhile, Gen down-regulated the Keap1 protein expression and up-regulated the Nrf2 and HO-1 protein expressions. Gen also increased the expressions of P53, P21 proteins, elevated the phosphorylation of PI3K and AKT proteins, and reduced the phosphorylation of ERK, P38 and JNK proteins. Gen could delay the cell senescence and improve the antioxidant capacity in H9c2 cells. The mechanism may be related to the up-regulation of Nrf2 through PI3K/AKT and MAPKs signaling pathways.
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