Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul, 156-756, Republic of Korea.
Naunyn Schmiedebergs Arch Pharmacol. 2011 Feb;383(2):191-201. doi: 10.1007/s00210-010-0594-6. Epub 2011 Jan 7.
Peroxisome proliferator-activated receptor-gamma (PPARγ) agonists have been reported to enhance antioxidant defenses by increasing levels of catalase and copper-zinc superoxide dismutase (Cu/Zn SOD) in oligodendrocyte progenitor cells. In this study, we investigated the effects of the PPARγ agonist, rosiglitazone, on hydrogen peroxide (H(2)O(2)) generation by acidified medium at pH 5.5 (AM5.5), which is in the pH range of duodenogastric refluxates, in primary cultured feline esophageal epithelial cells (EEC). Successful isolation of EEC was identified by immunocytochemistry. AM5.5- and rosiglitazone-induced cell viabilities were determined using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assays. The NAD(P)H oxidase activity was measured, and expression of catalase or SOD protein by AM5.5 in the absence and presence of rosiglitazone was assessed using western blotting analysis. PPARγ protein and mRNA were constitutively expressed in EEC. In the incubation with rosiglitazone alone, cell viability was shown more than 90% at 0-10 μM for 72 h. After exposure to AM5.5 for 8 h, intracellular H(2)O(2) was significantly generated. Treatment with rosiglitazone prior to and during exposure to AM5.5 inhibited the H(2)O(2) generation whereas the specific PPARγ antagonist GW9662 offsets the inhibitory action of rosiglitazone. H(2)O(2) generation was also prevented by a nonspecific ROS scavenger N-acetylcysteine or an inhibitor of NADPH oxidase diphenyleneiodonium. The enhanced AM5.5-induced NAD(P)H oxidase activity was not suppressed by rosiglitazone. Instead, the pretreatment of rosiglitazone enhanced the protein expression of catalase, Cu/Zn SOD, and Mn SOD, which are endogenous antioxidative enzymes. These findings indicate that rosiglitazone inhibits AM5.5-induced intracellular H(2)O(2) production, which occurs via NAD(P)H oxidase activation, by using a PPARγ-dependent pathway, and that the underlying mechanism involves an increase in the expression of catalase and SOD proteins.
过氧化物酶体增殖物激活受体-γ（PPARγ）激动剂已被报道通过增加少突胶质细胞前体细胞中过氧化氢酶和铜锌超氧化物歧化酶（Cu/Zn SOD）的水平来增强抗氧化防御。在这项研究中，我们研究了 PPARγ 激动剂罗格列酮对 pH 5.5 酸化培养基（AM5.5）产生的过氧化氢（H2O2）的影响，这是十二指肠反流物的 pH 范围，在原代培养的猫食管上皮细胞（EEC）中。通过免疫细胞化学鉴定成功分离出 EEC。使用 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐测定 AM5.5 和罗格列酮诱导的细胞活力。通过 Western 印迹分析评估 AM5.5 诱导的过氧化氢酶或 SOD 蛋白的 NAD（P）H 氧化酶活性和表达。EEC 中持续表达 PPARγ 蛋白和 mRNA。在单独与罗格列酮孵育的情况下，在 0-10 μM 下孵育 72 h 后，细胞活力显示超过 90％。在用 AM5.5 孵育 8 h 后，细胞内 H2O2 显著产生。在用 AM5.5 孵育之前和期间用罗格列酮处理可抑制 H2O2 的产生，而特异性 PPARγ 拮抗剂 GW9662 抵消了罗格列酮的抑制作用。非特异性 ROS 清除剂 N-乙酰半胱氨酸或 NADPH 氧化酶二苯基碘鎓抑制剂也可防止 H2O2 的产生。罗格列酮未抑制增强的 AM5.5 诱导的 NAD（P）H 氧化酶活性。相反，罗格列酮的预处理增强了过氧化氢酶，Cu/Zn SOD 和 Mn SOD 的蛋白表达，这些是内源性抗氧化酶。这些发现表明，罗格列酮通过 PPARγ 依赖性途径抑制 AM5.5 诱导的细胞内 H2O2 产生，这是通过 NAD（P）H 氧化酶激活发生的，其潜在机制涉及过氧化氢酶和 SOD 蛋白表达的增加。
