Song Hyun Ju, Shin Chang Yell, Oh Tae Young, Min Young Sil, Park Eon Sub, Sohn Uy Dong
Department of Pharmacology, College of Pharmacy, Chung Ang University, Korea.
Biol Pharm Bull. 2009 Apr;32(4):589-96. doi: 10.1248/bpb.32.589.
We previously reported that eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) extracted from Artemisia asiaitica, augmented the cellular antioxidant defense capacity through induction of the antioxidant protein heme oxygenase-1 (HO-1), thereby protecting ileal smooth muscle cells from nonsteroidal anti-inflammatory drug (NSAID)-induced intestinal toxicity. In the present study, we used cultured feline esophageal epithelial cells (EEC) to investigate the ability of eupatilin to induce expression of HO-1 and to analyze its cytoprotective effect against indomethacin-induced damage, since NSAID users have a higher risk of esophageal ulcers or esophagitis than non-NSAID users. A culture of EEC from cat was prepared. The identity of the cultures was confirmed by immunocytochemistry using cytokeratin antibodies. Western blot analysis showed a concentration- and time- dependent expression of HO-1 in response to eupatilin. Phosphorylation of extracellular regulating protein kinase (ERKs) and Akt, and nuclear translocation of nuclear related factor 2 (Nrf2) were induced by 150 microM eupatilin in a time-dependent manner. Eupatilin-induced HO-1 expression and Nrf2 were partly attenuated by MEK inhibitor PD98059 and almost completely by phosphatidyl-inactiol 3 kinase (PI3K) inhibitor LY294002, but not by c-Jun N-terminal kinase (JNK) inhibitor SP600125 or p38 mitogen activated protein kinase (MAPK) inhibitor SB202190. MTT assay showed that treatment with 2 mM indomethacin for 2 h decreased cell viability to about 41%. Pre-treatment of cells with eupatilin resulted in the dose-dependent inhibition of indomethacin-induced cell damage. We confirmed that ZnPP, an HO-1 inhibitor, repressed eupatilin-induced HO-1 activity and showed the protective effect of eupatilin against indomethacin-induced cell injury. The data suggested that HO-1 was partly responsible for the eupatilin-mediated protective action of esophageal epithelial cells against indomethacin via both ERKs and PI3K/Akt pathways as well as Nrf2 translocation.
我们之前报道过，从亚洲蒿中提取的灯盏乙素（5,7-二羟基-3,4,6-三甲氧基黄酮）通过诱导抗氧化蛋白血红素加氧酶-1（HO-1）增强细胞抗氧化防御能力，从而保护回肠平滑肌细胞免受非甾体抗炎药（NSAID）诱导的肠道毒性。在本研究中，我们使用培养的猫食管上皮细胞（EEC）来研究灯盏乙素诱导HO-1表达的能力，并分析其对吲哚美辛诱导损伤的细胞保护作用，因为NSAID使用者比非NSAID使用者患食管溃疡或食管炎的风险更高。制备了来自猫的EEC培养物。使用细胞角蛋白抗体通过免疫细胞化学确认培养物的身份。蛋白质印迹分析显示，灯盏乙素作用下HO-1呈浓度和时间依赖性表达。150μM灯盏乙素以时间依赖性方式诱导细胞外调节蛋白激酶（ERK）和Akt的磷酸化以及核相关因子2（Nrf2）的核转位。MEK抑制剂PD98059部分减弱了灯盏乙素诱导的HO-1表达和Nrf2，磷脂酰肌醇3激酶（PI3K）抑制剂LY294002几乎完全减弱了其表达，但c-Jun氨基末端激酶（JNK）抑制剂SP600125或p38丝裂原活化蛋白激酶（MAPK）抑制剂SB202190没有这种作用。MTT分析表明，用2 mM吲哚美辛处理2小时可使细胞活力降至约41%。用灯盏乙素预处理细胞导致对吲哚美辛诱导的细胞损伤呈剂量依赖性抑制。我们证实，HO-1抑制剂ZnPP可抑制灯盏乙素诱导的HO-1活性，并显示灯盏乙素对吲哚美辛诱导的细胞损伤具有保护作用。数据表明，HO-1部分负责灯盏乙素介导的食管上皮细胞通过ERK和PI3K/Akt途径以及Nrf2转位对吲哚美辛的保护作用。
