Liu Lie-Gang, Yan Hong, Zhang Wen, Yao Ping, Zhang Xi-Ping, Sun Xiu-Fa, Nussler Andreas K
Department of Nutrition and Food Hygiene, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China.
Biomed Environ Sci. 2004 Sep;17(3):315-26.
We investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage.
Dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe2+ formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supernatants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2E1 activity.
We first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h. Being similar increased protein expression started to what had been demonstrated with the mRNA level, at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fe2+ formation in human hepatocytes. Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyrin (ZnPP), an antagonist of HO-1 induction.
HO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human liver.
我们研究了乙醇暴露与人肝细胞中血红素加氧酶（HO-1）之间的关系，以确定HO-1的诱导是否可以预防乙醇诱导的细胞损伤。
本研究采用剂量依赖性（25-100 mmol/L）和时间依赖性（0-24小时）的乙醇暴露。分别通过PT-PCR和蛋白质印迹法检测HO-1 mRNA和蛋白表达。HO-1活性通过胆红素和Fe2+的生成来表示。通过培养上清液中乳酸脱氢酶（LDH）和天冬氨酸转氨酶（AST）水平以及细胞内丙二醛（MDA）的生成、细胞内谷胱甘肽（GSH）状态和CYP 2E1活性来研究细胞毒性。
我们首先证明了乙醇暴露与人肝细胞中HO-1 mRNA和蛋白表达之间存在剂量依赖性反应。我们进一步观察到，使用100 mmol/L乙醇时，HO-1 mRNA表达在乙醇暴露后30分钟开始呈时间依赖性增加，在3小时至9小时之间达到最大值。与之相似，蛋白表达在乙醇暴露后6小时开始增加，与mRNA水平的变化一致，并在18小时内持续升高。此外，我们发现乙醇暴露于肝细胞后，以人肝细胞中胆红素和Fe2+的生成来衡量，HO-1酶活性呈时间依赖性显著增加。我们的结果清楚地表明，乙醇暴露导致LDH、AST和MDA水平显著升高，而抗氧化剂GSH则呈时间依赖性降低。此外，我们证明预先给予钴原卟啉（CoPP）可诱导人肝细胞中的HO-1，并防止MDA增加和GSH减少。HO-1诱导拮抗剂锌原卟啉（ZnPP）可部分逆转这些作用。
细胞或器官中HO-1的表达可能为更好地预防和治疗乙醇诱导的人肝脏氧化损伤带来新的策略。
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