Hou W, Qiu P, Chen N J, Yao P, Liu S, Qin H
Department of Gastroenterology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Gan Zang Bing Za Zhi. 2020 Jun 20;28(6):504-508. doi: 10.3760/cma.j.cn501113-20200522-00269.
To study the protective effect and potential mechanism of heme oxygenase (HO-1)/carbon monoxide (CO)-mediated quercetin on alcoholic oxidative damage of primary rat hepatocytes. Primary rat hepatocytes were isolated and cultured by two-step collagenase technique. Ethanol exposed primary rat hepatocytes were simultaneously added with quercetin (100 μmol/L) and/or hemoglobin (100 μmol/L) or different doses of CO-releasing molecules (CORM-2, 5-50 μmol/L) for their combined action. After polling, LDH, AST activities and MDA and GSH levels were measured in the supernatant of cell culture. The alone or combined effects of quercetin, CORM-2, hemoglobin and zinc protoporphyrin IX exposed to ethanol were detected by the activity of CYP2E1 in liver microsomes. Statistical analysis of data was performed by analysis of variance (ANOVA) and intergroup comparison was done by SNK-test. Simultaneous addition of 100 μmol/L quercetin had significantly reduced ethanol-induced AST and LDH release, and GSH consumption and MDA elevation extent. Moreover, quercetin had not only lost the hemoglobin (CO blocker) protective effect but also had further exacerbated ethanol-induced lipid peroxidation. CORM-2 had reduced ethanol-induced AST and LDH release, and GSH consumption and MDA production in liver cells, and thus had dose-dependent protective effect. Ethanol had increased significantly CYP2E1 activity. Quercetin or CORM-2 had inhibited CYP2E1 activity, while hemoglobin or protoporphyrin IX had eliminated quercetin inhibitory effect and had increased the CYP2E1 activity. Quercetin, and CYP2E1 activity was constant as compared to ethanol group when CORM-2, zinc protoporphyrin IX and ethanol were incubated with hepatocytes, but the CYP2E1 activity was significantly decreased ( < 0.05), and the differences were statistically significant. CO/HO-1 metabolite mediates the protective effect of quercetin on alcoholic oxidative damage of hepatocytes, which may be related to the inhibition of CYP2E1 activity.
研究血红素加氧酶（HO-1）/一氧化碳（CO）介导的槲皮素对原代大鼠肝细胞酒精性氧化损伤的保护作用及潜在机制。采用两步胶原酶法分离培养原代大鼠肝细胞。将乙醇处理的原代大鼠肝细胞同时加入槲皮素（100 μmol/L）和/或血红蛋白（100 μmol/L）或不同剂量的CO释放分子（CORM-2，5 - 50 μmol/L）进行联合作用。取样后，检测细胞培养上清液中的乳酸脱氢酶（LDH）、天冬氨酸转氨酶（AST）活性以及丙二醛（MDA）和谷胱甘肽（GSH）水平。通过肝微粒体中细胞色素P450 2E1（CYP2E1）的活性检测槲皮素、CORM-2、血红蛋白和锌原卟啉IX单独或联合作用于乙醇处理细胞的效果。数据采用方差分析（ANOVA）进行统计分析，组间比较采用SNK检验。同时加入100 μmol/L槲皮素可显著降低乙醇诱导的AST和LDH释放以及GSH消耗和MDA升高程度。此外，槲皮素不仅失去了血红蛋白（CO阻断剂）的保护作用，还进一步加剧了乙醇诱导的脂质过氧化。CORM-2可降低乙醇诱导的肝细胞AST和LDH释放以及GSH消耗和MDA生成，具有剂量依赖性保护作用。乙醇显著增加了CYP2E1活性。槲皮素或CORM-2可抑制CYP2E1活性，而血红蛋白或原卟啉IX消除了槲皮素的抑制作用并增加了CYP2E1活性。当CORM-2、锌原卟啉IX与乙醇共同作用于肝细胞时，槲皮素组CYP2E1活性与乙醇组相比无变化，但CORM-2组CYP2E1活性显著降低（<0.05），差异具有统计学意义。CO/HO-1代谢产物介导了槲皮素对肝细胞酒精性氧化损伤的保护作用，这可能与抑制CYP2E1活性有关。
