Li Yunbo, Cao Zhuoxiao, Zhu Hong, Trush Michael A
Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USA.
Arch Biochem Biophys. 2005 Jul 1;439(1):80-90. doi: 10.1016/j.abb.2005.05.008.
4-hydroxy-2-nonenal (HNE) plays an important role in the pathogenesis of cardiac disorders. While conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST) has been suggested to be a major detoxification mechanism for HNE in target cells, whether chemically upregulated cellular GSH and GST afford protection against HNE toxicity in cardiac cells has not been investigated. In addition, the differential roles of chemically induced GSH and GST as well as other cellular factors in detoxifying HNE in cardiomyocytes are unclear. In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat H9C2 cardiomyocytes. Treatment of cardiomyocytes with D3T resulted in a significant induction of both GSH and GST as well as the mRNA expression of gamma-glutamylcysteine ligase catalytic subunit and GSTA. Both GSH and GST remained elevated for at least 72 h after removal of D3T from the culture media. Treatment of cells with HNE led to a significant decrease in cell viability and an increased formation of HNE-protein adducts. Pretreatment of cells with D3T dramatically protected against HNE-mediated cytotoxicity and protein-adduct formation. HNE treatment caused a significant decrease in cellular GSH level, which preceded the loss of cell viability. Either depletion of cellular GSH by buthionine sulfoximine (BSO) or inhibition of GST by sulfasalazine markedly sensitized the cells to HNE toxicity. Co-treatment of cardiomyocytes with BSO was found to completely block the D3T-mediated GSH elevation, which however failed to reverse the cytoprotective effects of D3T, suggesting that other cellular factor(s) might be involved in D3T cytotprotection. In this regard, D3T was shown to induce cellular aldose reductase (AR). Surprisingly, inhibition of AR by sorbinil failed to potentiate HNE toxicity in cardiomyocytes. In contrast, sorbinil dramatically augmented HNE cytotoxicity in cells with GSH depletion induced by BSO. Similarly, in BSO-treated cells, D3T cytoprotection was also largely reversed by sorbinil, indicating that AR played a significant role in detoxifying HNE only under the condition of GSH depletion in cardiomyocytes. Taken together, this study demonstrates that D3T can induce GSH, GST, and AR in cardiomyocytes, and that the above cellular factors appear to play differential roles in detoxification of HNE in cardiomyocytes.
4-羟基-2-壬烯醛（HNE）在心脏疾病的发病机制中起重要作用。虽然谷胱甘肽S-转移酶（GST）催化的与谷胱甘肽（GSH）的结合被认为是靶细胞中HNE的主要解毒机制，但化学上调的细胞内GSH和GST是否能保护心脏细胞免受HNE毒性尚未得到研究。此外，化学诱导的GSH和GST以及其他细胞因子在心肌细胞中对HNE解毒的不同作用尚不清楚。在本研究中，我们表征了3H-1,2-二硫杂环戊烯-3-硫酮（D3T）对GSH和GST的诱导作用，以及D3T提高的细胞防御对大鼠H9C2心肌细胞中HNE介导的毒性的保护作用。用D3T处理心肌细胞导致GSH和GST以及γ-谷氨酰半胱氨酸连接酶催化亚基和GSTA的mRNA表达显著上调。从培养基中去除D3T后，GSH和GST至少72小时仍保持升高。用HNE处理细胞导致细胞活力显著下降，HNE-蛋白质加合物形成增加。用D3T预处理细胞可显著保护细胞免受HNE介导的细胞毒性和蛋白质加合物形成。HNE处理导致细胞内GSH水平显著下降，这先于细胞活力的丧失。用丁硫氨酸亚砜胺（BSO）消耗细胞内GSH或用柳氮磺胺吡啶抑制GST均显著使细胞对HNE毒性敏感。发现用BSO与心肌细胞共同处理可完全阻断D3T介导的GSH升高，但这未能逆转D3T的细胞保护作用，表明可能有其他细胞因子参与D3T的细胞保护作用。在这方面，D3T被证明可诱导细胞醛糖还原酶（AR）。令人惊讶的是，用索比尼尔抑制AR未能增强心肌细胞中HNE的毒性。相反，索比尼尔显著增强了由BSO诱导的GSH耗竭的细胞中HNE的细胞毒性。同样，在BSO处理的细胞中，索比尼尔也在很大程度上逆转了D3T的细胞保护作用，表明AR仅在心肌细胞中GSH耗竭的条件下在HNE解毒中起重要作用。综上所述，本研究表明D3T可在心肌细胞中诱导GSH、GST和AR，并且上述细胞因子在心肌细胞中对HNE的解毒中似乎发挥不同作用。
