基于分子对接技术探讨木犀草素调控脂氧合酶途径抗H9c2心肌细胞缺氧缺糖/复氧复糖损伤的分子机制

Molecular mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion injury in H9c2 cardiomyocytes based on molecular docking

该研究旨在探讨木犀草素调控脂氧合酶途径抗H9c2心肌细胞缺氧缺糖/复氧复糖(oxygen-glucose deprivation/reperfusion,OGD/R)损伤的具体分子机制。首先利用Discovery Studio 2019软件对脂氧合酶途径3个关键酶脂氧合酶5(lipoxygenase 5,ALOX5)、脂氧合酶12(lipoxygenase 12,ALOX12)、脂氧合酶15(lipoxygenase 15,ALOX15)与木犀草素进行分子对接,结果显示木犀草素与三者均有较高的对接分数和与原配体相似的作用基团,据此推测木犀草素可能具有与原配体相类似的生物活性,对脂氧合酶途径有潜在的抑制作用。基于此,体外培养H9c2心肌细胞,采用缺氧缺糖8 h复氧复糖12 h诱导H9c2心肌细胞损伤模型,荧光倒置显微镜下观察H9c2心肌细胞形态,四甲基偶氮唑盐(tetrazolium,MTT)比色法检测细胞活力,乳酸脱氢酶(lactate dehydrogenase,LDH)比色法检测细胞上清LDH水平,结果显示与模型组相比,木犀草素可以明显保护H9c2细胞形态,显著提高OGD/R损伤模型H9c2心肌细胞存活率,显著降低细胞上清LDH水平,抑制细胞毒性,保护细胞膜的完整性。酶联免疫吸附试验检测上清中炎症因子白细胞介素6(interleukin-6,IL-6)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)水平,与模型组相比,木犀草素可显著降低炎症因子IL-6、TNF-α水平。蛋白免疫印迹法检测脂氧合酶途径关键酶ALOX5、ALOX12、ALOX15的蛋白表达水平,木犀草素干预后ALOX5、ALOX12、ALOX15的蛋白表达水平较模型组均显著降低。上述研究结果表明木犀草素可以通过抑制脂氧合酶途径的激活,抑制炎症因子IL-6、TNF-α释放,从而在OGD/R诱导的心肌细胞损伤模型中发挥保护作用。

The aim of this study was to investigate the mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion(OGD/R) injury in H9 c2 cardiomyocytes. First, Discovery Studio 2019 was used for the molecular docking of luteolin with three key enzymes including lipoxygenase 5(ALOX5), lipoxygenase 12(ALOX12), and lipoxygenase 15(ALOX15) in lipoxygenase pathway. The docking results showed that luteolin had high docking score and similar functional groups with the original ligand. From this, H9 c2 cardiomyocytes were cultured in vitro, and then the injury model of H9 c2 cardiomyocytes was induced by deprivation of oxygen-glucose for 8 h, and rehabilitation of oxygen-glucose for 12 h. Cell viability was detected by tetrazolium(MTT) colorimetry. H9 c2 cardiomyocytes were observed with a fluorescence inverted microscope, and colorimetry was used to detect the level of lactate dehydrogenase(LDH) in cell supernatant. The results showed that luteolin could significantly protect the morphology of H9 c2 cells, significantly improve the survival rate of H9 c2 cardiomyocytes in OGD/R injury model, reduce the level of LDH in cell supernatant, inhibit cytotoxicity, and maintain the integrity of cell membrane. The inflammatory cytokines interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with the model group, luteolin can significantly reduce the release of IL-6 and TNF-α. Western blot was employed to detect the protein levels of ALOX5, ALOX12, and ALOX15 in lipoxygenase pathway. After luteolin intervention, the protein levels of ALOX5, ALOX12, and ALOX15 were significantly down-regulated compared with those in model group. These results indicate that luteolin can inhibit the release of IL-6 and TNF-α by restraining the activation of lipoxygenase pathway, thereby playing a protective role in the cardiomyocyte injury model induced by OGD/R.