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    白皮杉醇对过氧化氢诱导的人角膜上皮细胞损伤的保护作用

    Protective Effects of Piceatannol on Hydrogen Peroxide-induced Damage to Human Corneal Epithelial Cells

      摘要
    • 摘要:
      目的  探究白皮杉醇对过氧化氢(H2O2)诱导的人角膜上皮细胞(human corneal epithelial cells，HCECs)损伤的保护作用及机制。
      方法 体外培养HCECs，采用CCK-8法检测不同浓度H2O2和白皮杉醇对HCECs活力的影响，筛选H2O2的适宜浓度，乳酸脱氢酶(lactate dehydrogenase，LDH)试剂盒检测不同浓度的白皮杉醇干预后HCECs中LDH释放水平并筛选白皮杉醇最佳作用浓度，细胞分成对照组、模型组、白皮杉醇40 μmol·L–1组和白皮杉醇80 μmol·L–1组；2′,7′-二氯荧光二乙酸酯(DCFH-DA)探针检测活性氧(reactive oxygen species，ROS)生成水平；划痕试验检测细胞迁移能力；蛋白免疫印迹法检测HCECs中Bcl-2、Bax、cleaved caspase-3蛋白表达水平变化，使用SIRT1激动剂SRT1720和抑制剂EX527验证白皮杉醇与SIRT1/FOXO3a/BNIP3信号通路的关系。
      结果 细胞增殖试验和细胞毒性试验结果表明，与对照组相比，H2O2处理后HCECs的活性下降，细胞中LDH释放水平升高，ROS水平升高，划痕愈合能力下降，Western blotting结果表明，H2O2处理后HCECs中Bcl-2的表达下降，Bax和cleaved caspase-3的表达升高；与模型组相比，白皮杉醇处理后HCECs的活性升高，LDH释放水平下降，细胞内ROS水平下降，划痕愈合能力增强。使用SIRT1激动剂SRT1720和抑制剂EX527验证发现，EX527和H2O2均可抑制HCECs中SIRT1、FOXO3a、BNIP3的表达，白皮杉醇逆转了这一作用，SRT1720和白皮杉醇能够协同使得SIRT1、FOXO3a、BNIP3的表达水平进一步升高。
      结论 白皮杉醇可能通过激活SIRT1/FOXO3a/BNIP3信号通路改善H2O2诱导的HCECs损伤。

       

      Abstract:
      OBJECTIVE  To investigate the protective effect and mechanism of piceatannol on hydrogen peroxide(H2O2)-induced injury of human corneal epithelial cells(HCECs).
      METHODS  HCECs were cultured in vitro, the effects of different concentrations of H2O2 and piceatannol on HCECs activity were detected by CCK-8 method. The working concentration of H2O2 was screened out. The lactate dehydrogenase(LDH) release levels in HCECs after intervention with different concentrations of piceatannol were measured using an LDH assay kit, and the optimal concentration of piceatannol was determined. The cells were divided into four groups: the control group, the model group, the 40 μmol·L–1 piceatannol group, and the 80 μmol·L–1 piceatannol group. Reactive oxygen species(ROS) generation was detected using the 2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA) probe. Cell migration ability was evaluated using the scratch assay. Changes in the expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins in HCECs were examined by Western blotting. The SIRT1 agonist SRT1720 and inhibitor EX527 were used to validate the relationship between piceatannol and the SIRT1/FOXO3a/BNIP3 signaling pathway.
      RESULTS  The results of cell proliferation assay and cytotoxicity assay showed that, compared with the control group, H2O2-treated HCECs exhibited reduced cell viability, increased LDH release, elevated ROS levels, and impaired scratch wound healing ability. Western blotting analysis showed that, H2O2 treatment downregulated the expression of Bcl-2 while upregulating the expression of Bax and cleaved caspase-3 in HCECs. In contrast, compared with the model group, piceatannol treatment enhanced the viability of HCECs, decreased LDH release, reduced intracellular ROS levels, and promoted scratch wound healing. By using the SIRT1 agonist SRT1720 and inhibitor EX527, it was found that both EX527 and H2O2 could inhibit the expression of SIRT1, FOXO3a and BNIP3 in HCECs, and this effect was reversed by piceatannol, which was synergistically SRT1720 further increased the expression levels of SIRT1, FOXO3a and BNIP3.
      CONCLUSION  Piceatannol may ameliorate H2O2-induced HCECs injury by activating the SIRT1/FOXO3a/BNIP3 signaling pathway.

       

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