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研究

Protective effects of Piceatannol on hydrogen peroxide-induced damage to human corneal epithelial cells

关键词:  白皮杉醇  过氧化氢  氧化应激损伤  沉默调节蛋白1  Bcl-2/腺病毒E1B 19kD相互作用蛋白3

Key words:  Piceatannol  H2O2  Oxidative stress injury  SIRT1  BNIP3

摘要:

摘要：目的 探究白皮杉醇(Piceatannol，PIC)对过氧化氢(H2O2)诱导的人角膜上皮细胞(human corneal epithelial cells，HCEC)损伤的保护作用及机制。方法 体外培养HCEC细胞，采用CCK-8法检测不同浓度H2O2和白皮杉醇对HCEC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HCEC细胞中LDH释放水平并筛选白皮杉醇最佳作用浓度，将实验分成对照组(CON组)、模型组(H2O2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；划痕实验检测细胞迁移能力；蛋白免疫印迹法Western blot检测HCEC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验证白皮杉醇与沉默信息调节因子1/叉头框转录因子O3a/BCL-2/腺病毒E1B 19kD相互作用蛋白3(SIRT1/FOXO3a/BNIP3)信号通路的关系。结果 细胞增殖实验和细胞毒性实验结果表明与正常对照组相比，H2O2处理后HCEC细胞的活性下降，细胞中LDH释放水平升高，ROS水平升高，划痕愈合能力下降，Western blot结果表明H2O2处理后HCEC细胞中BCL-2的表达下降，BAX和Cleaved caspase-3的表达升高；与模型对照组相比，白皮杉醇处理后HCEC细胞的活性升高，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HCEC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诱导的人角膜上皮细胞损伤。

Abstract:

ABSTRACT: OBJECTIVE The objective of this study is to investigate the protective effect and mechanism of piceatannol (PIC) on hydrogen peroxide (H2O2)-induced injury of human corneal epithelial cells (HCEC). METHODS HCEC cells were cultured in vitro, the effects of different concentrations of H2O2 and piceatannol on HCEC activity were detected by CCK-8 method. The working concentration of H2O2 was screened out. The LDH kit was employed to detect the LDH release level in HCEC cells after the intervention of different concentrations of piceatannol, and determine the optimal concentration of piceatannol; DCFH-DA probe was used to detect the level of reactive oxygen species (ROS);Scratch assay to detect cell migration ability; Western blot was adopted to detect the changes of BCL-2, BAX and Cleaved caspase-3 protein expression levels in HCEC, and the relationship between piceatannol and SIRT1/FOXO3a/BNIP3 signaling pathway was verified by SIRT1 agonist SRT1720 and inhibitor EX 527. RESULTS The results of cell proliferation assay and cytotoxicity assay showed that piceatannol could increase the activity and reduce the LDH release of HCEC cells after H2O2 treatment. The ROS results illustrated that piceatannol had a significant effect on the reduction of ROS level in HCEC cells after H2O2 treatment, and the scratch assay indicated that piceatannol could promote the wound healing of HCEC cells after H2O2 treatment. Western blot results demonstrated that piceatannol could promote the expression of BCL-2 and inhibit the expression of BAX and Cleaved caspase-3 in HCEC cells after H2O2 treatment. 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 HCEC, 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 human corneal epithelial cell injury by activating the SIRT1/FOXO3a/BNIP3 signaling pathway.