目的探讨褪黑素对过氧化氢(H 2O 2)诱导的人晶状体上皮细胞(HLECs)焦亡的影响及其机制。
方法取传代培养的HLECs分为5个组，其中正常对照组常规培养，模型对照组于100 μmol/L H 2O 2条件下培养，褪黑素组、维生素E组和维生素E溶剂组分别用1×10 -6 mol/L褪黑素、100 μmol/L维生素E和维生素E溶剂预处理后，用100 μmol/L H 2O 2继续培养，收集细胞待检测。为进一步探讨褪黑素的作用机制，将细胞分为7个组，其中正常对照组常规培养；模型对照组细胞于100 μmol/L H 2O 2条件下培养；核转录因子E2相关因子2短发夹RNA(shNrf2)阴性对照组和shNrf2组细胞分别用shNrf2阴性对照和shNrf2慢病毒转染，并于终浓度100 μmol/L H 2O 2条件下培养；褪黑素组细胞在终浓度1×10 -6 mol/L褪黑素条件下培养，然后加入终浓度100 μmol/L H 2O 2继续培养；褪黑素+shNrf2阴性对照组和褪黑素+shNrf2组细胞首先分别采用shNrf2阴性对照或shNrf2慢病毒转染，再于终浓度1×10 -6 mol/L褪黑素和终浓度100 μmol/L H 2O 2条件下培养，收集各组细胞。采用酶联免疫吸附测定(ELISA)法检测各组细胞培养上清液中乳酸脱氢酶(LDH)活性，白细胞介素(IL)-1β和IL-18浓度；采用流式细胞术检测各组细胞中活性氧簇(ROS)的荧光强度；采用Western blot法检测各组细胞中Nrf2、NLRP3、凋亡相关斑点样蛋白(ASC)、caspase-1 p20、GSDMD-N蛋白的表达。
结果ELISA法检测发现，与模型对照组相比，褪黑素组和维生素E组细胞培养上清液中LDH活性及IL-1β和IL-18浓度均有不同程度的下调，差异均有统计学意义(均 P<0.05)。流式细胞仪检测发现，褪黑素组和维生素E组细胞中ROS荧光强度分别为13 040.67±1 550.66、12 593.67±1 677.06，显著低于模型对照组的18 310.33±1 248.01，差异均有统计学意义(均 P<0.05)。Western blot结果显示，褪黑素组和维生素E组Nrf2蛋白相对表达量分别为4.24±0.44、3.73±0.38、较模型对照组的2.28±0.34明显上升(均 P<0.05)，褪黑素组和维生素E组NLRP3、ASC、caspase-1 p20及GSDMD-N蛋白相对表达量较模型对照组显著下降，差异均有统计学意义(均 P<0.05)。shNrf2组相较于shNrf2阴性对照组，褪黑素+shNrf2组相较于褪黑素+shNrf2阴性对照组，细胞培养上清液中LDH活性、IL-1β和IL-18浓度均显著上升，细胞中Nrf2蛋白相对表达量明显下降，细胞中ROS荧光强度、NLRP3、ASC、caspase-1 p20及GSDMD-N焦亡相关蛋白相对表达量明显升高，差异均有统计学意义(均 P<0.05)。
结论褪黑素可能通过激活Nrf2，减少NLRP3炎性体的释放，进而抑制HLECs焦亡。
ObjectiveTo investigate the inhibitory effect of melatonin on pyroptosis of lens epithelium cells (HLECs) induced by hydrogen peroxide (H 2O 2) and its mechanism.
MethodsThe cultured HLECs were divided into normal control group, model control group, melatonin group, vitamin E group, and vitamin E solvent group.Cells in melatonin group, vitamin E group and vitamin E solvent group were pre-cultured with 1×10 -6 mol/L melatonin, 100 μmol/L vitamin E or equal volume of vitamin E solvent, then cultured with 100 μmol/L H 2O 2, respectively, and the cells in the normal control group and model control group were cultured with normal condition or 100 μmol/L H 2O 2, respectively.The HLECs transfected with nuclear factor erythroid 2-related factor 2 short hairpin RNA (shNrf2) or shNrf2 negtive control lentivirus and following with 100 μmol/L H 2O 2 treatment were served as shNrf2 group and shNrf2 negative control group, respectively; and the transfected cells treated with 1×10 -6 mol/L melatonin and subsequent 100 μmol/L H 2O 2 treatment were served as melatonin+ shNrf2 group and melatonin+ shNrf2 negative control group.The activity of lactic dehydrogenase (LDH) and the expression levels of interleukin (IL)-1β and IL-18 in the culture supernatant were detected by the enzyme linked immunosorbent assay (ELISA) kit.The concentration of reactive oxygen species (ROS) was assessed by flow cytometry.The expression level of Nrf2, NLRP3, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1 p20 and GSDMD-N proteins were evaluated by Western blot.
ResultsCompared with model control group, the activity of LDH and the concentrations of IL-1β and IL-18 were significantly decreased in melatonin group and vitamin E group, showing statistically significant differences (all at P<0.05). The ROS fluorescence intensities were 13 040.67±1 550.66 and 12 593.67±1 677.06 in melatonin group and vitamin E group, respectively, which were significantly lower than 18 310.33±1 248.01 in model control group (both at P<0.05). The relative expression levels of Nrf2 protein were 4.24±0.44 and 3.73±0.38 in melatonin group and vitamin E group, respectively, which were significantly higher than 2.28±0.34 in model control group, and the relative expression levels of NLRP3, ASC, caspase-1 p20 and GSDMD-N in melatonin group and vitamin E group were significantly decreased than model control group (all at P<0.05). The relative expression level of Nrf2 protein in shNrf2 group and melatonin+ shNrf2 group was significantly reduced, and the expression levels of LDH, IL-1β, IL-18, ROS content, NLRP3, ASC, caspase-1 p20 and GSDMD-N were significantly increased in comparison with shNrf2 negative control group and melatonin+ shNrf2 negative control group, respectively (all at P<0.05).
ConclusionsMelatonin can inhibit the release of NLRP3 inflammasome by activating Nrf2, and has an inhibitory effect on the pyroptosis of HLECs.
杨鑫,刘旭辉,孟佳,等. 褪黑素抗人晶状体上皮细胞焦亡作用及其机制[J]. 中华实验眼科杂志,2022,40(05)：414-421.
DOI:10.3760/cma.j.cn115989-20210516-00307版权归中华医学会所有。
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杨鑫：酝酿和设计实验、实施研究、采集数据、分析和解释数据、撰写文章；刘旭辉、孟佳：实施研究、采集数据、分析和解释数据；方梦园：实施研究、采集数据、数据统计分析；张凤妍：酝酿和设计实验、对文章进行指导及定稿
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