摘要
目的探讨褪黑素(MT)联合锌(Zn)对H 2O 2诱导的人视网膜色素上皮(ARPE)细胞氧化损伤的保护作用。方法常规培养ARPE细胞株(ARPE-19),利用不同浓度H 2O 2处理ARPE-19细胞,将细胞存活率接近50%的H 2O 2浓度作为氧化损伤浓度。将ARPE-19细胞分为:不同浓度MT(10-5 mol·L^(-1)、10-6 mol·L^(-1)、10-7 mol·L^(-1))+ZnCl 2(15μmol·L^(-1))组,ZnCl 2组(采用15μmol·L^(-1) ZnCl 2培养细胞),H 2O 2组(不添加MT和ZnCl 2培养细胞),空白对照组(细胞不做任何处理)。采用流式细胞仪检测细胞凋亡情况。采用试剂盒测量各组细胞内活性氧(ROS)和丙二醛(MDA)的含量。ELISA检测各组细胞内白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的表达量。结果当H 2O 2浓度为300μmol·L^(-1)时,细胞存活率为(54.31±1.91)%,此时细胞存活率接近50%,后续实验中均选择该浓度作为氧化损伤浓度。ZnCl 2组、10-5 mol·L^(-1) MT+ZnCl 2组、10-6 mol·L^(-1) MT+ZnCl 2组、10-7 mol·L^(-1) MT+ZnCl 2组细胞凋亡率分别为3.05%、1.17%、1.50%、1.71%,与空白对照组(0)相比差异均无统计学意义(均为P>0.05)。H 2O 2组、ZnCl 2组、10-7 mol·L^(-1) MT+ZnCl 2组细胞内ROS含量分别为空白对照组的1.76倍、1.59倍、1.41倍,差异均有统计学意义(均为P<0.05);10-5 mol·L^(-1) MT+ZnCl 2组、10-6 mol·L^(-1) MT+ZnCl 2组细胞内ROS含量分别为空白对照组的1.13倍和1.25倍,差异均无统计学意义(均为P>0.05)。H 2O 2组细胞内MDA的含量较空白对照组显著提高(P<0.05);ZnCl 2组、10-5 mol·L^(-1) MT+ZnCl 2组、10-6 mol·L^(-1) MT+ZnCl 2组、10-7 mol·L^(-1) MT+ZnCl 2组细胞内MDA的含量均较空白对照组稍增加,差异均无统计学意义(均为P>0.05)。H 2O 2组、ZnCl 2组、10-7 mol·L^(-1) MT+ZnCl 2组细胞内IL-6和TNF-α的表达量均显著高于空白对照组,差异均有统计学意义(均为P<0.05);10-5 mol·L^(-1) MT+ZnCl 2组和10-6 mol·L^(-1) MT+ZnCl 2组细胞内IL-6和TNF-α的表达量均略高于空白对照组,差异均无统计学意义(均为P>0.05)。结论MT联合Zn能有效降低H 2O 2损伤所致ARPE-19细胞内ROS的含量,随之减少细胞内MDA的含量,同时也能抑制IL-6和TNF-α的表达量,从而起到保护RPE细胞的作用。
Objective To investigate the protective effects of melatonin(MT)and zinc(Zn)on retinal pigment epithelial(RPE)cells against H 2O 2-induced oxidative damage.Methods ARPE-19 cells were treated with H 2O 2 of different concentrations,and finally the H 2O 2 concentration that ensured the cell viability close to 50%was chosen to be used in other experiments.ARPE-19 cells were divided into the MT(10-5 mol·L^(-1),10-6 mol·L^(-1),and 10-7 mol·L^(-1))+ZnCl 2(15μmol·L^(-1))group,ZnCl 2 group(cells were cultured with 15μmol·L^(-1) ZnCl 2),H 2O 2 group(cells were cultured without MT and ZnCl 2),and blank control group(cells received no treatment).Cell apoptosis was detected by flow cytometry.The content of reactive oxygen species(ROS)and malondialdehyde(MDA)in the cells was determined by assay kits.The expression levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were measured by ELISA kits.Results When the concentration of H 2O 2 was 300μmol·L^(-1),the cell survival rate was(54.31±1.91)%,close to 50%.Thus,this concentration was selected in subsequent experiments for oxidative damage.The apoptosis rates in the ZnCl 2 group,10-5 mol·L^(-1) MT+ZnCl 2 group,10-6 mol·L^(-1) MT+ZnCl 2 group,and 10-7 mol·L^(-1) MT+ZnCl 2 group were 3.05%,1.17%,1.50%,and 1.71%,respectively,without statistically significant differences compared with the blank control group(all P>0.05).The ROS content in the H 2O 2 group,ZnCl 2 group,and 10-7 mol·L^(-1) MT+ZnCl 2 group was 1.76,1.59,and 1.41 times higher than that in the blank control group,respectively,and the differences were statistically significant(all P<0.05).The ROS content in the 10-5 mol·L^(-1) MT+ZnCl 2 group and 10-6 mol·L^(-1) MT+ZnCl 2 group were 1.13 and 1.25 times higher than that in the blank control group,respectively,but the differences were not statistically significant(all P>0.05).Compared with the blank control group,the MDA content in the H 2O 2 group was significantly increased(P<0.05),while in the ZnCl 2 group,10-5 mol·L^(-1) MT+ZnCl 2 group,10-6 mol·L^(-1) MT+ZnCl 2 group,and 10-7 mol·L^(-1) MT+ZnCl 2 group,the MDA content was slightly increased,without statistically significant differences(all P>0.05).The expression levels of IL-6 and TNF-αin the H 2O 2 group,ZnCl 2 group,and 10-7 mol·L^(-1) MT+ZnCl 2 group were significantly higher than those in the blank control group,and the differences were statistically significant(all P<0.05).The expression levels of IL-6 and TNF-αin the 10-5 mol·L^(-1) MT+ZnCl 2 group and 10-6 mol·L^(-1) MT+ZnCl 2 group were slightly higher than those in the blank control group,and the differences were not statistically significant(all P>0.05).Conclusion MT and Zn can effectively reduce the ROS and MDA content in ARPE-19 cells induced by H 2O 2 damage,and meanwhile inhibit the expression of IL-6 and TNF-α,thus protecting RPE cells.
作者
易彩霞
孙馨
印双红
黄啸
YI Caixia;SUN Xin;YIN Shuanghong;HUANG Xiao(School of Sports and Health Science,Tongren University,Tongren 554300,Guizhou Province,China)
出处
《眼科新进展》
CAS
北大核心
2022年第3期189-193,共5页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助(编号:31800839)
贵州省教育厅普通本科高校青年科技人才成长项目(编号:黔教合KY字[2022]069号)。
关键词
视网膜色素上皮细胞
褪黑素
锌
氧化损伤
炎症反应
retinal pigment epithelial cells
melatonin
zinc
oxidative damage
inflammatory response
