目的

探讨不同浓度硫柳汞对人结膜上皮细胞自噬调控的影响及水杨酸钠对其保护的机制。

方法

在细胞培养时，依据添加药品种类分为硫柳汞实验组、水杨酸钠组、硫柳汞+水杨酸钠组及对照组。硫柳汞实验组采用浓度为0.00001%、0.00005%、0.0001%、0.0005%及0.001%的硫柳汞处理30 min，随即清除药液；水杨酸钠组采用0.00001%、0.00005%、0.0001%、0.0005%及0.001%水杨酸钠预处理30 min；硫柳汞+水杨酸钠组采用0.001%硫柳汞+0.001%水杨酸钠预处理30 min；对照组仅含有培养基。采用二苯基四氮唑溴盐（MTT）法测定检测细胞存活率。采用免疫印迹法检测微管相关蛋白1轻链3（LC3）-Ⅰ型和LC3-Ⅱ蛋白在自噬相关通路中的表达。采用免疫荧光法法检测自噬相关通路中核因子κβ（NF-κβ）、LC3-Ⅰ及LC3-Ⅱ蛋白的表达。细胞存活率和蛋白的表达水平以±s表示，组内比较采用t检验，组间比较采用单因素方差分析。

结果

以对照组为基数，0.00001%、0.00005%、0.0001%、0.0005%及0.001%的硫柳汞实验组和水杨酸钠组细胞存活率分别为（97.06±1.71）%、（91.76±1.82）%、（89.46±4.67）%、（85.20±3.88）%、（77.93±2.72）%、（98.80±2.27）%、（97.98±3.17）%、（96.83±1.30）%、（96.08±2.40）%及（95.52±2.59）%，硫柳汞实验组内比较差异有统计学意义（F=30.66，P＜0.05）；各浓度水杨酸钠细胞存活率组内比较差异无统计学意义（F=1.86，P＞0.05）。0.001%水杨酸钠+0.001%硫柳汞细胞的细胞存活率为（89.65±1.98）%，与0.001%硫柳汞细胞的细胞存活率比较，差异有统计学意义（t=8.51，P＜0.01）。0.00001%、0.00005%、0.0001%、0.0005%及0.001%硫柳汞细胞LC3-Ⅰ蛋白相对表达分别为0.74±0.11、0.86±0.08、0.97±0.09、1.02±0.07及1.14±0.04；LC3-Ⅱ蛋白相对表达分别为0.56±0.10、0.73±0.09、0.97±0.09、1.05±0.08及1.17±0.08，组间比较的差异有统计学意义（F=8.31，22.33；P＜0.05）。对照组、0.00001%及0.001%硫柳汞组结膜细胞NF-κβ、LC3-Ⅰ及LC3-Ⅱ表达量分别108.23±5.88、138.00±3.60、164.46±7.60、90.07±3.48、102.93±2.72、127.43±6.96、107.70±4.20、128.36±7.07及154.01±6.24，比较的差异有统计学意义（F=67.51，47.65，45.39；P＜0.05）；对照组、0.001%水杨酸钠+0.001%硫柳汞组结膜细胞NF-κβ、LC3-Ⅰ及LC3-Ⅱ的表达量分别为164.66±9.29、175.66±12.2、195.33±10.07、141.67±8.50、133.00±15.52及169.33±12.66，比较的差异有统计学意义（t=3.16，3.74，2.78；P＜0.05）。

结论

硫柳汞能通过人结膜上皮细胞自噬，降低细胞存活率且0.001%的水杨酸钠能显著改善此情况。

Objective

The aim of this study is to investigate the effects of different concentrations of thiomersal on the regulation of autophagy in human conjunctival epithelial cells and the protective mechanism of sodium salicylate.

Methods

During cell culture，according to the type of added drugs，they were divided into thiomersal experimental group，sodium salicylate group，thiomersal+sodium salicylate group，and control group.The thiomersal experimental group was treated with thiomersal at concentrations of 0.00001%，0.00005%，0.0001%，0.0005% and 0.001% for 30 min，and then the solution was removed.The sodium salicylate group was pretreated with 0.00001%，0.00005%，0.0001%，0.0005% and 0.001% sodium salicylate for 30 minutes.Thiomersal+sodium salicylate group was pretreated with 0.001% thiomersal+0.001% sodium salicylate for 30 min.The control group contained medium only.The cell viability was measured by MTTassay.Microtubule-associated protein 1 light chain 3-Ⅰand microtubule-associated protein 1 light chain 3-Ⅱ（LC3-Ⅱ）in autophagy-related pathways was detected by Western blot.The protein expressions of nuclear factor kappa-β（NF-κβ），LC3-Ⅰand LC3-Ⅱin autophagy-related pathways were detected by immunofluorescence.The cell survival rate and protein expression level were expressed as±s，and compared by t test for intra-group comparison，and one-way ANOVA was used for inter-group comparison.

Results

With the control group as the base，The cell survival rates of 0.00001%，0.00005%，0.0001%，0.0005%and 0.001% thiomersal groups and sodium salicylate groups were（97.06±1.71）%，（91.76±1.82）%，（89.46±4.67）%，（85.20±3.88）% and（77.93±2.72）%，（98.80±2.27）%，（97.98±3.17）%，（96.83±1.30）%，（96.08±2.40）%，（95.52±2.59）%，respectively.There were significant differences in the thiomersal group（F=30.66，P＜0.05）.There was no significant difference in cell survival rate among different concentrations of sodium salicylate（F=1.86，P＞0.05）.The cell survival rate of 0.001% sodium salicylate+0.001% thiomersal cells was（89.65±1.98）%，which was higher than that of 0.001%thiomersal cells with statistically significant（t=8.51，P＜0.01）.The protein expression of LC3-Ⅰin 0.00001%，0.00005%，0.0001%，0.0005% and 0.001% thiomersal cells were 0.74±0.11，0.86±0.08，0.97±0.09，1.02±0.07 and 1.14±0.04，respectively.The protein expression of LC3-Ⅱwas 0.56±0.10，0.73±0.09，0.97±0.09，1.05±0.08，1.17±0.08，respectively，and the difference was statistically significant（F=22.33，P＜0.05）.The expressions of NF-κβ，LC3-Ⅰand LC3-Ⅱin the control group，0.00001% thimerosal group and 0.001% thimerosal group were 108.23±5.88，138.00±3.60，164.46±7.60，90.07±3.48，102.93±2.72，127.43±6.96，respectively.107.70±4.20，128.36±7.07，154.01±6.24，the differences were statistically significant（F=67.51，47.65，45.39；P＜0.05）.The expressions of NF-κβ，LC3-Ⅰand LC3-Ⅱin the control group and 0.001% sodium salicylate+0.001% thiomersal group were 164.66±9.29，175.66±12.2，195.33±10.07，141.67±8.50，133.00±15.52，169.33±12.66，respectively and the difference was statistically significant（t=3.16，3.74，2.78；P＜0.05）.

Conclusions

The lower the concentration of thiomersal，the higher the cell survival rate，while 0.001% sodium salicylate can significantly improve the cell survival rate and improve autophagy.