Abstract:

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.

Key words: Thiomersal, Autophagy, Microtubule-associated protein 1 light chain-Ⅰ, Microtubule-associated protein 1 light chain-Ⅱ