Effects Of Trichloroethylene Intake By Drinking Water On Regulatory T Cells In Mice

Backgroud Trichloroethylene(TCE) is mainly used as metal cleaning and degreasingin industry. With the rapid development of electronic industry, the usage increases yearafter year, TCE has become important professional poison and environmental pollutant.In addition to cause significant target organ toxicity, some studies show that TCEexposure is associated with immune correlation effect including auto-immunity, injuriedimmune system contributes to the occurrence and development of tissue injury. but themechanism that TCE exposure causes immune injury is unclear at present.Objective By detecting Treg cells number and Foxp3mRNA contents in spleen andIL-10contents in serum, to investigate effects of TCE intake by drinking water on Tregcells in mice; Combined with the detection of liver injury and pro-inflammatorycytokines in liver in mice, to analysis effects of TCE intake by drinking water on Tregin mice and the relationship with liver injury.Methods Female mice, which were6weeks old, were adopted at SPF animal room.After adaptive feeding a week, the mice were divided to blank control group, solvent(DMSO) control group,2.5mg/ml and5mg/ml TCE group in random. At2，4，8，12weeks, blood were collected, liver and spleen biopsies were taken. Treg cells number inspleen were measured by flow cytometry, Foxp3mRNA contents in spleen weremeasured by qRT-PCR, IL-10contents in serum were evaluation by ELISA; Serumwere also prepared and assayed hepatic functions, Sterile livers for pathologicalexamination, the levels of IL-1、IL-6and TNF-α in liver were evaluation by ELISA.Experimental results were analyzed with SPSS13.0. Results1General condition Each group of mice were in good condition throughout thetrial. No deaths occured because of infection. There were not significant differences inaverage daily water intake(F=0.89),The average daily water intake was0.13ml/(g·d),the average exposure levels of2.5mg/ml and5mg/ml TCE group were450mg/(kg·d)and730mg/(kg·d) respectively. There were not significant differences on growth ofweight and spleen organ coefficient at each time point and group.2Effects of TCE intake by drinking water on Treg2.1Effects on Treg cells number in spleen At2,4,8,12weeks, Treg cells were notsignificantly different between vehicle group and blank group; Compared with vehiclegroup and blank group, Treg cells in TCE treated groups were all significantlydecreased at2,4weeks（P<0.05）,and Treg cells in5mg/ml TCE groups decreasedlarger than that in2.5mg/ml TCE groups. this trend began to swing at8,12weeks, onlyTreg cells in5mg/ml TCE group at8weeks were significantly lower than blankgroup（P<0.05）, there were not significant difference in other groups.2.2Effects on Foxp3mRNA express in spleen At2,4,8,12weeks,,Foxp3mRNAexpress in spleen were not significantly different between vehicle group and blankgroup; Compared with vehicle group and blank group, Foxp3mRNA express in TCEtreated groups were all significantly decreased at2,4,8weeks（P<0.05）and wereminimum at4weeks. Foxp3mRNA express in5mg/ml TCE groups decreased largerthan that in2.5mg/ml TCE groups. This trend began to swing at8,12weeks.At12weeks, there were not significant difference in the four groups.2.3Effects on IL-10in serum At2,4,8,12weeks, IL-10levels in liver were notsignificantly different between vehicle group and blank group; Compared with vehiclegroup and blank group, IL-10levels in TCE treated groups were all significantlydecreased at2,4weeks（P<0.05）and were minimum at4weeks. there were not significant difference at8,12weeks.2.4The correlation between Treg cells number and Foxp3mRNA express inspleen, IL-10in serum Correlation analysis showed that Treg cells number werepositively significantly correlated with Foxp3mRNA express in spleen and IL-10inserum（r=0.630，0.593，P<0.01）.3Effects of TCE intake by drinking water on liver injury3.1Effects on liver function At2,4,8,12weeks, ALT and AST levels were notsignificantly different between vehicle group and blank group; Compared with vehiclegroup and blank group, ALT and AST levels in TCE treated groups were allsignificantly increased at2,4weeks（P<0.05）and were maximum at4weeks. This trendbegan to swing at8,12weeks,but ALT and AST levels in TCE treated groups were stillhigher than that in vehicle group and blank group.3.2Pathological test results in liver In the control group and vehicle control group,liver cells arranged in neat, with large round nuclei, clear nuclear membrane anduniform cytoplasm; Obvious inflammatory cells infiltration were seen in TCE treatedgroups and were most obvious at4weeks, inflammatory cells infiltration decreased at8,12weeks.3.3Effects on pro-inflammatory cytokines in liver3.3.1Effects on IL-1in liver At2,4,8,12weeks, IL-1levels in liver were notsignificantly different between vehicle group and blank group; Compared with blankgroup, IL-1levels in5mg/ml TCE treated groups were significantly increased at2,4week（sP<0.05）and were maximum at4weeks. this trend began to swing at8,12weeks,but IL-1levels in TCE treated groups were still higher than that in vehicle group andblank group.3.3.2Effects on IL-6in liver At2,4,8,12weeks, IL-6levels in liver were not significantly different between vehicle group and blank group; Compared with vehiclegroup and blank group, IL-6levels in5mg/ml TCE treated groups were all significantlyincreased at2,4,8weeks（P<0.05）and were maximum at4weeks, IL-6levels in2.5mg/ml TCE treated groups were also significantly increased at8weeks（P<0.05）.3.3.3Effects on TNF-α in liver At2,4,8,12weeks, TNF-α levels in liver were notsignificant different between vehicle group and blank group; Compared with vehiclegroup and blank group, TNF-α levels in TCE treated groups were all significantlyincreased at2,4,8weeks（P<0.05）and were maximum at4weeks.3.4The correlation between liver function and IL-1、IL-6、TNF-α in liverCorrelation analysis showed that liver function positively correlated with IL-1、IL-6、TNF-α in liver(r=0.526,0.510,0.614,0.518,0.525,0.630, P<0.01).4The correlation between liver function、IL-1、IL-6、TNF-α in liver and Tregcells number in spleen Correlation analysis showed that liver function、IL-1、IL-6、TNF-α in liver positively correlated with Treg cells number in spleen(r=0.723,0.632,0.525,0.419,0.567, P <0.01).Conclusion1TCE intake by drinking water has significant effects on Treg in mice. Treg cellsnumber、Foxp3mRNA expression and IL-10contents significantly decreased.2TCE intake by drinking water can cause liver injury, the levels of ALT、AST andpro-inflammatory cell increased, inflammatory cell infiltration were seen.3Effects of TCE intake by drinking water on Treg in mice significantly correlatedwith liver injury.