NF-κB Activation Induced By MAC Aggravates The Liver Immune Injury In Trichloroethylene-sensitized Mice

BackgroundTrichloroethylene is one of the common organic solvent in industry,which is widely used as degreasing detergent and decontamination agent for raw materials and electronic components in electrodeposition,hardware,electronic and other industries.Workers who are exposed to TCE for a period of time may suffer from ODMLT.The main symptoms of ODMLT are severe rashe,fever,superficial lymph nodes and liver damage and severe liver injury is the leading cause of inpatient death.Previous studies find activation of complement,deposition of MAC,and no significant cracking death of liver cells in the immune liver injury of TCE-sensitized mice.MAC may occur at sublytic level to mediate immune liver injury of TCE-sensitized mice.MAC at sublytic level activates signalling pathways,acting as a drive to inflammation.MAC can active NF-κB pathway in retinal pigment epithelial cells,endothelial cells and smooth muscle cells.It is worthy to study whether MAC mediate immune liver injury of TCE-sensitized mice by activation of NF-κB pathway.ObjectiveWe used TCE sensitization animal model as the fundamental and adopted i.p.injection of sCD59-Cys to inhibit deposition of MAC.We deteced the expression of CD59a,deposition of MAC,activation of NF-κB pathway and damage of liver,to investigate the role of MAC in immune liver injury of TCE-sensitized mice and explore its mechanism.MethodsAfter 1 week of adaptive feeding,6-8 week female specific pathogen free BALB/c mice(n=89)were randomly assigned to blank control group(n=5),solvent control group(n=5),sCD59-Cys control group(n=5),TCE treatment group(n=50),TCE+sCD59-Cys treatment group(n=24).A model of TCE-sensitization was generated in the BALB/c mice.24 h after the last challenge,according to the sores of allergic reactions in mouse skin,mice were classified to sensitization positive group and sensitization negative group.Mice were sacrificed at 24 h,72 h and 7 d after the last challenge and serum and liver were harvested.Liver function was evaluated by ALT and AST in conjunction with histopathological characterizations.The transcriptions of TNF-αand IL-1βwere analyzed by real time RT-PCR.The expression of CD59a,P-IκBαand and nuclear NF-κB p65 were studied by western blotting.The distribution of CD59a and deposition of MAC were examined by immunohistochemistry.Results1.Sensitization rate of miceNo mice in the blank control group,solvent control group,and sCD59-Cys control group exhibited erythema and edema.Sensitization rates were 38%(19 positive in 50mice)in the TCE treatment group and 41.7%(10 positive in 24 mice)in the TCE+sCD59-Cys treatment group.2.The liver was damaged in trichloroethylene-sensitized mice.HE staining showed that blank and solvent control groups had no significant abnormalities in liver structure.However,significant liver cell oedema was found in the TCE~+groups at 24 h and 72 h.Especially at 72 h,ballooning degeneration of liver cells was obvious.Liver structure returned to normal in the TCE~+group at 7 d.Results of liver function detection showed there were no significant differences in serum ALT and AST levels between the blank and solvent control groups(P>0.05).The levels of ALT and AST were significantly increased in the TCE~+groups at 24 h and 72 h compared with the solvent control group(P<0.05).The peaks of serum ALT and AST appeared at 72 h in the TCE~+group.At 7 d,both of levels of ALT and AST in the TCE~+group returned to normal(P>0.05).3.Liver inflammatory cytokines were up-regulated in trichloroethylene-sensitized mice.Real time RT-PCR showed that liver TNF-αand IL-1β,two inflammatory cytokine genes,were markedly up-regulated at 24 h(P<0.05),remained increased at 72 h(P<0.05)and recovered at 7 d(P>0.05)in the TCE~+groups compared with the solvent control group.The peaks of TNF-αand IL-1βmRNA levels both appeared at 72 h.There were no significant differences in liver TNF-αand IL-1βmRNA levels between the blank and solvent control groups(P>0.05).4.CD59a was decreased in the liver of trichloroethylene-sensitized mice.The distribution of CD59a was detected by immunohistochemistry.The results showed CD59a was widely distribution in liver tissue in the blank and solvent control groups.However,the expression of CD59a was reduction in the TCE~+groups.The lowest level was appeared in the TCE~+group at 72 h and the expression of CD59a was recovered at7 d.Protein level of liver CD59a was investigated by western blotting.There was no significant difference in CD59a protein level between the blank and solvent control groups(P>0.05).The expression of CD59a protein was decreased at 24 h and 72 h in the TCE~+group(P<0.05)and recovered at 7 d(P>0.05).The lowest expression was in the TCE~+group at 72 h.5.MAC deposition was increased in the liver of trichloroethylene-sensitized mice.Immunohistochemistry was performed to detect MAC deposition in the liver tissues of trichloroethylene-sensitized mice at different time points.There was no visible MAC deposition in the blank control group and solvent control group(P>0.05).However,compared with the solvent control group,deposition of MAC occurred at 24 h in the TCE~+group,peaked at 72 h(P<0.05)and recovered at the 7th day(P>0.05).6.sCD59-Cys alleviated immunological liver injury in the trichloroethylene-sensitized mice.Liver structure in the sCD59-Cys control group was normal.The TCE+sCD59-Cys~+group had a smaller degree of liver cell oedema compared with the TCE~+group.The levels of ALT and AST were not significantly different between the solvent control group and sCD59-Cys control group(P>0.05).For the TCE+sCD59-Cys~+group at 72 h,the levels of ALT and AST were higher than the solvent control group(P<0.05).However,the levels of ALT and AST in the TCE+sCD59-Cys~+group were significantly lower than corresponding TCE~+group(P<0.05).7.sCD59-Cys alleviated MAC deposition in the liver of trichloroethylene-sensitized mice.The results of immunohistochemistry showed there was no visible MAC deposition in the sCD59-Cys control group.Although MAC deposition in the TCE+sCD59-Cys~+group at 72 h was still visible,reduced compared with the TCE~+group(P<0.05).8.sCD59-Cys suppressed NF-κB activation and inflammatory cytokines in the liver of trichloroethylene-sensitized mice.The levels of phosphorylated IκBαand nuclear NF-κB p65 at 72 h after the last challenge were examined using western blotting.We found the levels of phosphorylated IκBαand nuclear NF-κB p65 were signifcantly increased in the TCE~+group and TCE+sCD59-Cys~+group compared with the solvent control group(P<0.05).However,phosphorylated IκBαand nuclear NF-κB p65 in the TCE+sCD59-Cys~+group were significantly decreased compared with the TCE~+group(P<0.05).Compared with solvent control group,TNF-αand IL-1βmRNA expressions in the sCD59-Cys control group showed no significant difference(P>0.05).When compared with the solvent group,expressions of TNF-αand IL-1βmRNA at 72 h in the TCE+sCD59-Cys~+group were increased(P<0.05).TNF-αand IL-1βmRNA expressions in the TCE+s CD59-Cys~+group at 72 h were lower than TCE~+group(P<0.05).Conclusions1.CD59a is involved in the process of immune liver injury in trichloroethylene-sensitized mice.2.Activation of NF-κB pathway occurs in immune liver injury in trichloroethylene-sensitized mice.3.sCD59-Cys alleviates MAC deposition further to inhibit activation of NF-κB pathway to play the protective role in immune liver injury in trichloroethylene-sensitized mice.