Inhibition Of Kupffer Cells Activity Affects IL-6/STAT3 Signaling Pathway In Immunological Liver Injury In Trichloroethylene Sensitized Mice

BackgroundTrichloroethylene is a colorless liquid which is easily oxidized,volatile,transparent,and has strong solvency.It is a good solvent that often used as a dry cleaning agent,and it is widely used in production and life.Occupational exposure to trichloroethylene can be absorbed into workers through the skin,digestive tract,and respiratory tract.It can trigger occupational trichloroethylene drug-like dermatitis（OMLDT）which can also cause severe liver damage.In addition to the drug-like skin lesions,it makes a serious threat to the health status of the relevant occupational groups.Some studies found that liver damage induced by various causes,and it is closely related with hepatocyte apoptosis.Therefore,hepatocyte apoptosis and the expressions of apoptosis-related molecules are important for revealing the molecular mechanism of TCE induced liver injury.In the progression of liver injury and hepatocyte necrosis,KCs secretes a large number of inflammatory mediators,for example,IL-6.IL-6 is a cytokine which widely present in body,it can mediate a large number of biological responses and activate STAT3 protein.IL-6 can also regulate the expressions of apoptosis-related molecules and play an important biological role in the process of hepatocyte apoptosis.This study inhibited KCs activation by intraperitoneal injection of GdCl₃,and explored the mechanism of hepatocyte apoptosis in a mouse model of trichloroethylene sensitization.ObjectiveBased on the model of trichloroethylene-sensitized mice,a part of mice were intraperitoneally injected with GdCl₃（KCs inhibitor）.The apoptosis mechanism of IL-6/STAT3 signaling pathway in liver cell of trichloroethylene-sensitized mice was investigated by detecting the expression of IL-6/STAT3 related protein and apoptosis-related molecular in the liver of different groups.MethodA total of 36 BALB/c mice were purchased from the animal room of Anhui Medical University.The mice were Female,6-8 weeks old,randomly divided into four groups,as follows:blank control group（5 mice）,solvent（olive oil and acetone）control group（5 mice）,TCE treatment group（11 mice）,TCE+GdCl₃ combined treatment group（15mice）,established TCE sensitized BALB/c mouse experimental model,24 hours after the last challenge,the TCE treatment group and the inhibitor group mice were divided into the sensitization group and the non-sensitization group according to the skin reaction score.Mice were sacrificed on the 72 hours after the last challenge and the desired biomaterial was taken.The apoptosis of liver cells was observed by TUNEL staining.The mRNA levels of IL-6R and IL-6 were detected by RT-PCR.The levels of IL-6R,STAT3,P-STAT3,Bcl-2,Bax,Caspase-3 and Caspase-9 were detected by WB.The levels of IL-6,IL-6R and CD68 in the liver were detected by immunohisto-chemistry.Results1.Sensitization:The skin of the back of the blank control and solvent control mice showed no visible damage.The sensitization rate was 5/11（45.45%）in the TCE treatment group,5/15（33.33%）in the TCE+GdCl₃ treatment group,and the overall sensitization rate in the mice was 42.31%.2.TCE-sensitized mice with liver damageThe results of TUNEL staining showed that there were a small amount of liver cell apoptosis in the liver tissue of the blank control and solvent control mice,and a large number of hepatocyte apoptosis in the TCE sensitized group.GdCl₃ attenuated the hepatocyte apoptosis rate in the TCE sensitized group,and the difference was statistically significant（P<0.05）.3.GdCl₃ inhibits CD68 expression in TCE-sensitized groupThe expression of CD68 in liver was detected by immunohistochemistry.CD68 was highly expressed in TCE sensitized group.The expression of CD68 was lower in TCE+GdCl₃ sensitized group than that in TCE sensitized group.4.GdCl₃ inhibits IL-6 and IL-6R expression in TCE-sensitized groupCompare to blank,solvent and TCE negative groups,IL-6 and IL-6R gene expression levels were increased in TCE sensitized group,IL-6 and IL-6R expression in TCE+GdCl₃ sensitized group was lower than the TCE sensitized group,and the difference was statistically significant（P<0.05）.The results of immunohistochemistry isconsistent with the result of RT-PCR.The WB results showed that the expression of IL-6R in the TCE sensitized group was higher than that in the blank control group,the solvent control group and the negative group,and the expression of IL-6 and IL-6R in the TCE+GdCl₃ sensitized group were decreased when compared with TCE sensitization group（P<0.05）.5.STAT3 and P-STAT3 expression levels in the liverThe expressions of STAT3 and P-STAT3 in liver were detected by WB.The expression of STAT3 in each group were consistent and there was no statistical difference（P>0.05）.The expression of P-STAT3 in TCE sensitized group was reduced when compare with TCE+GdCl₃ sensitized group（P<0.05）.6.Expression of apoptosis-related proteins in the liverWB was used to detect the expressions of apoptosis-related proteins in the liver.The expressions of Bax,Caspase-3 and Caspase-9 in the TCE sensitized group were significantly higher than that in the blank group and the solvent control group,and the expressions in the inhibitor sensitized group were significantly down regulated（P<0.05）.The expression of Bcl-2 in the TCE sensitization group was significantly lower than that in the vehicle control group（P<0.05）.The value of Bcl-2/Bax in the TCE sensitization group was lower than that in the other groups（P<0.05）.Conclusions1.GdCl₃ effectively inhibits KCs activation in the liver which caused by TCE;2.IL-6/STAT3 was activated during the immunological liver injury in TCE-sensitized mice;3.GdCl₃ may down-regulate the activation of KCs and decrease the level of IL-6,then inhibit the activation of IL-6/STAT3 signaling pathway to protect liver cells from TCE-sensitized mice.