Intracellular Complement Activation Involve In Liver Damage Of Trichloroethylene-sensitized BALB/c Mice

BackgroundTrichloroethylene（TCE）,molecular formula for C₂HCl₃,boiling point 86.7℃,is the industry commonly used halogenated hydrocarbons,colorless transparent liquid,soluble in water,with a variety of organic solvents miscibility.Due to its strong solubility,it is commonly used in industrial production as metal detergent,refrigerator refrigerant,insecticide and extractant.Some workers exposure to trichloroethylene will have health damage effects on the body,its characteristic is similar to the severe drug allergy reaction caused by systemic skin disease,also can happen at the same time the whole body fever,liver and kidney disease,and clinical manifestations such as superficial lymph node enlargement,serious and even lead to multiple organ failure and death,Including acute,chronic and subacute effects.Occupational medicamentosa-like dermatitis due to trichloroethylene（ODMLT）is a systemic allergic disease mainly manifested by acute skin inflammatory response,accompanied by multiple organ injuries.ODMLT can occur after a considerable number of workers are exposed to TCE.The disease has a high mortality rate and rapid development of the disease,which has brought considerable health hazards to the workers in occupational contact,among which liver damage is an important cause of death.Why the harm of ODMLT T is so serious,studies have found that in addition to late type IV allergy,the complement system also plays an important role in the pathogenesis of the disease.Our previous studies have shown that complement activation may play a key role in the generation andprogressionofimmune-mediatedhepaticinjurybyexposureto trjchloroethylen（TCE）.Studies have shown that C3 activation occurred intracellular and protease Cathepsin L（CTSL）processe C3 into biologically active C3a and C3b.The activated product can bind to the receptor of the corresponding complement on lysosome and produce a series of biological effects.We examined the role of CTSL and complement activation in the liver injuries using a mouse model of TCE-induced sensitization by skin sensitized TCE.ObjectiveIn this study,a trichloroethylene-sensitized mouse model was used to intraperitoneally inject CTSL inhibitor and kupffer cell inhibitor gadolinium trichloride（GdCl₃）into mice of different groups.Through the observation of the skin sensitization,liver C3,CTSL,liver function indexes and liver pathological sections of different groups of mice,the role of complement activation in liver cells mediated by CTSLin the liver injury induced by trichloroethylene was investigated.MethodsIn the experiment,130 female mice of BALB/c were selected,TCE treatment group,TCE+CTSL inhibitor combined treatment group（later called CTSL inhibitor treatment group）,and TCE+GdCl₃ inhibitor combined treatment group（later called GdCl₃inhibitor treatment group）.Animals of the corresponding group were killed at 24h,72h and 7d after the last excitation,and corresponding biological samples were collected.To observe the skin sensitization of mice,and observe the liver histology sections of mice under the microscope.Use the alanine aminotransferase（ALT）and aspertate aminotransferase（AST）kit to detect serum ALT and AST level,immunohistochemical detection of renal expression of complement and CTSL,immunofluorescence C3 and CTSL expression in the detection of liver cells,real time quantitative PCR detection of C3 and CTSL expression in liver.Results1.Sensitization rate of miceIn this sensitization experiment,the sensitization rate of TCE treatment group of mouse sensitization model was 32.6%;The sensitization rate of mice treated with cathepsin L inhibitor was 30.4%and that of mice treated with GdCl₃ was 28.3%.In the blank control group and the solvent control group,there was no obvious abnormality such as erythema and edema.Liver HE staining showed that the local area of mouse liver in TCE sensitized group showed cell edema.Mice induced by TCE+CTSL inhibitor showed significantly reduced liver cell edema and decreased damage.2.The organ coefficien and the appraisal of liver gross appearanceNo obvious abnormal liver lesions were found in the blank control group and the solvent control group.No obvious abnormalities were found in the liver of the non-sensitized TCE group.Liver enlargement was observed in TCE sensitized positive group,and organ coefficient was significantly higher than that in blank and solvent control group（P<0.05）.3.Result of liver functionCompared with the serum ALT and AST levels of the solvent control group and the blank control group,P>0.05.The expression levels of ALT and AST in the TCE sensitized positive 24h group were higher（P<0.05）.ALT and AST expression levels were significantly increased in the tce-sensitized 72h group（P<0.05）,while ALT and ASTincreased7 day（P<0.05）.The serum levels of ALT and AST in the CTSL inhibitor sensitization group were also increased compared with the solvent control group and the blank control group at each time point,but the elevation was decreased compared with the TCE sensitizationP<0.05.The serum ALT and AST levels of the sensitized group of GdCl₃ treatment group at each time point were also increased compared with the solvent control group and the blank control group,but the elevation was decreased compared with the TCE sensitized group at the corresponding time point.Compared with the solvent control group,ALT and AST expression levels of the non-sensitized group at the corresponding time points in each treatment group showed no statistically significant difference P>0.05.In addition,according to the liver function indexes,all the indexes reached the peak at 72h after the last stimulation,and then the indexes would gradually recover to normal values.4.The expression of C3 fragments in liverImmunohistochemical results showed that C3 was expressed in all TCE sensitized groups,but mainly in the liver of TCE sensitized positive group 72h,which was consistent with the trend of liver function,The expression level decreased in the CTSL inhibitor sensitization positive group 72h,while no significant expression was found in the blank control group,solvent control group and the two non-sensitized groups;In the GdCl₃ treatment group,the expression level also decreased in the 72h positive group.According to the immunofluorescence test,C3 was mainly expressed in the TCE sensitized 72h group,CTSL inhibitor sensitized 72h group and C3 was decreased in the GdCl₃ treatment group,while C3 was not significantly expressed in other groups.the blank control group and the solvent control group.The C3 expression level in the CTSL inhibitor sensitized 72h group was significantly lower than that in the TCE sensitized72h group.5.The expression of CTSL in liverImmunofluorescence detection showed that CTSL was expressed in liver of each group,but the expression level was significantly different between the groups.Among them,CTSL expression was significantly increased in the TCE sensitized positive group 72h,the blank group and the solvent group,while CTSL expression was significantly decreased in the CTSL inhibitor group.Meanwhile,CTSL expression was also significantlydecreasedintheGdCl₃treatmentgroup.Theresultsof immunohistochemistry showed that CTSL was also expressed in the liver of each group,in which the expression of CTSL was significantly increased in the tce-sensitized positive group 72h,the blank group and the solvent group,and significantly decreased in the CTSL inhibitor group.Meanwhile,the expression of CTSL was also significantly decreased in the GdCl₃ treatment group.6.The expression of C3 and CTSLmRNA in liver tissueCTSLmRNA levels of the blank control group and the solvent control group were not statistically significant（P>0.05）.Compared with the solvent control group,CTSLmRNA expression level in the TCE sensitized 24h group was increased,both indicators expression level in the TCE sensitized 72h group was significantly increased,and CTSLmRNA expression level in the TCE sensitized 7d group was increased,all differences were statistically significant（P<0.05）.However,partial indexes of the CTSL inhibitor sensitization group also increased compared with that of the solvent control group at each time point,but the elevation was decreased compared with that of the TCE sensitization group;The sensitization group of GdCl₃ treatment group also showed an increase in the index at each time point compared with the solvent control group,but the increase was decreased（P<0.05）.There was no statistically significant difference between C3 and CTSLmRNA in the non-sensitized group and the blank group and the solvent control group（P>0.05）.7.Result of liver histopathologyNo pathological lesions were found in the liver of the non-sensitized groups.Liver lesions occurred in the TCE sensitized 24 h group and the 72 h group,common balloon degeneration,which was more severe in the 72h group,including liver cell degeneration,liver cell balloon degeneration,and liver cell edema was significantly reduced in the TCE+CTSL inhibitor sensitization group and the GdCl₃ treatment group.TCE sensitized7 d group gradually returned to normal liver tissue.Conclusions1.the sensitization rate in this experiment was 32.6%.2.Activation of intracellular complement may be involved in liver injury.3.i.p.injection of CTSL inhibitor and Kupffer cells inhibitor GdCl₃ before challenge significantly reduced the liver injury induced by TCE sensitization;4.In the immunological liver injury of BALB/c mice induced by TCE,CTSL may mediate complement activation in liver cells.5.CTSL can regulate liver complement activation and play an important role in immune liver injury induced by TCE.6.Complement activation in hepatic kupffer cells may be an important cause of hepatic injury,providing new ideas for the selection of targeted drugs for clinical treatment.