Effects Of Mitochondrial Reactive Oxygen Species-induced NLRP3 Inflammasome Activation On Trichloroethylene-mediated Kidney Immune Injury

BackgroundTrichloroethylene is widely used in industry because of its good lipid solubility,the Occupational disease caused by"Occupational Medicamentosa-like dermatitis due to trichloroethylene（OMLDT）"threat to Occupational health,In severe cases,acute kidney failure can occur,threatening the patient’s life.Previous studies of our group found that NLRP3 inflammasome was activated in the kidney tissues of TCE-sensitized mice,but its specific activation mechanism was not cleared.ObjectiveA TCE-sensitized mouse model was established to investigate whether mitochondrial reactive oxygen species played a role in NLRP3 inflammatory body activation.Mitochondrial reactive oxygen species in renal tubular epithelial cells were reduced by intraperitoneal injection of mitochondrial reactive oxygen scavengers Mito TEMPO in mice.Kidney injury and NLRP3 activation were detected in mice of each group,in order to illustrate whether the activation of NLRP3 inflammatory bodies was mediated by mitochondrial reactive oxygen species and explore its specific mechanism.Methods1.The trichloroethylene sensitized mouse model was established and divided into blank control group,solvent control group,TCE treatment group,TCE+Mito TEMPO treatment group and TCE+sCD59-Cys treatment group,and further divided into TCE sensitized negative group,TCE sensitized positive group,TCE+Mito TEMPO sensitized negative group,TCE+Mito TEMPO sensitized positive group,TCE+sCD59-Cys sensitized negative group and TCE+sCD59-Cys sensitized positive group according to the sensitization situation.2.The urineα1-MG andβ2-MG levels were detected by ELISA to reflect the renal function of mice.HE staining was used to observe the pathological damage of kidney in mice.Ultrastructure of renal tubule epithelial cells was observed by transmission electron microscopy.The deposition of C5b-9 was detected by immunohistochemistry.3.The renal tubular epithelial cells were isolated by enzyme digestion method and identified by immunofluorescence method of CK18.Fluo-8 fluorescent probe was used to detect intracellular Ca²⁺concentration.Mito SOX fluorescent probe was used to detect intracellular mitochondrial reactive oxygen species.Mito Scene Green Ⅱ fluorescent probe was used to detect the number of mitochondria in cells.Mitochondrial membrane potential was detected by JC-1 fluorescence probe.4.Wstern blot was used to detect the expression levels of NLRP3,Caspase-1,ASC,IL-1β,IL-18 and MAVS proteins.Results1.Sensitization rate of mice in blank control group and solvent control group was 0%;and 36.4%in TCE treatment group,33.3%in TCE+Mito TEMPO treatment group,42.1%in TCE+sCD59-Cys treatment group.2.The levels ofα1-MG andβ2-MG in urine of mice were the highest in TCE sensitized positive group,followed by TCE+Mito TEMPO sensitized positive group,and there was no significant difference between blank control group,solvent control group and negative groups.HE staining results showed that a small area of renal tubules were dilated in TCE+Mito TEMPO sensitized positive group,and a large area of renal tubules were dilated and vacuolated in TCE sensitized positive group.TEM results showed that there were a large number of normal mitochondria in the renal tubular epithelial cells of blank control group,solvent control group and negative groups.The mitochondria in renal tubular epithelial cells of TCE sensitized positive group were swollen,crest broke and deformed,and the mitochondria in TCE+Mito TEMPO sensitized positive group were less abnormal.3.Immunofluorescence results of renal tubular epithelial cell identification showed that CK18 protein almost completely overlapped with DAPI.4.The content of mitochondrial reactive oxygen species in the renal tubule epithelial cells of mice in TCE sensitized positive group was higher,and the mitochondrial reactive oxygen species were effectively eliminated after Mito TEMPO was applied.JC-1fluorescence results showed that the mitochondrial membrane potential in TCE sensitized positive group was lower than that in solvent control group,JC-1 green fluorescence was significantly increased,JC-1 red/JC-1 green was significantly decreased.In addition,compared with the solvent control group,the number of mitochondria in TCE sensitized positive group was significantly reduced,but the mitochondrial damage caused by TCE sensitization could be alleviated after Mito TEMPO was applied.5.The expression level of C5b-9 in mouse kidney was detected by immunohistochemistry,and the results showed that there was a large amount of C5b-9 deposition in renal tubules of mice in TCE sensitized positive group,and less deposited in TCE+sCD59-Cys sensitized positive group.The concentration of Ca²⁺was the highest in TCE sensitized positive group,followed by TCE+sCD59-Cys sensitized positive group.The lowest values were found in blank control group,solvent control group and sensitization negative group.6.Western blot results showed that NLRP3,Caspase-1,ASC,IL-1β,IL-18 and MAVS were increased in TCE sensitized positive group compared to the solvent control group,Mito TEMPO significantly inhibited the expressions of NLRP3,Caspase-1,ASC,IL-1βand IL-18.ConclusionsThe activation of NLRP3 in renal tubular epithelial cells of TCE sensitized mice is related to mitochondrial reactive oxygen species（ROS）.Injection of Mito TEMPO can reduce the activation of NLRP3 and alleviate renal tubular injury to a certain extent.