| BackgroundTrichloroethylene(TCE)is a non-flammable,volatile,colorless and fat-rich organic solvent.TCE is widely used in the industrial field,mainly used in metal surface treatment agents,electroplating,pre-painting detergents,dry cleaning and textile industry chemical intermediates and extractors,and used as a component of many consumer products.The heavy use of TCE causes some workers who are exposed to TCE to experience extensive skin necrosis and peeling similar to drug rash-like dermatitis.The occupational trenchtype dermatitis diagnostic standard has named the injury "occupational t-chloroethylene drug-like dermatitis"(OMLDT).As a serious systemic occupational skin disease,TCEinduced OMLDT not only causes different degrees of skin damage,but also causes multiorgan damage mainly of liver and kidney damage,which is often one of the causes of death in OMLDT patients.The kidney damage of OMLDT patients tends to occur 3-7 days after liver damage occurs,however,in the progression of OMLDT disease and treatment process,kidney damage is often not paid enough attention.Early research of our group had found that in the process of TCE sensitivity,the sub-lysis level of C5b-9 was able to activate NLRP3 inflammatory small bodies,raise the level of inflammatory factors such as IL-1β and IL-18,and acted on the epithelial cells of the renal tube,aggravated the inflammatory damage of the renal tubular epithelial cell.As a form of inflammatory cell death,pyroptosis can involve the development of a variety of kidney diseases by activating inflammatory small bodies,releasing inflammatory factors.ObjectiveThe main purpose of this study is to explore the mechanism by which complement C5b-9 induces the pyroptosis of renal tubular epithelial cell in the course of renal tubular injury caused by TCE by establishing a model of TCE sensitized BALB/c mice.By Inhibiting the synthesis of C5b-9 by celiac injection of C5b-9 inhibitors(s CD59-cys)and detect kidney pathology,kidney function,expression of C5b-9,inflammatory media and GSDMD proteins,the active of the proteins GSDMD,Caspase-1 and Caspase-11 associated with pyroptosis signaling path in the local kidneys,to further explore the specific mechanism of renal inflammatory damage in TCE-sensitive mice.MethodsSixty-six BALB/c mice were adaptively reared for one week and were randomly divided into blank control group(n=5),vehicle control group(n=5),TCE treatment group(n=30)and TCE+CD59 combined treatment group(n=26),based on the previous research of our research group to establish a TCE percutaneously sensitized BALB/c mouse model.30 minutes before the last challenge on day 19,mice in the TCE+CD59 combined treatment group were intraperitoneally injected with 10μl of phosphate buffer containing 25μg of s CD59-Cys.24 hours after the last challenge,the mice’s back reaction was scored.According to the back skin reaction scoring standard,the skin reaction score≥1 point is judged as the sensitization positive group,and the score of 0 points is judged as the sensitization negative group.According to the skin sensitization score,the TCE treatment group was further divided into TCE positive group and TCE negative group.TCE+CD59 combined treatment components were TCE+CD59 positive group and TCE+CD59 negative group.72 hours after the last challenge,extract relevant biological materials.The corresponding biological materials were collected under aseptic conditions for subsequent experiments.Serum is used to detect mouse creatinine(Cre),urea nitrogen(BUN),albumin(ALB),α1 microglobulin(α1-MG),β2 microglobulin(β2-MG).Take part of the kidneys and make paraffin sections for the detection of pathological structural damage in the kidneys of mice.Double immunofluorescence labeling to localize the expression of GSDMD in the kidney.Immunohistochemical method was used to detect the deposition levels of C5b-9,GSDMD and inflammatory factors(interleukin-1β)IL-1β and IL-18 in the kidneys of mice.Part of the primary renal tubular epithelial cell homogenate was extracted for Western Blot to detect GSDMD,GSDMD-C,Caspase-1,Caspase-1 p20,Caspase-11,Caspase-11 p20 protein levels and q RT-PCR to detect mouse kidney GSDMD,Caspase-1,Caspase-11 gene level.Results1 The sensitization rate of mice in the blank control group and the vehicle control group was 0%.The sensitization rate of the mice in the TCE treatment group was 26.78%.The sensitization rate of the mice in the TCE+CD59 combined treatment group was 26.92%.2 The pathological results of mouse kidneys showed that no obvious pathological damage was found in the kidneys of the mice in the blank control group,the vehicle control group,the TCE negative group and the TCE+CD59 negative group.The kidneys of the mice in the TCE positive group could be observed obvious renal tubule expansion,renal tubule epithelial cells and renal interstitial edema,TCE+CD59 positive group mice kidney injury was significantly reduced.3 The serum CRE,BUN,ALB,α1-MG and β2-MG levels of mice in the TCE positive group and TCE+CD59 positive group were significantly increased,while the levels in the TCE+CD59 positive group were significantly lower than those in the TCE positive group.4 Transmission electron microscopy results of mouse kidney tissue showed that the brush borders of renal tubular epithelial cells in the vehicle control group were neatly arranged,the mitochondrial cristae were clearly visible,the desmosome structure was clear,and no obvious organelle damage was seen.Obvious mitochondrial edema,vacuolar degeneration,disordered and broken internal ridge structure,unclear desmosome structure,and large reduction of organelles were seen in the renal tubular epithelial cells of the TCE-sensitized mice.The damage of renal tubular epithelial cells of mice in the TCE+CD59 positive group was significantly less than that in the TCE positive group.5 The immunohistochemical results of C5b-9 and inflammatory factors IL-1β and IL-18 showed that the damaged renal tubules of mice in the TCE positive group had a large amount of C5b-9,IL-1β,and IL-18 deposition,and no significant deposition of C5b-9,IL-1β,IL-18 was seen in the blank control group,vehicle control group,and TCE negative group.Compared with the TCE positive group,C5b-9,IL-1β,and IL-18 deposition in the TCE+CD59 positive group were significantly reduced.6 The results of the pyroptosis executive protein GSDMD test were found that: double immunofluorescence labeling of renal tubular epithelial cell marker protein CK18 and GSDMD protein showed that in the TCE positive group,GSDMD was mainly deposited on renal tubular epithelial cells,but almost nothing could be found on the glomerulus.Immunohistochemical detection of the expression level of GSDMD protein in the kidney of each group showed that the TCE positive group showed a large amount of GSDMD deposition in the damaged renal tubules,while the blank control group,vehicle control group,and TCE negative group did not see obvious GSDMD deposition.Compared with TCE positive group,GSDMD deposition was significantly reduced in TCE+CD59 positive group.Western blotting was performed to detect the expression levels of the pyroptosis executive proteins GSDMD and GSDMD-C in the renal tubular epithelial cells of mice in each group.The results showed that the protein expression level of GSDMD and its spliced body GSDMD-C in the mouse renal tubular epithelial cells in the TCE positive group was significantly increased,but the blank control group,vehicle control group,and TCE negative group did not see obvious expression levels of GSDMD and GSDMD-C.Compared with the TCE positive group,the expression levels of GSDMD and GSDMD-C in the TCE+CD59 positive group was significantly reduce.The real-time quantitative PCR results also showed that the expression level of GSDMD mRNA in the blank control group,vehicle control group and the corresponding TCE negative group was not statistically different.The GSDMD mRNA expression in the TCE positive group was significantly increased,and was higher than the TCE+CD59 positive group.7 Detection results of proteins related to the pyroptosis pathway in mouse renal tubular epithelial cells were found: Western blot results in the classical pathway showed the protein expression levels of Caspase-1 and its spliced Caspase-1 p20 in mouse renal tubular epithelial cells in the TCE-sensitized group were significantly increased.The expression level of Caspase-1 and Caspase-1 p20 in the blank control group,vehicle control group and TCE negative group was not significantly increased.Compared with the TCE positive group,the expression levels of Caspase-1 and Caspase-1 p20 were significantly reduce in the TCE+CD59 positive group.The real-time quantitative PCR results also showed that there was no statistically significant difference in Caspase-1 mRNA expression between the blank control group,vehicle control group and the corresponding TCE negative group.The expression level of Caspase-1 mRNA in the TCE positive group was significantly higher than the TCE+ CD59 positive group.However,the results of Western blot detection in the non-classical pathway showed that the expression of the Caspase-11 protein in the renal tubular epithelial cells had no significant difference among the groups.The real-time quantitative PCR results also showed that the expression levels of Caspase-11 mRNA in mouse renal tubular epithelial cells were not statistically significant in each group.ConclusionsIn the process of renal tubular inflammatory injury caused by TCE sensitization in mice,the sub-lysis level C5b-9 induces renal tubular epithelial cell pyroptosis by activating the classical Caspase-1 pathway,and aggravates the renal inflammatory injury. |
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