| BackgroundTrichloroethylene（TCE）is an organic solvent with good lipid cleaning ability,which has been widely used in the industrial field.Few workers exposed to TCE manifested a large area of epidermal necrosis and peeling of the skin,similar to drug eruption,accompanied by multiple organs damage such as liver and kidney,which was defined as a legal occupational skin disease in our country,named "occupational trichloroethylene drug rash dermatitis"（OMDT）.Kidney injury often affects the progression of OMDT and aggravates the severity of the disease.However,the mechanism has not been elucidated.Previous studies have found that TNF-α plays a key role in TCE-induced renal injury,and the specific mechanism is unclear.TNF-α is involved in many biological processes of cell survival and death through apoptosis,necroptosis and NF-κB signaling pathways.The results of electron microscopy and HE staining showed that the renal tubular epithelial cells of TCE sensitized mice had the morphological characteristics of necrosis.necroptosis is a new form of immunogenic programmed cell death.TNF-α induce cell membrane rupture and inflammatory response via activating the RIPK3/MLKL necroptosis signaling pathway.Recent studies have shown that necroptosis plays an important role in the amplification of renal tubule inflammation in renal diseases.However,it is worth to investigate whether TNF-α mediates TCE-sensitized renal injury by initiating tubular cell necroptosis.ObjectiveThe main purpose of this study was to investigate whether the TNF-α/TNFR1 axis mediates TCE-sensitized kidney injury by initiating tubular epithelial cell necroptosis.TCE-sensitized BALB/c mouse model was established,and then intraperitoneal injection of R7050（TNF-α receptor antagonist）pretreatment was used to inhibit the downstream molecules of TNF-α/TNFR1.Renal pathology,renal function,electron microscopy,and the expression levels and localization of TNF-α,TNFR1,RIPK3,MLKL,and HMGB1 in the kidney were investigated to explore the mechanism of renal tubule inflammatory injury in TCE-sensitized mice.Urine α1-MG,β2-MG,NGAL and serum TNF-α levels in OMDT patients were detected to provide further support for animal experiments.MethodsA total of 7 OMDT patients and 21 normal controls were enrolled as subjects in Guangdong Occupational Disease Prevention and Control Hospital from January 2017 to January 2021.OMDT patients received symptomatic treatment including anti-infection,liver protection and yellow removal,plasmapheresis,anti-allergy,and glucocorticoid combined with gamma globulin after admission.Serum and urine of patients before and after treatment and normal controls were collected for biological detection.Urine α1-MG,β2-MG and NGAL levels were detected to evaluate the renal tubular injury of OMDT patients,and the expression level of serum TNF-α was also detected.After adaptive feeding,71 BALB/ C female mice were randomly divided into four groups according to body weight,namely blank control（6 mice）,solvent control（6 mice）,TCE treatment（30mice）and TCE+R7050 combined treatment group（29 mice）.According to the previous modeling method,TCE-sensitized BALB/ C mouse model was established.TCE+R7050group mice was intraperitoneal injected 100μ L phosphate buffer containing R7050（12mg/kg）2 h before the challenge on days 17 and 19.The dorsal response of the mice was scored 24 hours after the last challenge.The positive ＆ negative group were determined according to the back skin-scored standard,named as TCE sensitization positive ＆negative group,and TCE+R7050 sensitization positive ＆ negative group.Serum and kidney were obtained from mice 72 h after the last challenge.Serum was used to detect creatinine（Cre）,urea nitrogen（BUN）,albumin（ALB）,α1-microglobμlin（α1-MG）,andβ2-microglobμlin（β2-Mg）.Part of the kidney was made into paraffin sections for the detection of renal pathological damage in mice.Double immunofluorescent staining revealed co-localization of P-RIPK3 and P-MLKL with renal tubular epithelial cells marker.The deposition levels of TNF-α,TNFR1,and HMGB1 were determined by immunohistochemistry in the kidney of mice.The protein expression levels of TNF-α,TNFR1,RIPK3/P-RIPK3 and MLKL/P-MLKL were detected by Western Blot.Results1 ELISA results showed that higher level of urinary NGAL,α1-MG,and β2-MG protein were found in OMDT patients before clinical treatment than after clinical treatment and normal control.2.After the establishment of TCE sensitization model,the sensitization of mouse skin was evaluated.The skin of mice in blank and solvent control group had no significant change,but the skin of the sensitized mice showed different degrees of erythema and edema.The sensitization rates of mice in TCE treatment group and TCE+R7050combined treatment group were respectively 33.33% and 31.03% and there was no significant difference（P > 0.05）.3.HE staining results of mouse skin showed that the epidermal cell space was widened,the epidermal layer was significantly thickened,and a lot of inflammatory cells infiltrated the dermis in the TCE-sensitized group.However,no obvious pathological damage was found in the blank,solvent control and TCE sensitization negative group,and no obvious thickening of the epidermis layer was found.4.Renal HE staining results showed that no obvious pathological damage was found in the kidney of mice in blank control,solvent control,TCE sensitization negative and TCE+R7050 negative group.Renal tubule epithelial cell swelling,vacuolar degeneration and renal interstitial edema were observed in the kidney in TCE sensitization positive group mice.The pathological damage of TCE+R7050 sensitized mice was reduced.5 The serum levels of CRE,BUN,ALB,α 1-Mg and β 2-Mg in TCE+R7050 sensitization group and TCE+R7050 sensitization group were significantly increased,and the serum renal function（renal tubule）in TCE+R7050 sensitization group was recovered to a certain extent compared with that in TCE+R7050 sensitization group.6.Transmission electron microscopy（TEM）results of mouse kidney tissue showed that the mitochondrial structure of renal tubule epithelial cells in blank and solvent control groups and TCE sensitization negative group were normal,with clear mitochondrial crest and neat brush edge.And,renal tubular epithelial cells were characterized by cell membrane rupture,endoplasmic reticulum expansion,mitochondrial edema,vacuolar degeneration,and other morphological characteristics of necroptosis in the TCE sensitized positive group.7 The expression of RIPK3/MLKL-mediated necroptosis pathway in mice was detected by immunofluorescence and western blot.Double immunofluorescent results of renal tubular epithelial cell marker protein（CK18）with p-RIPK3 and P-MLKL proteins showed that p-RIPK3 and P-MLKL were mainly expressed in renal tubular epithelial cells in the TCE-sensitized group,while was significantly reduced in the TCE+R7050sensitized group（P<0.05）.Western blotting results showed that the expression of PRIPK3 and P-MLKL in TCE+R7050 sensitization group was significantly increased,while the expression in TCE+R7050 sensitization group was significantly decreased compared with TCE sensitized group（P<0.05）.And,no significant expression was observed in other groups（P>0.05）.8 immunohistochemistry resμlts showed that HMGB1 nuclear cytoplasmic shift occurred in damaged renal tubules of TCE-sensitized mice,while HMGB1 nuclear cytoplasmic shift was significantly reduced in TCE+R7050 sensitized tubules compared with TCE sensitized tubules.HMGB1 in blank control group,solvent control group,TCE sensitized negative group and TCE+R7050 sensitized negative group was mainly highly expressed in the nucleus.ELISA results of serum HMGB1 in mice showed that there was no significant difference in HMGB1 expression between blank control group,solvent control group and TCE sensitized negative group（P>0.05）.The expression level of HMGB1 in TCE sensitization positive group and TCE+R7050 sensitization positive group was significantly increased,while the expression level of HMGB1 in TCE+R7050sensitization positive group was decreased to a certain extent compared with that in TCE sensitization positive group（P<0.05）.9 The expression of TNF-α/TNFR1 axis in mice was detected by immunohistochemistry and western blot.Immunohistochemical results showed that large amounts of TNF-α and TNFR1 deposits were found in the damaged renal tubules of mice in the TCE sensitization positive group.The deposition of TNF-α and TNFR1 in TCE+R7050 sensitized group was significantly reduced compared with TCE sensitized group（P < 0.05）.The expression of TNF-α and TNFR1 in the TCE+R7050 sensitized group was significantly increased,and the expression of TNF-α and TNFR1 in the TCE+R7050 sensitized group was significantly decreased compared with that in the TCE+R7050 sensitized group（P<0.05）.While no significant expression was observed in other groups（P>0.05）.10 HE staining results of mouse kidney showed that the morphological changes of renal tubular cells in TCE+R7050 sensitized group were improved compared with that in TCE sensitized positive group.Compared with TCE+R7050 sensitization group,the serum levels of CRE,BUN,ALB and β2-MG in TCE+R7050 sensitization group were decreased（P<0.05）.Conclusions1 RIPK3/MLKL-mediated RTECs necroptosis may be involved in renal inflammatory injury induced by TCE sensitization,and TNF-α/ TNFR1 may be a key trigger of RTECs necroptosis.2 To some extent,our results support that TNF-α/TNFR1 axis as a clinical therapeutic target for OMDT.R7050 may be a new targeted drug with potential clinical application. |
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