Oxidative Stress Mediates Nrf2/HO-1 And JAK1/STAT3 Pathways And Participates In The Liver Injury Of Mice Sensitized By Trichloroethylene

BackgroundTrichloroethylene(TCE),as a commonly used traditional industrial organic solvent,is widely considered as an occupational and environmental pollutant.It is often used as an extractant,refrigerant,dry cleaning agent,organic synthesis,and pesticide production.Long-term exposure to TCE can cause damage to the nervous system,internal organs and skin.Some serious patients suffer from occupational medicamentosa-like dermatitis due to TCE(OMLDT).The treatment cycle of this disease is long and the organs are affected.Injury is a common disease,and liver failure is one of the main causes of death.In clinical observation of OMLDT patients,abnormal liver function was found.Bultrasound examinations of some patients showed liver enlargement.The previous research of the research group found that TCE-sensitized animals also had liver damage,and TCE-sensitized animals had obvious oxidative reactions.The surge level has increased.Oxidative stress mediates related organ damage,and intraperitoneal injection of tetramethylpiperidine(4-Hydroxy-TEMPO,Tempol)can relieve the body’s peroxidation and reduce oxidative damage.Studies have shown that oxidative stress is closely related to nuclear factor E2-related factor 2(NF-E2-related factor 2,Nfr2)and hemeoxygenase 1(HO-1)proteins,and can mediate the release of inflammatory factors.Body injury.Stimulation of cytokines such as ROS in vivo can mediate the activation of Janus kinase(Janus.protein.tyrosine.kinase,JAKs)and induce the activation of downstream signal activators of transcription(STAT).The JAK/STAT signaling pathway is in the cell It plays an important role in proliferation,differentiation,apoptosis,and immune response.ObjectiveIn this study,based on the TCE sensitization model,this study was compared by intraperitoneal injection of the antioxidant Tempol in mice to analyze the liver oxidative stress levels and the expression levels of Nrf2/HO-1,JAK1/STAT3 signaling pathwayrelated proteins and cytokines in each group of mice To explore the mechanism of oxidative stress in immune liver injury induced by TCE in mice.MethodsForty female SPF Balb/c mice were randomly divided into blank group(5),solvent group(5),TCE group(15)and TCE+Tempol group(15),using the classic TCE of the research group The sensitization model continues to be studied.The animals in the TCE+Tempol group were intraperitoneally injected with Tempol solution(100mg/kg)before the last challenge on the 19 th day,and 24 hours after the last challenge,the skin sensitization score was scored according to the skin reaction on the back of the mouse.All mice were divided into sensitization-positive group and sensitization-negative group.72 hours after the last challenge,the eyeballs were removed for blood,and the mice were sacrificed by cervical dislocation.The tissue samples were collected under sterile conditions.HE staining to observe liver histopathology,kit to detect serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels,kit to detect MDA and ROS content in liver tissue,Western Blot to detect Nrf2/HO-1,JAK in liver tissue/STAT signal pathway related proteins,NLRP3,Caspase-3 and IL-1β expression levels,RT-PCR detection of Nrf2,HO-1 and NLRP3 gene level differences,IHC detection of Caspase-3,IL-1β expression levels in liver tissue.Results1.Skin scoreThere was no obvious abnormality in the skin of the mice in the blank group and the solvent group.Some mice in the TCE group and TCE+Tempol group showed different degrees of erythema and edema in the experimental parts of the mouse.The sensitization rate of the TCE group was 40%,and the TCE+Tempol group caused The sensitivity rate was 33.3%.2.Liver functionSerum AST and ALT activity values were not statistically different between blank group,solvent group and TCE sensitization negative group;compared with solvent group and TCE sensitization negative group,the TCE sensitization positive group and the TCE+Tempol sensitization positive group both increased AST and ALT,and the difference was statistically significant(P < 0.05);the AST and ALT values of the TCE+Tempol sensitization positive group were down-regulated compared with the TCE sensitization positive group,and the difference was statistically significant(P<0.05).3.Liver pathology testThere were no obvious pathological changes in the liver cells of the blank group,solvent group,and sensitization-negative group.There was vacuolar degeneration in the liver cells of the TCE-sensitized positive group,the cells were obviously edema,and the cytoplasm was loose.TCE+Tempol sensitized The positive group showed hepatocyte edema-like manifestations in some tissues,which was improved compared with the TCE sensitization positive group.4.Liver MDA and ROS contentThe contents of MDA and ROS in the liver of mice were not statistically different between blank group,solvent group and TCE sensitization negative group;compared with solvent group and TCE sensitization negative group,the contents of MDA and ROS in the TCE-sensitized positive group increased,and the difference was statistically significant(P<0.05);TCE+ The contents of MDA and ROS in the Tempol sensitized positive group were lower than those in the TCE sensitized positive group,and the difference was statistically significant(P<0.05).5.The expression level of Nrf2/HO-1 and JAK/STAT signaling pathway related proteins in liver tissueThere was no statistical difference in the gray value of each protein band in blank group,solvent group and TCE sensitization negative group;JAK-1 and STAT-1expression levels were not statistically different between the groups;TCE sensitized mice liver tissue p-JAK1,p-STAT3,NLRP3,Caspase-3 and IL-1β The protein content was significantly increased compared with the solvent group and TCE sensitization negative group,and the protein content of Nrf2 and HO-1 was down-regulated(P<0.05);TCE+Tempol sensitized positive group p-JAK1,p-STAT3,NLRP3,Caspase-3 and IL The-1β protein content was lower than the TCE sensitization positive group,and the Nrf2 and HO-1 protein content increased,and the difference was statistically significant(P<0.05).6.Nrf2,HO-1,and NLRP3 gene expression levels in liver tissuesThere was no statistically significant difference in Nrf2,HO-1,and NLRP3 gene levels between blank group,solvent group and TCE sensitization negative group;compared with solvent group and TCE sensitization negative group,Nrf2 in the TCE sensitized positive group The transcription level of HO-1 was down-regulated,and the transcription level of NLRP3 was increased(P<0.05);compared with the TCE sensitized positive group,the transcription level of Nrf2 and HO-1 increased in the TCE+Tempol sensitized positive group,and the transcription level of NLRP3 decreased,and the difference There is statistical significance(P<0.05).7.The amount of Caspase-3 and IL-1β deposition in liver tissueIHC results showed that there was a large amount of Caspase-3 and IL-1βexpression in the liver tissue of the TCE sensitization positive group,and the Caspase-3and IL-1β deposition in the TCE+Tempol sensitization positive group were relatively reduced,there was no obvious expression in the blank group,solvent group,and sensitization negative group.Conclusions1.The immune liver injury induced by TCE is related to oxidative stress,and Nrf2/HO-1 and JAK1/STAT3 signaling pathways are involved in this immune liver injury.2.Tempol can significantly reduce the level of oxidative stress in this animal model,thereby regulating the Nrf2/HO-1 and JAK1/STAT3 signaling pathways and the release of related cytokines,and alleviating the immune liver injury caused by TCE.