| BackgroundTrichloroethylene（TCE）,as an excellent industrial organic solvent,is widely used in metal surface treatment,paraffin extraction and the production of refrigerants and dry cleaning agents.Occupational trichloroethylene drug eruptive dermatitis caused by occupational exposure to TCE in Southeast Asia and Guangdong,China seriously threatens the health of the occupational population;and its extensive industrial application has caused a large number of TCE to enter the environment,seriously polluting the air,water and soil.TCE in the environment can enter the body through bioaccumulation and exert toxic effects.The damage to the skin and organs of the human body and other organisms has been widely confirmed.Among them,the liver is the most common organ involved.Studies have shown that TCE and its metabolites can cause epigenetic changes,thereby altering immune function and promoting liver inflammation.Liver macrophages can be rapidly activated and polarized in different directions during the process of inflammation.According to the stimulation of different components in the microenvironment,liver macrophages are mainly polarized into two subtypes,including the pro-inflammatory M1 type and anti-inflammatory M2 type.M1-type hepatic macrophages secrete tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）and other cytokines,and M2-type hepatic macrophages secrete interleukin-10,Transforming growth factor-β and other cytokines.Epigenetic regulation related to M1 or M2 activation mainly relies on histone modification in the promoter region of inflammation-related genes,which plays an important role in the signal regulation and conduction of macrophage polarization.A variety of histone modification enzymes can catalyze the post-translational modification of arginine and lysine in histone tails,recruit the binding of effector proteins,influence downstream proteins,and then participate in specific cellular processes.Among them,histone lysine demethylation KDM4 A is related to the activation of macrophages.ObjectiveThe purpose of this study is to establish a TCE oral exposure mouse model and a RAW264.7 cell exposure model,combined with the KDM4 A inhibitor JIB-04,to detect the expression of KDM4 A and histone H3K9 methylation index H3K9me3/2/1,macrophage marker F4/80,M1 type macrophage marker CD11 c,i NOS and cytokines TNF-α and IL-1β in the liver during the oral exposure of TCE in mice,and explore the activation of β-catenin,a key signaling pathway of macrophage pro-inflammatory,to further explore the mechanism of TCE-induced liver injury.Methods96 SPF BALB/c mice,female,6-8 weeks old,after 1 week of adaptive feeding,the mice were randomly divided into 4 groups: 2.5 mg/ml TCE group,5.0 mg/ml TCE group,vehicle control Group,blank control group.The selection of materials was completed at the four time points of the second,fourth,eighth,and twelfth weeks according to the experimental requirements.Take mouse serum to determine liver function（indicators of ALT and AST）.Take the mouse liver tissue to make paraffin sections,and observe the pathological damage of the liver by hematoxylin-eosin staining（HE staining）;immunofluorescence double labeling to detect the expression and overlap of F4/80 and CD11 c in the mouse liver;immunohistochemical method for detection the deposition of i NOS in rat liver.The protein stock solution was extracted from mouse liver tissue,and the protein expression levels of mouse liver KDM4 A,H3K9me3/2/1,CD11 c,TNF-α,IL-1β,and β-catenin were detected by Western blotting experiment.The cell experiment set up a blank control group,a vehicle control group,a JIB-04 inhibitor control group,a TCE treatment group,and a TCE+JIB-04 inhibitor group.After treatment and culture for 24 hours,the cells were scraped and the cell culture supernatant was collected.The cell protein stock solution was extracted,and the expression levels of KDM4 A,H3K9me3/2/1,CD11 c,and β-catenin protein in the cells were detected by Western blotting experiment.Take the cell culture supernatant and detect the levels of TNF-α and IL-1β in the cell supernatant by ELISA.Results1.During the experiment,the mice in each group were in good condition.There were no individuals who died due to TCE poisoning.The average daily water consumption was 0.13 ml/g.The average exposure of the 2.5 mg/ml TCE group and5.0 mg/ml TCE group was 357.03 mg/ kg/d and 657.62 mg/kg/d.There was no statistically significant difference in the weight gain and liver coefficient of mice at each time point.2.The results of HE staining of mouse liver showed that the liver cells of the blank control group and the vehicle control group were arranged neatly,with large and round nuclei,obvious nucleoli,uniform cytoplasm,and no inflammatory changes;while the 2.5 mg/ml TCE group In the 5.0 mg/ml TCE group,there was obvious inflammatory cell infiltration around the portal duct of the liver tissue,and vacuolar degeneration of cells,a little cell degeneration or necrosis,loose cytoplasm,and abnormal cell morphology were seen in some areas.3.The results of transmission electron microscope observation of mouse liver showed that the blank control group and vehicle control group had no obvious histomorphological changes.In the 2.5 mg/ml TCE group and 5.0 mg/ml TCE group,a large number of vacuoles appeared in the hepatocytes,and the glycogen granules decreased.Mitochondrial edema,disorganized internal cristae,and decreased numbers.The endoplasmic reticulum is discrete and fragmented into fragments of different sizes.4.The statistical analysis of the results of the liver function measurement of mice showed that there was no significant difference in serum AST and ALT levels of the vehicle control mice compared with the blank control group at 2,4,8,and 12 weeks;at the second week,2.5 mg/ml TCE group AST and ALT levels were significantly higher than the blank control group;in the 4th and 8th weeks,the 5.0 mg/ml TCE group had higher AST and ALT levels than the blank control group and vehicle control group,and the AST and ALT levels of the 5.0 mg/ml TCE group were higher than those of the 2.5mg/ml TCE group;at 12 weeks,the AST and ALT levels of the 2.5 mg/ml TCE group and 5.0 mg/ml TCE group decreased,but still higher than the control group.5.Co-localization of F4/80,an important indicator of macrophage activation,and M1 macrophage surface marker CD11 c by immunofluorescence double-labeling,the results showed: at 2,4,8,and 12 weeks,the expression of F4/80 and CD11 c was less in blank control group and vehicle control group,and there were more positive areas of F4/80 and CD11 C in the 2.5 mg/ml TCE group and 5.0 mg/ml TCE group.6.The protein expression level of CD11 c was verified again by western blotting experiment.The results showed that compared with the blank control group,the expression level of CD11 c protein in the 2.5 mg/ml TCE group and 5.0 mg/ml TCE group increased,and the difference was statistically significant.7.The expression of M1 macrophage marker i NOS was detected by immunohistochemistry.The results showed that the blank control group and vehicle control group had very little i NOS expression,and there were large deposits in the 2.5mg/ml TCE group and 5.0 mg/ml TCE group.8.The protein expression levels of TNF-α and IL-1β in the liver of mice were detected by Western blotting experiment.The results showed that compared with the blank control group,the protein expression level of TNF-α and IL-1β in the 2.5 mg/ml TCE group and 5.0 mg/ml TCE group increased,and the difference was statistically significant.9.The protein expression levels of KDM4 A and H3K9me3/2/1 in mouse liver were detected by western blotting experiment.The results showed that: compared with the blank control group,the protein expression level of KDM4 A was increased in the2.5 mg/ml TCE group and the 5.0 mg/ml TCE group,while H3K9 was desylated（the protein expression level of H3K9me3/2 decreased in the 2.5 mg/ml TCE group and the5.0 mg/ml TCE group,while the protein expression level of H3K9me1 increased）,the difference was statistically significant.10.The expression of β-catenin in mouse liver was detected by western blotting experiment.The results showed that the protein expression level of β-catenin in the 2.5mg/ml TCE group and 5.0 mg/ml TCE group increased compared with the blank control group,the difference was statistically significant.11.The protein expression levels of KDM4 A,H3K9me3/2/1,CD11 c andβ-catenin in mouse macrophages RAW264.7 were detected by Western blotting experiment.The results showed that: compared with the blank control group and the vehicle control group,the protein expression level of KDM4 A was increased in the TCE-treated group,while H3K9 was desylated（the protein expression level of H3K9me3/2 in the TCE treatment group decreased,while the protein expression level of H3K9me1 increased）,and the difference was statistically significant.In the presence of the KDM4 A inhibitor JIB-04,the expression level of KDM4 A protein can be reduced,and the overall demethylation of H3K9me3/2 has been rescued to a large extent.12.The levels of M1-type pro-inflammatory cytokines such as TNF-α and IL-1βin the supernatant of mouse RAW264.7 macrophages were detected by double-antibody sandwich method.The results showed: there was no statistically significant difference in the levels of TNF-α and IL-1β in blank control group,vehicle control group and the JIB-04 inhibitor control group.Compared with the above three groups,the expression of TNF-α and IL-1β in the TCE treatment group increased.In the TCE+JIB-04 inhibitor group,the expression of TNF-α and IL-1β decreased,and the difference was statistically significant.Conclusions1.In the process of oral exposure of TCE to induce M1 type polarization of mouse liver macrophages and promote the expression of liver inflammatory factors,the expression level of histone lysine demethylase KDM4 A protein increased,and H3K9 was demethylated,the β-catenin signaling pathway was activated.2.Exogenous KDM4 A inhibitor（JIB-04）can inhibit TCE-induced H3K9 demethylation,inhibit the M1 polarization of macrophages and the activation ofβ-catenin signaling pathway. |
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