| BackgroundTrichloroethylene(TCE)is an organic solvent widely used in industrial and agricultural production at home and abroad.It is non-flammable and volatile in the air,and is a common occupational poison and environmental pollutant.TCE can enter the human body through air inhalation,skin contact and digestive system of occupational workers during work,causing occupational trichloroethylene drug eruptive dermatitis(OMLDT),also known as TCE allergic syndrome(THS).The main manifestations of the disease are fever,skin rash,liver dysfunction and superficial lymph node enlargement.OMLDT patients are often accompanied by different degrees of liver damage.In severe cases,acute liver failure can develop rapidly.Liver failure is also one of the important causes of death.In OMLDT patients,immune function indicators such as Ig M,Ig G,Ig A are abnormally expressed,and the expression levels of cytokines such as TNF-α,IL-10 and IL-4 are significantly increased.Animal experiments have found that the levels of cytokines such as TNF-α and IL-6 in the liver of TCE-sensitized mice have changed significantly.The immune inflammatory response mediated by cytokine storm may be the main cause of liver damage caused by TCE.Tumor Necrosis Tumor Necrosis Factor-α(TNF-α)is a multifunctional cytokine,which plays an essential role in cell apoptosis and survival,immune response and inflammatory response.In our previous study,we established a Balb/c mouse model sensitized by trichloroethylene and found that TCE induces immune liver damage,promotes the M1 polarization of Kupffer cells in the liver,and the serum and liver TNF-a Expression increased significantly.TNF-α may be the key node in the occurrence and development of skin and liver lesions in OMLDT patients.However,the mechanism of TNF-α in TCE-induced immune liver injury remains unclear.This study used TCE to smear the back skin of mice to stimulate sensitization,and intraperitoneally injected TNF-α inhibitor R7050 to explore the mechanism of TNF-α/TNFR1 regulating Kupffer cell polarization-mediated liver injury induced by trichloroethylene.ObjectiveTCE is an organic solvent with strong industrial practical performance.It can enter the human body through air inhalation,skin contact and gastrointestinal digestive system when workers work,causing occupational trichloroethylene drug-like dermatitis(OMLDT).Patients are often accompanied by immune liver injury,and the expression of TNF-α in the body is increased,but the pathogenic mechanism is still unclear.This study established a mouse trichloroethylene skin sensitization model and intervened by intraperitoneal injection of TNF-α inhibitor R7050,aiming to investigate whether TNF-α and its receptors regulate Kupffer cell polarization during TCE sensitization and downstream inflammation signaling pathways,and further study the mechanism of TCE-mediated immune liver injury.MethodIn this study,45 SPF Balb/c female mice,6-8 weeks old,were selected to establish a TCE transdermal sensitization model.In the experiment,TNF-α/TNFR1 inhibitor R7050 was used and divided into four treatment groups.According to the skin reaction score,they were divided into TCE sensitized group and TCE non-sensitized group.Serum was taken to detect liver function indexes ALT and AST;mouse liver tissues were embedded in slices and then pathological staining(HE staining)was performed to observe liver damage;immunohistochemistry(Immunohistochemistry,IHC)and western blotting(Western Blotting,WB)Detect the expression of F4/80,CD11 C and CD16/32,and judge the activation and polarization of KCs.Detect the expression of liver TNF-α and TNFR1 by histochemistry,WB and qRT-PCR;detect the expression of NF-κb signaling pathway protein p65 and phosphorylated p65,and detect the expression of STAT3 and phosphorylated STAT3;by WB,IHC,qRT-PCR detects the expression of downstream inflammatory factors IL-1β and IL-6 from different levels.Results1.Skin sensitization in miceAccording to the mouse skin reaction score,the mice were divided into skin sensitization positive(TCE positive)group and mouse skin sensitization negative(TCE negative)group.The sensitization rate of TCE group was 38.89%(7/18),and that of TCE + R7050 group was sensitization.The rate is 35.29%(6/17).There was no obvious skin damage on the back of the mice in the control group,and the damage such as edema and erythema was mild.2.Liver function testSerum liver function indicators of mice were detected.There was no significant difference in the expression levels of ALT and AST between the blank and solvent control groups(P>0.05).The serum ALT and AST levels in the TCE positive group were higher than the corresponding negative group and solvent control group(P <0.05),the serum vitality value of TCE+R7050 group was relatively reduced and higher than the normal level,the difference between the two was statistically significant(P<0.05).3.F4/80 immunohistochemical staining results and WB protein deposition levelF4/80 deposited in the liver of the TCE positive group.The F4/80 deposition level in the TCE+R7050 positive group was lower than that in the TCE positive group(P<0.05);the WB result was consistent with the immunohistochemical result.The expression level in the positive group increased,and the expression in the TCE+R7050positive group was lower than that in the TCE positive group,the difference was statistically significant(P<0.05).4.Mouse liver CD11 c and CD16/CD32 immunohistochemical staining results and WB protein deposition levelThe results of WB and IHC showed that the expression levels of CD11 c and CD16/CD32 proteins in the blank and solvent control groups and TCE negative groups were lower,the expression levels of TCE positive groups were significantly increased(P<0.05),and the expression levels of inhibitor TCE+R7050 positive groups Lower than the TCE positive group,the difference was statistically significant(P<0.05).5.Mouse liver TNF-α immunohistochemical staining results and WB protein deposition levelThe results of WB and IHC showed that the expression level of TNF-α protein in the blank and solvent control groups and the TCE negative group was lower,and the expression level of the TCE positive group increased,while the expression of TNF-αin the TCE+R7050 positive group decreased.Statistically significant(P<0.05).6.Mouse liver TNFR1 WB protein deposition levelWB results showed that the expression level of TNFR1 protein in the blank and solvent control groups and the TCE negative group was lower,the expression level of the TCE positive group was increased(P<0.05),while the protein expression of the TCE+R7050 positive group was reduced,and the difference was statistically significant.(P<0.05).7.Real-time quantitative PCR detection results of TNF-α and TNFR1The results of qRT-PCR showed that the m RNA expression levels of TNF-α and TNFR1 in the blank and solvent control groups and TCE-negative groups were lower,while the expression levels in the TCE positive group were increased.The expression levels of the above indicators in the TCE+R7050 positive group were relatively decrease,the difference is statistically significant(P<0.05).8.CD11 c and TNF-α fluorescence double labeling resultsImmunofluorescence was used to detect the expression of CD11 c and TNF-α in the liver of mice.CD11 c and TNF-α were deposited in the liver of mice in the TCE positive group.The deposition levels of CD11 c and TNF-α in the TCE+R7050 positive group were relatively.As a result,the fluorescence distribution is reduced.9.TNF-α and TNFR1 fluorescence double standard resultsImmunofluorescence was used to detect the expression of TNF-α and TNFR1 in the liver of mice.TNF-α and TNFR1 were deposited in large amounts in the livers of the TCE positive mice,and the levels of TNF-α and TNFR1 in the TCE+R7050-positive mice were relatively high.As a result,the fluorescence distribution is reduced.10.Protein deposition levels of P65 and P-P65 in mouse liver and real-time quantitative PCR detection resultsWB results showed that there was no difference in the expression level of p65 among the groups(P>0.05);while the expression level of p-p65 protein after phosphorylation was lower in the blank and solvent control group and the TCE negative group,but in the TCE positive group.The expression level increased,while the protein expression of TCE+R7050 positive histone was relatively reduced,and the color of the band was lighter,the difference was statistically significant(P<0.05).The results of qRT-PCR detection of mouse liver m RNA are consistent with the results of WB.11.Mouse liver STAT3 and P-STAT3 protein deposition levels and real-time quantitative PCR detection resultsWB results showed that there was no difference in the expression level of STAT3 between the groups(P>0.05);the phosphorylated p-STAT3 protein expression level was lower in the blank and solvent control group and the TCE negative group,but it was in the TCE positive group.The expression level increased,while the protein expression of TCE+R7050 positive histone was relatively reduced,the color of the band was lighter,and the difference was statistically significant(P<0.05).The results of q RTPCR detection of mouse liver m RNA detection are consistent with WB results.12.IL-1β protein deposition level in mouse liver,immunohistochemistry and real-time quantitative PCR test resultsThe results of WB and IHC showed that the expression level of IL-1β was lower in the blank and solvent control group and the TCE negative group,but the expression level increased in the TCE positive group,while the TCE+R7050 positive histone expression was relatively reduced,and the band color Shallow,the difference is statistically significant(P<0.05).The qRT-PCR results showed that the expression level of IL-1β m RNA in the blank and solvent control groups and the TCE negative group was lower,the expression level of the TCE positive group was increased,and the expression of the TCE+R7050 positive group was relatively reduced,and the difference was statistically significant(P <0.05).13.The protein deposition level of IL-6 in mouse liver,immunohistochemistry and realtime quantitative PCR test resultsThe results of WB and IHC showed that the expression level of IL-6 was low in the blank and solvent control group and the TCE negative group,but the expression level increased in the TCE positive group,while the TCE+R7050 positive histone expression was relatively reduced,and the band color Shallow,the difference is statistically significant(P<0.05).The results of qRT-PCR showed that the expression level of IL-6 m RNA in the blank and solvent control groups and the TCE negative group was lower,and the expression level of the TCE positive group was increased,while the expression of the TCE+R7050 positive group was relatively reduced,and the difference was statistically significant(P <0.05).Conclusion1.In TCE-induced immune liver injury,there is KCs activation and polarization to the M1 subtype.TNF-α,as one of the key nodes in the pathogenesis,can aggravate immune liver injury.2.NF-α/TNFR1 can regulate the NF-κB and STAT3 signaling pathways,mediate the inflammatory response of Kupffer cells,increase the expression of downstream inflammatory factors such as IL-1β and IL-6,and aggravate the liver cytokine storm.3.After using TNF-α inhibitor R7050,it effectively reduces liver damage. |
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