Wnt5a/JNK-Mediated Activation Of Kupffer Cells In Trichloroethylene Immune Liver Injury

BackgroundTrichloroethylene(TCE)is a volatile,colorless liquid that is widely used as a chlorinated organic solvent in industrial production and processing industries.In the process of production and use,TCE enters the human body mainly through the respiratory and dermal routes,and some workers who are occupationally exposed to TCE may develop systemic diseases with drug-like allergic clinical manifestations.OMDT can cause damage to the skin,kidneys,liver and other organs,as well as various nerve damage,of which the liver is one of the fatal causes.OMDT is often treated with high-dose glucocorticoid shock therapy,but the application of high-dose hormones may cause more adverse effects and serious complications,increasing the risk of patient death.Nowadays,TNF-α is mostly used for clinical treatment.The group found that TNF-α is highly expressed in the liver of TCE-sensitized mice and promotes the activation of Kupffer cells,while blocking TNF-α/TNFR1 results in decreased expression of Wnt5 a,suggesting that Wnt5 a may be involved in liver injury in TCEsensitized mice.The Wnt signaling pathway can achieve hepatic homeostasis and help maintain liver function.It was found that Wnt5 a was significantly expressed in the liver of TCE-sensitized mice,and the fluorescence was localized in Kupffer cells,which are thought to be the key to signal transduction of oxidative stress in the liver,and Kupffer cells can play an important role in liver injury by causing hepatocyte death through the JNK pathway.The group’s previous study showed that polarization of Kupffer cells occurs during TCE-sensitized liver injury,which stimulates Kupffer cells to secrete large amounts of cytokines and chemokines,further aggravating liver injury.PurposeThe purpose of this study was to observe whether the Wnt5a/JNK pathway mediates the activation of Kupffer cells affecting the release of chemokines and cytokines involved in liver injury in immune liver injury due to TCE exposure.MethodsAnimal experiments: 70 SPF-grade BALB/c female mice fed for one week in an acclimatized environment were randomly divided into blank control(n= 10),solvent control(n= 10),TCE-treated(n= 30),and IWP-2 + TCE-treated(n= 20)groups.The TCE-treated group was then further divided into TCE-sensitized positive(TCE+)and TCE-sensitized negative(TCE-)groups based on subsequent skin scoring;the IWP-2+TCE-treated group was further divided into IWP-2+TCE-sensitized positive(IWP-2+)and IWP-2+TCE-sensitized negative(IWP-2-)groups;70 SPF-grade BALB/c female mice fed for one week in an acclimatized environment were randomly divided into blank control(n= 10),solvent control(n= 10),TCE-treated(n= 30),and SP600125+TCE-treated(n= 20)groups.The TCE-treated group was further divided into TCEsensitized positive group(TCE+)and TCE-sensitized negative group(TCE-)according to the subsequent skin score;the SP600125+ TCE-treated group was further divided into SP600125+TCE-sensitized positive group(SP600125+)and SP600125+TCEsensitized negative group(SP600125-).The liver lobes of mice in each group were prepared into paraffin sections,and some of the livers were kept for Western blot,and Kupffer cells were extracted from the livers of mice for Western blot.Results1.Elevated Wnt5 a expression in the liver of TCE-sensitized mice: WB results showed that the expression of Wnt5 a and Ror2 were significantly elevated in the TCEsensitized positive group(P<0.05),and the fluorescence co-localization results of Wnt5 a and F4/80 showed that the expression of Wnt5 a was significantly enhanced in the TCE-sensitized positive group,while F4/80 was seen to overlap with Wnt5 a.2.Sensitization rate of mice: 11 of 30 mice in the TCE-treated group showed skin erythema and edema,and the sensitization rate was 36.7%.In the TCE+IWP-2 treated group,7 out of 20 mice showed skin reaction,and the sensitization rate was 35%.The differences in sensitization rates between the TCE-treated and TCE+ IWP-2-treated groups were not statistically significant(P>0.05).The difference in sensitization rate between the treated groups and the blank control group was statistically significant(P<0.05).3.IWP-2 reduced the liver injury and inhibited the activation of Kupffer cells in TCE-sensitized mice: in the TCE-sensitized group,the expression levels of serum AST and ALT were significantly increased(P<0.05).H&E staining showed that the TCEsensitized group showed significant loss of hepatic cord structure,swelling of hepatocytes and inflammatory cell infiltration.4.IWP-2 intervention inhibited the activation of Kupffer cells:immunohistochemical results showed that Kupffer cell activation was significant in the TCE-sensitized positive group,and Kupffer cell activation was reduced after IWP-2intervention.5.IWP-2 inhibited Wnt5a/JNK expression in mouse Kupffer cells: WB results showed that hepatic Wnt5 a,Ror2,and JNK protein expression were significantly downregulated after IWP-2 administration(P<0.05).6.IWP-2 inhibited the expression of cytokines and chemokines in Kupffer cells:WB results showed that the expression of CXCL-1,CCL3,MCP-1,CXCL-10,in Kupffer cells of TCE sensitization positive group was significantly increased(P<0.05),and after Wnt5 a was inhibited CXCL-1,CCL3,MCP-1,CXCL-10 expression downregulation was significantly reduced(P< 0.05).7.Sensitization rate of mice: According to the skin score of mice,there was no significant skin reaction in the blank control group and solvent control group(20 mice in total).10 of 30 mice in the TCE-treated group showed skin erythema and edema,and the sensitization rate was 33.3%.6 of 20 mice in the TCE+SP600125-treated group were sensitized,and the sensitization rate was 30%.Statistical analysis showed that the differences in sensitization rates between the TCE-treated and TCE+SP600125-treated groups were not statistically significant(P>0.05).The difference in sensitization rate between each treatment group and the blank control group was statistically significant(P<0.05).8.SP600125 intervention reduced liver injury in TCE-sensitized mice: in the TCEsensitized group,the expression levels of serum AST and ALT were significantly increased(P<0.05),and the expression levels of AST and ALT decreased after the down-regulation of JNK expression.inflammatory cell infiltration,and the pathological damage of liver was alleviated after SP600125 intervention.9.SP600125 intervention inhibited the activation of Kupffer cells:immunohistochemical results showed that the activation of Kupffer cells was increased in the TCE-sensitized positive group,and the activation of Kupffer cells was significantly reduced after SP600125 inhibition of JNK.10.SP600125 inhibited the expression of cytokines and chemokines in mouse liver:WB results showed that the expression of CXCL-1,CCL-3,MCP-1,CXCL-10,in the liver of TCE-sensitized positive group was significantly increased(P<0.05),and CXCL-1,CCL-3,MCP-1,CXCL-10,expression was down-regulated after JNK signaling was inhibited(P< 0.05).q RT-PCR results showed that the expression of CXCL-1,CCL3,MCP-1 and CXCL-10 in the liver of TCE-sensitized mice was significantly increased(P<0.05).CXCL-1,CCL3,MCP-1,CXCL-10 expression decreased after JNK was inhibited(P<0.05).11.SP600125 inhibited the expression of chemokines in mouse Kupffer cells:extracted mouse Kuppfer cells,WB results showed that the expression of CXCL-1,CCL3,MCP-1,CXCL-10,was significantly increased(P < 0.05),and after JNK was inhibited CXCL-1,CCL3,MCP-1,CXCL-10 expression was downregulated(P<0.05).Conclusion1.Wnt5 a pathway regulates the degree of Kupffer cell activation during TCE sensitization and is involved in liver injury in TCE-sensitized mice2.The Wnt5a-mediated JNK pathway regulates the activation of Kupffer cells and promotes the release of chemokines and cytokines,which are involved in liver injury in TCE-sensitized mice.