| Background and objective: Macrophages are important immune cells in the body and are involved in all stages of the inflammatory response.Studies have shown that Peritoneal macrophage(PMO)plays an important role in inflammatory diseases such as Severe acute pancreatitis(SAP).Regulating macrophage polarization has achieved good effects in the treatment of a variety of diseases.Our previous study has found that T cell immunoglobulin-domain and mucin-domain protein-3(Tim-3)/Galectin-9(Gal-9)is involved in the regulation of inflammatory response in the early stage of SAP,by downregulating the expression of Tim-3/Gal-9,the PMO of mice is induced to M1 polarization,which promotes the release of inflammatory factors and aggravates the inflammatory response in mice with SAP,but the mechanism is still unclear.Janus kinase2(JAK2)/Signal transducer and activator of transcription 3(STAT3)signaling pathway is an important pathway for M2 macrophage polarization,studies have shown that Tim-3/Gal-9 cross-interacts with STAT3 in macrophage polarization.Therefore,this study aimed to explore the role and mechanism of Tim-3/ Gal-9 in regulating the polarization of PMO in mice.Methods: PMO was extracted from normal BALB/c mice and divided into two groups:(1)Control: PMO extracted from normal mice;(2)Induction group(IN): induced with LPS and IFN-γ to simulate the inflammatory environment in vivo.The phenotypes of PMO were identified by immunofluorescence before and after induction.IL-6 and iNOS were selected as phenotypic markers of M1 macrophages,CD206 and Arg-1 were selected as phenotypic markers of M2 macrophages.Western blot was used to detect the expression of Tim-3 and Gal-9 in PMO before and after induction.In order to further explore the relationship between Tim-3/Gal-9,JAK2/STAT3,macrophage polarization and inflammatory response,the subsequent experiments were divided into six groups:(1)Induction group(IN): PMO was induced with LPS and IFN-γ;(2)Tim-3 down-regulation group(IN+Tim-3 siRNA): after induction with LPS and IFN-γ,PMO was transfected with Tim-3 siRNA;(3)Gal-9 down-regulation group(IN+Gal-9 siRNA): after induction with LPS and IFN-γ,PMO was transfected with Gal-9 siRNA;(4)Gal-9 up-regulated group(IN+p Gal-9): after induction with LPS and IFN-γ,PMO was transfected with Gal-9 cDNA;(5)Gal-9 up-regulation and JAK2/STAT3 Inhibitor group(IN+p Gal-9+JAK2/STAT3-in): After induction with LPS and IFN-γ,Gal-9 cDNA was transfected and JAK2/STAT3 inhibitor was added;(6)Gal-9 down-regulation and JAK2/STAT3 Inhibitor group(IN+Gal-9 siRNA+JAK2/STAT3-in): after induction with LPS and IFN-γ,Gal-9 siRNA was transfected and JAK2/STAT3 inhibitor was added.CCK8 experiment was used to detect the cell viability of each group,RT-PCR was used to detect the mRNA expression of Tim-3,Gal-9,IL-6,TNF-α,CD206 and IL-10.The protein expressions of Tim-3,Gal-9,IL-6,CD206,p-JAK2 and p-STAT3 were detected by Western blot.Results:1.Compared with the Control group,the expression of IL-6 and iNOS were increased(P<0.05),the expression of CD206 and Arg-1 were decreased(P<0.05),and the PMO of mice were polarized to M1 type in IN group.2.Compared with the Control group,the protein expressions of Tim-3 and Gal-9 were decreased in IN group(P<0.05);3.CCK8 assay showed that there was no significant difference in cell viability among each group(P>0.05),indicating that transfection had little effect on cell viability,and subsequent related experiments could be carried out.4.Compared with IN group,the mRNA levels of Tim-3 and Gal-9 in IN+Tim-3 siRNA and IN+Gal-9 siRNA groups were significantly decreased,indicating that the transfection was successful,and the mRNA levels of IL-6 and TNF-α were increased(P<0.05),the mRNA levels of CD206 and IL-10 were decreased(P<0.05).Compared with IN group,the mRNA levels of IL-6 and TNF-α were decreased,and the mRNA levels of CD206 and IL-10 were increased in the IN+p Gal-9 group(P<0.05).Compared with IN+p Gal-9group,the mRNA levels of CD206 and IL-10 were decreased(P<0.05),the mRNA level of TNF-α was increased(P<0.05)in IN+p Gal-9 + JAK2/STAT3-in group.Compared with IN+Gal-9 siRNA group,the mRNA levels of IL-6 and TNF-α were increased(P<0.05)in IN+Gal-9 siRNA+JAK2/STAT3-in group.5.Compared with the IN group,the protein expression levels of Tim-3 and Gal-9 were significantly decreased,the protein expression level of IL-6 was increased,and the protein expression levels of CD206,p-JAK2 and p-STAT3 were decreased in IN+Tim-3 siRNA group and IN+Gal-9 siRNA group(P<0.05),while the protein expression level of IL-6was decreased,the protein expression levels of CD206 and p-JAK2 and p-STAT3 were increased in IN+p Gal-9 group(P<0.05).Compared with IN+p Gal-9 group,the protein expressions of p-JAK2 and p-STAT3 were significantly inhibited in IN+p Gal-9+JAK2/STAT3-in group(P<0.05),and the protein expression level of IL-6 was increased(P<0.05),the protein expression level of CD206 was decreased(P<0.05).Compared with IN+Gal-9 siRNA group,the protein expression level of IL-6 was increased,and the protein expression level of CD206 was decreased in IN+Gal-9siRNA+JAK2/STAT3-in group(P<0.05).Conclusion:Tim-3/Gal-9 regulates the polarization of PMO to M2 type through JAK2/STAT3 signaling pathway,reduces the production of inflammatory factors,and thus alleviates inflammatory response. |
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