An Experimental Study Of Regulating Macrophage Polarization Against Schistosomiasis Hepatic Fibrosis In Mice

Part I SEA and Gamma-secretase Blockers on the Effect of Macrophage Polarization Reaction and Regulation of Notch Signaling PathwayBackground/purpose Cultivate macrophage RAW264.7 to explore Schistosomiasis soluble egg antigen(SEA)and gamma-secretase blocker DAPT on its polarization direction,and the activation of Notchl/Jaggedl signal pathway and analyzes the role of SEA time and dose dependent.Methods SEA stimulated RAW264.7 cells with different concentrations and at different times,then detected the expression of Notch1,Jaggedl,Hes1,he Notch signaling pathways related genes by PCR,Western blot as well as IL-10,IL-12,Arg-1,NOS-2 polarization direction of RAW264.7 cells relevant indicators by Elisa and Western blot.SEA continue to function in cells after Gamma-secretase blocker DAPT to RAW264.7 cells,detected the Notch signaling pathway and the cell polarization direction indicator by ditto methods,respectively.Results SEA acted in RAW264.7 cells:(1)Notchl/Jaggedl signaling pathways related indicators such as Notch1 Jagged1,Hesl were significant higher than the control group in mRNA expression and protein level with time-dose dependent,and the index increased the most obvious in the SEA levels of 40 ug/ml,for 2 hours;(2)in cell polarization direction,the results showed that the polarization direction indicators of the M2,such as Arg-1,IL-10 were expressed higher than the control group.Gamma-secretase blocker DAPT acted in RAW264.7 cells:(3)Notch1/Jagged1 signaling pathways related indicators such as Notch1 Jagged1,Hesl expressed in decline than the control group;(4)in cell polarization direction,the results showed that the polarization direction indicators of the M2,such as IL-10 and Arg-1 were expressed in decline than the control group.Conclusion SEA could activate Notch1/Jagged1 signaling pathways of RAW264.7 cells,and promote cell polarization in the direction of the M2,furthermore,its role was in time-dose dependent.Gamma-secretase blocker DAPT could block Notch 1/Jagged1 signaling pathways of RAW264.7 cells,and could reverse cell polarization in the direction of M2.Part II Established the model of Schistosomiasis liver fibrosis in murine and to explore the effect of gamma-secretase blockers on model murine liver granuloma and fibrosisBackground/purpose Schistosomiasis liver fibrosis in murine model was established to detect the polarization direction of the infiltrated macrophages,and to explore the effect of gamma-secretase blockers on model murine liver granuloma and fibrosis as well as its main mechanism.Methods(1)Schistosomiasis liver fibrosis in mice model was established.16 healthy adult female Balb/c mice were divided into two groups,a group of model group,percutaneous infected with schistosoma japonicum cercaria,another group as control,treated with PBS.Two groups of mice were sacrificed after being infected 8 weeks,and then examined the expression of F4/80,Arg-1,CD206 in the two groups of mice liver tissue by using laser confocal method.(2)24 healthy adult female Balb/c mice were randomly assigned to 3 groups of 8 mice each as follows:the model group,the DAPT treated group and control group.In the model and DAPT-treated groups,each mouse was infected percutaneously with 25 Schistosoma japonicum cercariae for 12 weeks.In the DAPT treated group,each mouse was given DAPT(10 mg/kg)8 weeks post-infection with cercariae by intraperitoneal injections daily for 4 weeks.Mice in the control group were neither infected with cercariae nor treated with DAPT,but were administered the same volume of solven.Hematoxylin eosin staining and Masson trichromatic staining were used to observe each experimental mice liver tissue section,including the average size of a egg granuloma(mm)and the expression of collagen fiber by using the MOD semi-quantitative method;detected the expression of F4/80 and Arg-1 in the liver tissue of mice by Laser confocal method;The hydroxyproline kits was used to measure the hydroxyproline content in mice liver tissue.Results(1)The numbers of F4/80+ cells were markedly increased in granulomata in the liver tissues relative to that of normal mice.Moreover,the F4/80+ cells also showed co-expression of Arg-1 or CD206 in the murine schistosomiasis model;(2)The expression of Arg-1 decreased obviously in the infiltrated macrophages in liver tissues of the DAPT-treated mice compared to those in control group.Meanwhile,the mean liver granuloma diameter,hydroxyproline content,and collagen deposition was significantly decreased in the DAPT-treated group in compared with those of the untreated schistosomiasis model group.Conclusion The infiltrated F4/80+ macrophages exhibited the M2-polarized phenotype in the murine schistosomiasis model;the inhibition of Notch1/Jagged1 signaling could regulate the status of macrophage polarization and attenuate liver granulomata and fibrosis in murine schistosomiasis.