The Molecular Mechanisms Of Wnt Signaling On Regulating Macrophage Activation During Renal Fibrosis

Objective:To observe the impact of TGF-?1 on bone marrow derived macrophages,exploring whether Wnt signaling play a regulative role in the process of fibrosis or not.Methods:Bone marrow derived cells(BMCs)purified from the C57 mice,which are treated with bone marrow colony stimulating factor(M-CSF).After cultured in 37 ? and 5% CO2 incubator for 7 days,the cell morphological changes were observed by inverted phase contrast microscope,and the purity of BMDM was determined by flow cytometry.Then all the cells are treated by transforming growth factor(TGF-beta 1)as a intervention factor.We set them into 2 groups respectively,the control group(group A: alpha MEM sugar complete medium + M-CSF),and the experimental group(group B: alpha MEM sugar complete medium + M-CSF + TGF-beta 1).The concentration of M-CSF is 60ng/ml,and the TGF-beta1 is 5 ng/ml.Treating alpha-SMA as the marker of myofibroblasts,F4/80 as macrophages.3 days later,the alpha SMA+ F4/80+ cells are determined by flow cytometry,and the expressions of Wnt3 a,DVL(Dishevelled),beta-catenin protein are detected by Western imprinting method.SPSS19.0 statistical software was used for data processing.Results:1.When stimulated by M-CSF(60ng/ml)for 7 days,low cytometry of ?-SMA+ F4/80+cells isolated from the bone marrow accounted for(94.44±6.11)%;2.All the bone marrow cells are cultiveted by M-CSF for 7d.And then 3 days later after stimulating by TGF-?1(5ng/ml),low cytometry of ?-SMA+F4/80+ cells are significantly incereased in the experimental group(t=6.365,P=0.0007);3.All the bone marrow cells are cultiveted by M-CSF for 7d.And then 3 days later after stimulating by TGF-?1(5ng/ml).The protein levels of Wnt3 a,DVL and ?-cateninare significantly increased in the experimental group.(t Wnt3a=12.06,P<0.001;t DVL=8.168,P<0.001,t?-catenin=6.752,P<0.001).Conclusion1.Bone marrow der-ived macrophages transformed into myofibroblasts with the help of TGF-?1;2.Wnt/?-catenin signaling pathway takes part in the process of this transtion in vitro.A further research will provide a potential way to renal fibrosis.