NF-кb And P38MAPK May Contribute To The Differentiation Of BMSCs Into Hepatocyte-like Cells

Objective:To investigated the nexus between P38 MAPK and NF-κB signaling pathway in the process of the hepatocellular differentiation.As the upstream pathway of NF-κB,whether P38 MAPKinvolved inthe activation of NF-κB pathway in the hepatocellular differentiation.Methods: BMSCs were isolated from femurs and tibias of male SD rats with 4weeks old.The whole bone marrow adherent culture method was used to obtained the cells.This method used the different adherence time among different cells to screen the target cell.The separated cells were cultured in the DMEM low glucose medium containing10%FBS.Whole liquid exchange at the first time can remove most of cells which attached loosely or in bad condition.The culture medium were exchanged every 3 days.Cells were subcultured in a new culture flask in 1:2 with collagenase digestion when the cell fusion rate reached 70% to 80%.The third generation of BMSCs were induced into hepatocyte-like cells by using HGF.Cells were divided into four main groups in this experiment: induction group,NF-кB inhibition group,p38 inhibition group and negative control group.hepatic differentiation was induced with hepatocyte growth factor(HGF)in10% fetal bovine serum..The inhibitors of p38(SB203580)and NF-κB(BAY 11-7082)were added to the differentiation medium for inhibition of signaling molecular activities.morphological characteristics were observed under microscope and transferring function of the differentiated cells were examined by indocyanine green(ICG)uptake assay.Immunohistochemical staining was used to evaluate the protein expression position of NF-кB.And western blot analysis was used to detect the protein expression of several markers,including the specifc markers of hepatocytes(AAT),phosphorylated-P38(p-P38)and NF-кB.Results:After 20 days of induction,most of the cells in induction group were round or oval shape.Indocyanine green test shows that these cells can uptake of ICG,and excreted ICG in a certain period of time.Western blot showed the expression of the specific maker of liver cells,alpha 1-antitrypsin(AAT).Immunohistochemistry showed that NF-кB were observed transferred into nuclear in the induction group at 7th day and 20 th day.The respective inhibitors inhibited the expressions of NF-кB and p-P38 effectively.Compared to the induction group,the ability of uptaking ICG and expressions of specifc marker,AAT,were decreased visibly in the p38 and NF-κB inhibitor-treated groups.Notably,expression of NF-κB were significantly decreased in the p38 inhibitor-treated group.Concludions: Both NF-κB and P38 MAPK pathway participate in the hepatocellular differentiation of BMSCs,P38 MAPK could affect the regulation of NF-κB to this process of differentiation.