Effects Of Dose-and Time-dependent Of Intestinal Flora Co-metabolite Deoxycholic Acid On Human Umbilical Cord Mesenchymal Stem Cells’ Function

ObjectivesDeoxycholic acid is intestinal flora co-metabolite,human umbilical cord mesenchymal stem cells are a kind of stem cells that have the potential of self-regeneration and external amplification,and are easy to develop in vitro.The effects of dose-and time-dependent of deoxycholic acid on the proliferation,cellular cycle,inflammatory factor,surface markers and osteo-genic differentiation in the external environment were studied in mesenchymal stem cells,in order to understand the effect of deoxycholic acid on the stem cells,and to further understand the value of intestinal flora cometabolite deoxycholic acid.Methods1.The hUC-MSCs were cultured for 24h,48h and 72h in α-MEM stem cell medium with 11 different concentrations of deoxycholic acid(0.00μmol/L,1.56μmol/L,3.13μmol/L,6.25μmol/L,12.50μmol/L,25.00μmol/L,50.0Oμmol/L,100.00μmol/L,200μmol/L,400.00μmol/L,800.00μmol/L),and then the cell proliferation was detected by CCK8.2.The hUC-MSCs were cultured for 24h,48h and 72h in a-MEM stem cell medium with 4 different concentrations of deoxycholic acid(0.00μmol/L,1.56μmol/L,25.00μmol/L,400.00μmol/L),and then the cell cycle changes of stem cells were detected by flow cytometry.3.The hUC-MSCs were cultured for 24h,48h and 72h in a-MEM stem cell medium with 6 different concentrations of deoxycholic acid(0.00μmol/L,1.56μmol/L,6.25μmol/L,25.00μmol/L,100.00μmol/L,400.00μmol/L),and then the inflammatory cytokines interleukin-6(IL-6)was detected by ELASA.4.The hUC-MSCs were cultured for 24h,48h and 72h in a-MEM stem cell medium with 4 different concentrations of deoxycholic acid(0.O0μmol/L,1.56μmol/L,25.00μmol/L,400.0Oμmol/L),and then the cell surface markers of mesenchymal stem cells were detected by flow cytometry.5.The hUC-MSCs were cultured for 24h in a-MEM stem cell medium with 4 different concentrations of deoxycholic acid(0.00μmol/L,1.56μmo1/L,25.00μmol/L,400.00μmol/L),and then the osteogenic differentiation induced by stem cells was detected by osteoinductive agent and alizarin red S staining.Results1.The effect of deoxycholic acid on the proliferation of hUC-MSCs was reflected in dose-and time-dependent of deoxycholic acid(F=6.622,P<0.001).When the concentration was constant,cell proliferation increased with time;When the treatment time was fixed,the cell proaliferation decreased with the increase of concentration.2.When the concentration was constant,the proportion of hUC-MSCs arrested in G0/G1 phase increased with time(P<0.0001);The cell cycle of hUC-MSCs treated by DC A was blocked in G0/G1 and S phases in the low concentration group(1.56μmol/L)while S phase and G2/M phase in the high concentration group(400.00μmol/L)(P<0.0001).3.At 24h,48h and 72h,the secretion of IL-6 in hUC-MSCs decreased gradually with the increase of deoxycholic acid concentration,especially in the high concentration group(400.00μmol/L).When the concentration of deoxycholic acid was constant,the secretion of IL-6 increased with time.4.After 24h,48h and 72h,three concentrations of deoxycholic acid had no statistical significance on the expression of hUC-MSCs’ antigen molecules.While high concentration of deoxycholic acid(400.00μmol/L)decreased the expression of CD 105,CD73.5.After alizarin red S staining,the cells in the negative control group showed calcium salt deposition under the microscope,which was stained red,indicating that hUC-MSCs were successfully induced.The low concentration(1.56μmol/L)and medium concentration(25.00μmol/L)deoxycholic acid groups showed the same red bone calcium nodules as the negative control group,while the high concentration group(400.00μmol/L)showed patchy red around the punctated cell structure,without clumpy red nodules.Neither the black control nor the three concentrations of deoxycholic acid a-MEM groups were stained red.Conclusions1.In vitro,the effects of deoxycholic acid on the proliferation,cell cycle and IL-6 expression of hUC-MSCs were related to the dose-and time-dependent.2.The low and medium dose-and time-dependent of deoxycholic acid did not affect the expression of CD90,CD105,CD73,CD45,CD34,CD19,CD11b and HLA-DR molecules on the surface of hUC-MSCs.However,high concentration of deoxy-cholic acid could decrease the expression of CD105,CD73.3.Only high concentration of deoxycholic acid could inhibit the osteogenic differentiation of hUC-MSCs,while low and medium concentration had no effect on the osteogenic differentiation of hUC-MSCs.