Effects Of High Glucose On Osteogenic Differentiation And Related Signaling Pathways And Epigenetic Changes

Diabetes is a metabolic disease characterized by hyperglycemia.At present,the mechanism of bone metabolism disorder in diabetic patients has not been clarified.It is of great significance to study the mechanism of hyperglycemia on osteoblastic differentiation of different cells in clinical application.Abnormal bone metabolism in diabetic patients may also be related to inflammatory environment.IL-10 is a potent anti-inflammatory cytokine,and it is of great significance to study its influence on osteogenic differentiation.However,it is not clear how high glucose and IL-10 regulate osteoblastic differentiation,and how much they might change in microenvironment to affect epigenetic modification during osteoblastic differentiation.Therefore,it is necessary to study the effect of high glucose environment on osteoblastic diferentiation,regulatory mechanism and epigenetic expression.Objective:to study the effects,mechanism and epigenetic expression of high glucose on osteogenic differentiationMethods:Part one:Effects of high glucose environment on osteogenic differentiation of Primary Osteoblastic Cells and Adipose Stem CellsIsolated and cultured Mouse Primary Osteoblastic Cells(MPC),Brown Adipose Stem Cells(B-ASCs),White Adipose Stem Cells(W-ASCs)and the resuscitated MC3T3-E1 cell lines were respectively stimulated with different concentrations of glucose(5.5mmol/L,25mmol/L and 55mmol/L).After osteoblastic differentiation,alkaline phosphatase(ALP)staining was used to observe the osteoblastic differentiation of different cells.Primary Osteoblastic Cells were taken for osteoblast differentiation.The expression level of markers of osteoblastic differentiation was detected by qPCR.Western blot was used to detect the expression of osteogenic related proteins.Part two:Effect of high glucose environment on osteogenesis inhibition induced by IL-10 low expressionWT MPC and IL-10tml/tml MPC were isolated and cultured.Osteogenic differentiation was induced for 7d with different concentrations of glucose(5.5mmol/L,25mmol/L and 55mmol/L).Alkaline phosphatase(ALP)staining was used to observe the osteogenic differentiation of different cells.The cranial tissues of WT mice and IL-10tml/tml mice were taken,and the differences of cranial development were analyzed by microCT and quantitative PCR.Part three:High glucose environment regulated osteogenic differentiation via STAT3 signaling pathway and affected the expression of epigenetic related proteinsWT MPC was stimulated with glucose at different concentrations(5.5mmol/L,25mmol/L and 55mmol/L)for 7 days to induce osteogenic differentiation,and the expression of osteogenic pathway-related proteins and epigenetic related proteins was detected by Western Blot.WT MPC and IL-10tml/tml MPC were respectively stimulated with 25mmol/L glucose,and osteogenic differentiation was induced for 7d.Western Blot was used to detect the expressions of osteogenic pathway-related proteins and epigenetic related proteins.Results:Part one:Effects of high glucose environment on osteogenic differentiation of Primary Osteoblastic Cells and Adipose Stem Cells1.The osteogenic differentiation ability of MC3T3-E1,MPC and W-ASCs enhanced with the increase of glucose concentration,while the osteogenic differentiation ability of B-ASCs was inhibited.2.High glucose promoted the osteogenic differentiation ability of MPC.After 3d and 7d of osteogenic induction culture,the expression of genes and proteins related to osteogenesis markers increased with the increase of glucose concentration.Part two:Effect of high glucose environment on osteogenesis inhibition induced by IL-10 low expression1.Compared with WT MPC,the osteogenic differentiation ability of IL-10 tml/tml MPC in vitro was significantly decreased.2.Compared with WT mice,IL-10 10tml/tml mice showed severer osteoporosis in the skull and lower expression of osteogenic marker genes in the skull tissue.3.The addition of 25mmol/L glucose during the induction of IL-10 10tml/tml MPC can partially restore its ability of osteogenic differentiation.Part three:High glucose environment regulated osteogenic differentiation via STAT3 signaling pathway and affected the expression of epigenetic related proteins1.The expression of p-STAT3 in MPC was inhibited with the increase of glucose concentration,while the total expression of STAT3 was not significantly different.The expressions of H3K4me3 and H3K9ac were increased,while the expression of H3K27me3 was decreased.2.p-STAT3 and STAT3 were significantly activated when IL-10 expression was inhibited.3.After the supplementation of 25mmol/L glucose,the p-STAT3 expression of WT MPC and IL-1010tml/tml MPC decreased significantly.At the same time,the expression of H3K4me3 and H3K9ac increased,while the expression of H3K27me3 decreased.Conclusion:High glucose environment on osteoblastic differentiation has different effects in different cells.High glucose promoted the osteogenic diferentiation of mouse primary osteoblastic cells and partially rescued the osteoblastic inhibition caused by IL-10 low expression.High glucose inhibited the expression of p-STAT3 and promoted the osteogenic differentiation of MPC through the STAT3 pathway.IL-10 low expression activated the expression of p-STAT3,indicating that IL-10 regulates the process of osteoblastic differentiation through the STAT3 pathway.High glucose affected the expression of epigenetic modification proteins during the osteoblastic differentiation of mouse primary osteoblastic cells.