The Effect Of Teriparatide On Osteoblast Differentiation Under High Glucose Condition And Its Action Mechanism

Objectives Teriparatide（TPTD）is the first choice for the treatment of severe osteoporosis（OP）,which can promote bone formation and increase bone mass.Diabetic osteoporosis（DOP）as a secondary bone metabolic disease can effect the stability of bone system.The purpose of this study was to explored the effects of teriparatide on osteoblasts proliferation,cytoskeleton and differentiation under high glucose,and the regulation of Wnt3a/β-catenin pathway in it.We hope to provide a experimental basis for clinical research on TPTD as a potential treatment for diabetic osteoporosis.Methods 1 To explore the effect of TPTD onMC3T3-E1 cells proliferation under normal glucose condition,the experiment was divided into five groups:blank group（without TPTD）,10^-10 mol/L TPTD group,10^-9 mol/L TPTD group,10^-8 mol/L TPTD group,10^-7 mol/L TPTD group,and the cell proliferation was detected by CCK-8 method after 24h,48h and 72h culture.2 To explore the effects of TPTD and Wnt3a inhibitor G244-LM on proliferation activity,number and cytoskeleton of MC3T3-E1 cells under high glucose condition,experiments were performed in five groups:normal glucose group（5.5 mM glucose）,normal glucose+TPTD group（5.5 mM glucose+10 nM TPTD）,high glucose group（16.5 mM glucose）,high glucose+TPTD group（16.5 mM glucose+10nM TPTD）,high glucose+TPTD+Wnt3a inhibitor G244-LM group（16.5 mM glucose+10nM TPTD+0.1μM G244-LM）.CCK-8 was used to determine the absorbance value of cells in each group after 24h,48h and 72h culture;Calcein-AM was used to detect the number of viable cells in each group after 72h culture;immunofluorescence staining was performed to observe the formation of cytoskeleton after 72h culture.3 To explore the effect of TPTD on osteoblast differentiation through Wnt3a/β-catenin signaling pathway under high glucose condition,the experiment consisted of five groups:normal glucose group（5.5 mM glucose）,normal glucose+TPTD group（5.5 mM glucose+10nM TPTD）,high glucose group（16.5 mM glucose）,high glucose+TPTD group（16.5 mM glucose+10nM TPTD）,and high glucose+TPTD+Wnt3a inhibitor G244-LM group（16.5 mM glucose+10nM TPTD+0.1μM G244-LM）.After 7d of cell culture,osteoblast differentiation was determined by ALP staining and protein activity;the mineralization and deposition of osteoblasts was detected by alizarin red S staining after 21d of cell culture;Real-time PCR and Western blot were used to detect the expression of osteoblast-related factors in each group:Wnt3a,β-catenin,Tcf1,OPG,COL I mRNA and protein expression.Results 1 There was no significant difference in the effect of TPTD on the proliferation of MC3T3-E1 cells in the concentration range of 10^-10 to 10^-7 mol/L TPTD solution.（P>0.05）.2 TPTD and Wnt3a inhibitor had no significant effect on the proliferative activity of MC3T3-E1 cells under high glucose（P>0.05）.3 High glucose inhibited the clarity of MC3T3-E1 cytoskeleton and cell size,the addition of TPTD improved the clarity of cytoskeleton and cell morphology under high glucose condition;after Wnt3a inhibitor was added,the clarity of cytoskeleton and cell size decreased significantly.4 High glucose inhibited the differentiation of osteoblasts,and the addition of TPTD promoted the osteoblasts differentiation under high glucose condition;Wnt3a inhibitor inhibited osteoblast differentiation in high glucose condition（P<0.05）.5 Compared with the high glucose group,the mRNA and protein expression of Wnt3a,β-catenin,Tcf1,OPG,and COL I increased significantly after adding TPTD under high glucose condition,with the addition of Wnt3a inhibitor,the expression of Wnt3a,β-catenin,Tcf1,OPG,and COL I mRNA and protein expression were significantly down-regulated（P<0.05）.Conclusions 1 10^-10～10^-7mol/L TPTD and Wnt3a inhibitor don’t affect the proliferation of MC3T3-E1 cells.After adding TPTD in high glucose condition,MC3T3-E1 cells are significantly enlarged with clear contour and morphology,Wnt3a inhibitor significantly reduces the clarity of cytoskeleton and cell contour,and affects cell morphology and size in high glucose+TPTD group.2 TPTD can improve the expression of pathway-related factors Wnt3a,β-catenin,Tcf1,OPG and COL I by regulating Wnt3a/β-catenin signaling pathway,antagonize the inhibitory effect of high glucose and promote the differentiation of osteoblasts in high glucose environment.Figure 14;Table 11;Reference 128...