The Effect Of High Glucose And PTHrp On Osteoblastic And Canoniacal Wnt Signaling Pathway In Osteoblastic

[Background]:The incidence of osteoporosis in diabetic patients were significantly higher than that in patients without diabetic.And the risk of fractures increase significantly than without diabetic patients.Bone in these patients often exhibits bone formation reduced not bone resorption increased.Studies have shown that inhibition of osteoblastic by high glucose can be reversed by parathyroid hormone-related Protein(PTHrp).But the potential mechanism of it is unclear.[Objective]:To demonstrate the effect of high glucose,endogenous or exogenous PTHrp in function of osteoblastic and classical Wnt signaling pathway in osteoblastic under the background of diabetes.[Methods]:The osteoblasts from WT or PTHrp heterozygotes(PTHrp+/-)were isolated from calvaria of 1-2 day-old mice and passaged cultured in contained 5mM glucose DMEM medium(NG).After the second generation grow to 70%,These osteoblasts were cultured in 5 mm glucose concentration(NG)and 25 mm glucose DMEM medium(HG)respectively.At the same time,the Wnt signaling pathway inhibitor XAV939 and different concentrations of exogenous PTHrp were joined in these osteoblasts.The CCK-8 and alizarin red staining were used for detected osteoblast proliferation and osteoblast mineralization in vitro respectively.The content of beta-catenin in these groups were tested by immunofluorescence.The Wnt signaling pathways related gene and protein expression level were analysis by RT-PCR and Western Blot.[Results]:Compared with WT+NG group,the expression level of beta-catenin,Lef-1,ALP,OPG and Runx2 genes or proteins in WT+HG and WT+HG +PTHrp(10-8)+XAV939(2 mu mol/L)were all decreased(p<0.05);And the expression of these index in WT+ HG + PTHrp group were raised obviously(P<0.05).Compared with WT+NG group,high glucose concentration of WT or PTHrp+/-osteoblastic proliferation,calcified nodule number were decreased significantly(P<0.05);In addition,for deficiency of endogenous PTHrp,the osteoblast proliferation and mineralization ability will be further hindered(P<0.05).Compared with WT group,Wnt signaling pathways of two genotypes of osteoblast in high glucose,the beta-catenin,p-GSK3 beta,Lef-1 factor and RunX2,OPG and other downstream cytokines significantly lowered(p<0.05).[Conclusion]:The data above shows that HG can deteriorate the ability of osteoblast proliferation,differentiation and mineralization.And then inhibition of the canonical Wnt pathway by HG can be reversed by exogenous PTH in Osteoblastic.In addition,HG may inhibit different part of canonical Wnt pathway to degradation of beta-catenin in osteoblast;and results in canonical Wnt signaling pathways blocked and affect the normal cell function in the osteoblast.Endogenous PTHrp secretion deficiency lead to canonical Wnt signaling pathways blocked worse.These results from vitro that exogenous PTHrp can increase bone formation in patients with diabetes mellitus osteoporosis can provides a new evidence.