| Postmenopausal osteoporosis (PMO) is a metabolic bone disease characterized by a decrease of bone mass after menopause. Normal bone remodeling depends upon a balance between the osteoblast (OB)-mediated bone formation and osteoclast-mediated bone resorption. Under the appropriate inducing condition, MSC can differentiate into osteobalst or adipocyte. After surgical and natural menopause, the bone resorption exceeds the bone formation, which leads to a decrease in bone mass. Skeletal system homeostasis is greatly influenced by endocrine and immune regulation. Recent study presents an association of these pathogenetic changes with disturbances of the endocrine system, especially sex steroid hormones, and immune system. Up to now, approaches to managing PMO have been directed towards prevention of bone resorption, for example the estrogen replacement therapy. The mechanism of actions of different treatment on the differentiation and biological functions of osteoblast is still unclear.Dehydroepiandrosterone, DHEA, is among the most plentiful circulating adrenal hormones, and exerts biological actions that cannot be attributed to its metabolites. In recent years, we have found that DHEA level is decreased in postmenopausal women compared to that of the fertile women, which suggests the potential clinical benefit of DHEA as therapeutics of PMO. However, both the molecule and biochemical mechanisms by which DHEA exerts its effect on bone metabolism remains shrouded in mystery. The present study is to elucidate the mechanisms of MSC differentiation into osteoblast, the regulation of immune cells on the differentiation of MSC to osteoblast, and the actions of DHEA or BSNXD, a Chinese traditional medicine recipe, on OB differentiation in vitro and in vivo. Part I The differentiation mechanism of MSC into osteoblastObjective To investigate the mechanisms of the bone marrow-derived MSC differentiating into osteoblast.Methods The bone marrow-derived MSC was isolated and cultured under the osteoblast differentiation condition. Real time RT-PCR was used to analyze the specific transcription factors Runx2, Osterix, Osteocalcin and specific marker Collagenl mRNA expression; osteoblast was identified by ALP staining, and the ALP activity was by ALP Assay Kit; Alizarin Red staining was used to detect the bone nodules. The MSCs were pretreated with IGF-1, LY294002(inhibitor of PI3K pathway) or rapamycin (inhibitor of mTOR pathway), respectively. The MSC was cultured with the adipocyte differentiation condition; real time RT-PCR analysis was for specific marker PPARy mRNA expression; Oil Red O staining was used to calculate adipocyte number.Results Under the OB differentiation condition, the expression of specific marker Collagenl, and transcription factors Runx2, osteocalcin, Osterix mRNA expression were increased. MSCs were differentiated into osteoblast, and then formed bone nodules. The ALP activity could be enhanced by IGF-1, and the blocker of PI3K and mTOR could inhibit the effect of IGF-1. Under the adipocyte differentiation condition, the specific transcription factor PPARy mRNA expression increased, and then mature adipocyte formed. The formation of adipocyte was not influenced by IGF-1, but increased by LY294002or rapamycin treatment.Conclusions The OB differentiation condition can increase the specific transcription factor Runx2, Osterix, Osteocalcin and specific marker Collagenl mRNA expression, which results in MSC differentiated into osteoblast. IGF-1promotes OB differentiation through PI3K-mTOR signaling pathway. Part II Regulation of immune cells on the MSC differentiating into osteoblastObjective To investigate the regulation of immune cells on the MSC differentiating into osteoblast. Methods The MSC and BMM from bone marrow was isolated, and cultured under the osteoblast differentiation culture condition; real time RT-PCR analysis was for transcription factor Runx2, Osterix, Osteocalcin, and specific marker Collagen1mRNA expression; ALP staining and ALP Assay Kit were used to analyze ALP or its activity in osteoblast; Alizarin Red staining was used to detect the bone nodules. IGF-1, LY294002(inhibitor of PI3K pathway) or rapamycin (specific inhibitor of mTOR pathway) were used to investigate the regulation on the MSC differentiation into osteoblast. MSCs were co-culture with BMM or Treg under the adipocyte differentiation culture condition; real time RT-PCR analysis for specific marker PPARr mRNA expression, Oil Red O staining for the calculation of adipocyte numbers. Treg cells from spleen were isolated and co-cultured with MSC under the OB differentiation medium to detect the ALP activity and bone nodules number. The anti-IL-10, anti-TGFβ, anti-CTLA-4or transwell were used to investigate the correspondent molecular mechanism.Results Under the OB differentiation culture condition, co-culture of BMM with MSC reduced the OB production, and the ration of RANKL/OPG increased, the expression of specific marker Collagen1, Osterix mRNA decreased, and ALP activity declined, bone nodules number decreased. Under the adipocyte inducing condition, co-culture of MSC with BMM did not affect PPARy mRNA expression and change the adipocyte number. Treg promoted MSC differentiating into osteoblast, ALP activity was enhanced, and bone nodules number increased. Ant-IL-10, TGF-β,CTLA-4and transwell could inhibit the regulation of Treg on OB formation. Treg could not influence adipocyte differentiation from MSC.Conclusions BMMs inhibit OB differentiation from MSC by downregulating of osterix, Collagen1mRNA expression, in turn decrease the ALP activity and bone nodule number. Treg promotes ALP activity in OB formation by cell-cell direct contact and secretion of IL-10, TGF-β, CTLA-4. Neither BMM nor Treg can affect adipocyte differentiation from MSC.Part Ⅲ The Modulation of DHEA on the MSC Differentiating into OsteoblastObjective To investigate the mechanism of DHEA on the osteoblast differentiation from MSC.Methods Under the OB differentiation inducing condition, DHEA were added to the cell culture system, including MSC, co-culture of BMM with MSC, Treg with MSC, MSC with BMM and Treg. Real time RT-PCR analysis was for Runx2, Collagenl, Osterix, Osteocalcin, RANKL mRNA expression, ALP activity was for OB quantity, and Alizarin Red O was for adipocyte number. IGF-1, PI3K and mTOR inhibitor were used to investigate the regulation on the differentiation. Under the adipocyte differentiation inducing condition, DHEA were added to the MSC culture, or the correspondent co-culture of MSC with BMM. C57BL/6mice were ovariectomized for foundation of osteoporosis, and then were afforded with DHEA (OVX+DHEA group),17β-estradiol (OVX+E2group) and Saline (OVX group), respectively. The femur and spleen were collected in12weeks of the administration. Femur were measured for morphometry of bone tissue by micro-CT and bone tissue chemical method. The immune cells from spleen were analyzed by flow cytometry (FCM).Results DHEA increased OB differentiation from MSC; in the co-culture of BMM with MSC, DHEA decreased the inhibition of BMM to the OB differentiation from MSC; in the co-culture of MSC with Treg, DHEA enhanced the upregulation of Treg on the OB differentiation from MSC; in the co-culture of MSC with BMM and Treg, DHEA increased the OB differentiation from MSC. DHEA raised the expression of Runx2, osterix, collagenl, osteocalcin mRNA, and decreased the RANKL mRNA expression, and then increased the ALP activity in OB, and bone nodule number. Under the adipocyte differentiation inducing condition, both in the MSC culture or the co-culture, DHEA promoted MSC differentiating into adipocyte; PPARγ expression in adipocyte increased. With the same number of MSCs, in either the OB differentiation or adipocyte differentiation inducing condition, DHEA increased the ration of osteoblast number to adipocyte number, which implies that the effect of DHEA on OB differentiation is dominant. DHEA promotes the bone tissue status; DHEA decreased the percent of CD19+B cells, CD14+monocytes, CD8+T cells in spleen, and increased the percent of FoxP3+Treg, but had no effect on the percent of NK cells.Conclusions DHEA promotes MSC differentiating into OB; DHEA antagonists the downregulation of BMM on MSC differentiating into OB; DHEA enhances the upregulation of Treg on the MSC differentiating into OB. DHEA increases the expression of Collagen1, Runx2, osteocalcin, OSX, and decreases the expression of RANKL mRNA expression; ALP activity of OB increases and bone nodules number increases. DHEA actions are enhanced by IGF-1and is independent of PI3K-Mtor signal pathway. DHEA could improve the bone tissue morphometry of the PMO model, and increase the percent of FoxP3+Treg.Part Ⅳ The Modulation of BSNXD on the Promotion of MSC Differentiating into OsteoblastObjective To investigate the mechanism of BSNXD on the osteoblast differentiation from MSC.Methods Under the OB differentiation inducing condition, BSNXD serum were added to the cell culture system, including culture of MSCs, co-culture of BMM with MSC, Treg with MSC, MSC with BMM and Treg. IGF-1, PI3K and mTOR inhibitor were used to investigate the regulation of BSNXD. Under the adipocyte differentiation inducing condition, BSNXD serum were added to the MSC mono-culture, and MSC and BMM co-culture system. C57BL/6mice were ovariectomized for foundation of osteoporosis, and then were afforded with BSNXD (OVX+BSNXD), DHEA (OVX+DHEA),17β-estradiol (OVX+E2) or Saline (OVX), respectively. The femur and spleen of each group were collected in12weeks of the administration. Femur were measured for morphometry of bone tissue by micro-CT and bone tissue chemical method. The immune cells of spleen were analyzed by flow cytometry (FCM).Results BSNXD increased OB differentiation from MSC; in the co-culture of BMM with MSC, BSNXD decreased the inhibition of BMM to the OB differentiation from MSC; in the co-culture of MSC with Treg, BSNXD enhanced the upregulation of Treg on the OB differentiation from MSC; in the co-culture of MSC with BMM and Treg, BSNXD increased the OB differentiation from MSC. BSNXD raised the expression of Runx2, osterix, collagenl, osteocalcin mRNA and suppressed the RANKL mRNA expression, and then increased the ALP activity in OB, and bone nodule number. BSNXD promoted the bone tissue status; BSNXD decreased the percent of CD19+B cells, CD14+monocytes, CD8+T cells in spleen, and increased the percent of CTLA-4+Treg, but had no effect on the percent of NK cells. Compared to DHEA, BSNXD only promoted the osteoblast formation, but had no effect on adipocyte production in vitro, and BMD level after treated with BSNXD was higher than that of DHEA.Conclusions BSNXD promotes MSC differentiating into OB; BSNXD presents antagonist to the downregulation of BMM on MSC differentiating into OB; BSNXD enhances the effect of Treg on the MSC differentiating into OB through increasing the expression of Collagenl, Runx2, osteocalcin, OSX and decreasing the expression of RANKL mRNA expression. The action of BSNXD may be promoted by IGF-1via PI3K-mTOR signal pathway, which improves the bone tissue morphometry of the PMO model. BSNXD increases further the percent of CTLA-4+Treg. BSNXD surpasses DHEA on bone metabolism. |
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