Glucagon-like peptide-1 receptor agonist Liraglutide has anabolic bone effects in ovariectomized rats without diabetes.
ABSTRACT: Recently, a number of studies have demonstrated the potential beneficial role for novel anti-diabetic GLP-1 receptor agonists (GLP-1RAs) in the skeleton metabolism in diabetic rodents and patients. In this study, we evaluated the impacts of the synthetic GLP-1RA Liraglutide on bone mass and quality in osteoporotic rats induced by ovariectomy (OVX) but without diabetes, as well as its effect on the adipogenic and osteoblastogenic differentiation of bone marrow stromal cells (BMSCs). Three months after sham surgery or bilateral OVX, eighteen 5-month old female Wistar rats were randomly divided into three groups to receive the following treatments for 2 months: (1) Sham + normal saline; (2) OVX + normal saline; and (3) OVX + Liraglutide (0.6 mg/day). As revealed by micro-CT analysis, Liraglutide improved trabecular volume, thickness and number, increased BMD, and reduced trabecular spacing in the femurs in OVX rats; similar results were observed in the lumbar vertebrae of OVX rats treated with Liraglutide. Following in vitro treatment of rat and human BMSCs with 10 nM Liraglutide, there was a significant increase in the mRNA expression of osteoblast-specific transcriptional factor Runx2 and the osteoblast markers alkaline phosphatase (ALP) and collagen ?1 (Col-1), but a significant decrease in peroxisome proliferator-activated receptor ? (PPAR?). In conclusion, our results indicate that the anti-diabetic drug Liraglutide can exert a bone protective effect even in non-diabetic osteoporotic OVX rats. This protective effect is likely attributable to the impact of Liraglutide on the lineage fate determination of BMSCs.
Project description:Cortex Eucommiae is used worldwide in traditional medicine, various constituents of Cortex Eucommiae, such as chlorogenic acid (CGA), has been reported to exert anti-osteoporosis activity in China, but the mechanism about their contribution to the overall activity is limited. The aims of this study were to determine whether chlorogenic acid can prevent estrogen deficiency-induced osteoporosis and to analyze the mechanism of CGA bioactivity. The effect of CGA on estrogen deficiency-induced osteoporosis was performed in vivo. Sixty female Sprague-Dawley rats were divided randomly among a sham-operated group and five ovariectomy (OVX) plus treatment subgroups: saline vehicle, 17?-ethinylestradiol (E2), or CGA at 9, 27, or 45 mg/kg/d. The rats' femoral metaphyses were evaluated by micro-computed tomography (?CT). The mechanism of CGA bioactivity was investigated in vitro. Bone mesenchymal stem cells (BMSCs) were treated with CGA, with or without phosphoinositide 3-kinase (PI3K) inhibitor LY294002. BMSCs proliferation and osteoblast differentiation were assessed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and alkaline phosphatase, with or without Shp2 interfering RNA (RNAi). The results display that CGA at 27 and 45 mg/kg/day inhibited the decrease of bone mineral density (BMD) that induced by OVX in femur (p< 0.01), significantly promoted the levels of bone turnover markers, and prevented bone volume fraction (BV/TV), connectivity density (CoonD), trabecular number (Tb.N), trabecular thickness (Tb.Th) (all p< 0.01) to decrease and prevented the trabecular separation (Tb.Sp), structure model index (SMI)(both p< 0.01) to increase. CGA at 1 or 10 ?M enhanced BMSC proliferation in a dose-dependent manner. CGA at 0.1 to 10 ?M increased phosphorylated Akt (p-Akt) and cyclin D1. These effects were reversed by LY294002. CGA at 1 or 10 ?M increased BMSC differentiation to osteoblasts (p< 0.01), Shp2 RNAi suppressed CGA-induced osteoblast differentiation by decreasing Shp2, p-Akt, and cyclin D1. This study found that CGA improved the BMD and trabecular micro-architecture for the OVX-induced osteoporosis. Therefore, CGA might be an effective alternative treatment for postmenopausal osteoporosis. CGA promoted proliferation of osteoblast precursors and osteoblastic differentiation of BMSCs via the Shp2/PI3K/Akt/cyclin D1 pathway.
Project description:Osteoporotic patients often suffer from bone fracture but its healing is compromised due to impaired osteogenesis potential of bone marrow-derived mesenchymal stem cells (BMSCs). Here we aimed to exploit adipose-derived stem cells from ovariectomized rats (OVX-ASCs) for bone healing. We unraveled that OVX-ASCs highly expressed miR-214 and identified 2 miR-214 targets: CTNNB1 (?-catenin) and TAB2. We demonstrated that miR-214 targeting of these two genes blocked the Wnt pathway, led to preferable adipogenesis and hindered osteogenesis. As a result, OVX-ASCs implantation into OVX rats failed to heal critical-size metaphyseal bone defects. We further engineered the OVX-ASCs with a novel Cre/loxP-based hybrid baculovirus vector that conferred prolonged expression of miR-214 sponge. Gene delivery for miR-214 sponge expression successfully downregulated miR-214 levels, activated the Wnt pathway, upregulated osteogenic factors ?-catenin/Runx2, downregulated adipogenic factors PPAR-? and C/EBP-?, shifted the differentiation propensity towards osteogenic lineage, enhanced the osteogenesis of co-cultured OVX-BMSCs, elevated BMP7/osteoprotegerin secretion and hindered exosomal miR-214/osteopontin release. Consequently, implanting the miR-214 sponge-expressing OVX-ASCs tremendously improved bone healing in OVX rats. Co-expression of miR-214 sponge and BMP2 further synergized the OVX-ASCs-mediated bone regeneration in OVX rats. This study implicates the potential of suppressing miR-214 by baculovirus-mediated gene delivery in osteoporotic ASCs for regenerative medicine.
Project description:Estrogen is very important to the differentiation of B lymphocytes; B lymphopoiesis induced by OVX was supposedly involved in osteoporosis. But the effects of B lymphocytes on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) are not clear. In this study, we detected bone quality and bone loss in a trabecular bone by electronic universal material testing machine and microcomputed tomography (micro-CT) in OVX and splenectomized-ovariectomy (SPX-OVX) rats. Additionally, changes in lymphocytes (B lymphocyte, CD4+ and CD8+ T lymphocytes, and macrophages) in the bone marrow were analyzed by flow cytometry. The osteogenesis of BMSCs cocultured with normal and LPS-pretreated B lymphocytes was detected by BCIP/NBT and Alizarin red S staining. Measurement of the Notch2, Notch4, Hey1, Hey2, Hes1, and runt-related transcription factor 2 (Runx2) expression in BMSCs cocultured with B lymphocytes was done using real-time PCR. The effects of dexamethasone and DAPT (inhibitor of Notch signaling) on osteogenesis of BMSCs were detected by BCIP/NBT, Alizarin red S staining, and real-time PCR. Osteoporosis happened in OVX rats, more serious in SPX-OVX rats, B lymphocytes increased in OVX rats, and sharply higher in SPX-OVX rats. Osteoporosis did not happen in SPX rats which is still companied with a high increase of B lymphocytes. LPS-pretreated B lymphocytes suppressed the osteogenesis of BMSCs, but the normal B lymphocytes could not. The LPS-pretreated B lymphocytes upregulated the expression of Notch4, Hes1, and Hey2 and downregulated the expression of Runx2 in BMSCs. Dexamethasone and DAPT could downregulate the high expression of Notch4, Hes1, Hey2 and upregulate the low expression of Runx2 in BMSCs which cocultured with LPS treated B lymphocytes, the inhibited ALP and Alizarin red staining in BMSCs which cocultured with LPS treated B lymphocytes also partly restored.
Project description:Osteoporosis is characterized by low bone density and quality with high risk of bone fracture. Here, we investigated anti-osteoporotic effects of natural plants (Lycii Radicis Cortex (LRC) and Achyranthes japonica (AJ)) in osteoblast and osteoclast cells in vitro and ovariectomized mice in vivo. Combined LRC and AJ enhanced osteoblast differentiation and mineralized bone-forming osteoblasts by the up-regulation of bone metabolic markers (Alpl, Runx2 and Bglap) in the osteoblastic cell line MC3T3-E1. However, LRC and AJ inhibited osteoclast differentiation of monocytes isolated from mouse bone marrow. In vivo experiments showed that treatment of LRC+AJ extract prevented OVX-induced trabecular bone loss and osteoclastogenesis in an osteoporotic animal model. These results suggest that LRC+AJ extract may be a good therapeutic agent for the treatment and prevention of osteoporotic bone loss.
Project description:Osteoporosis is caused by pathologic factors such as aging, hormone deficiency or excess, inflammation, and systemic diseases like diabetes. Bone marrow stromal cells (BMSCs), the mesenchymal progenitors for both osteoblasts and adipocytes, are modulated by niche signals. In differential pathologic states, the pathological characteristics of BMSCs to osteoporoses and functional differences are unknown. Here, we detected that trabecular bone loss co-existed with increased marrow adiposity in 6 osteoporotic models, respectively induced by natural aging, accelerated senescence (SAMP6), ovariectomy (OVX), type 1 diabetes (T1D), excessive glucocorticoids (GIOP) and orchidectomy (ORX). Of the ex vivo characteristics of BMSCs, the colony-forming efficiency and the proliferation rate in aging, SAMP6, OVX, GIOP and ORX models decreased. The apoptosis and cellular senescence increased except in T1D, with up-regulation of p53 and p16 expression. The osteogenesis declined except in GIOP, with corresponding down-regulation of Runt-related transcription factor 2 (RUNX2) expression. The adipogenesis increased in 6 osteoporotic models, with corresponding up-regulation of Peroxisome proliferator activated receptor gamma (PPAR?) expression. These findings revealed differential characteristics of BMSCs in a common shift from osteoblastogenesis to adipogenesis among different osteoporoses and between sexes, and provide theoretical basis for the functional modulation of resident BMSCs in the regenerative therapy for osteoporosis.
Project description:We evaluated the efficacy of platelet-rich plasma (PRP) in combination with allogeneic bone marrow mesenchymal stem cells (BMSCs) for the treatment of osteoporotic bone defects in an ovariectomized rat model. By day 42 after injury, in vivo microcomputed tomography (micro-CT) imaging revealed that bone defects of control rats and ovariectomized rats treated with PRP and BMSCs were completely repaired, whereas those of ovariectomized rats treated with PRP or BMSCs alone exhibited slower healing. Histological data were consistent with these results. We also assessed changes to bone trabeculae in the proximal tibial growth plate. In ovariectomized rats treated with PRP or with a combination of PRP and BMSCs, the trabecular connectivity densities (Conn.D), bone volume ratios (BV/TV), and numbers (Tb.N) in the defect areas increased significantly from day 7 to day 42. These results indicate that PRP treatment enhances bone microarchitecture in osteoporosis. Moreover, expression levels of osteogenesis-specific marker genes including RUNX2, OSX, and OPN were significantly upregulated in rats treated with PRP and BMSCs compared to those of other groups. Thus, we conclude that treatment with PRP combined with BMSCs significantly promotes healing of osteoporotic bone defects. This study provides an alternative strategy for the treatment of osteoporotic bone loss.
Project description:Increased subchondral trabecular bone turnover due to imbalanced bone-resorbing and bone-forming activities is a hallmark of osteoarthritis (OA). Wnt5a/Ror2 signaling, which can derive from bone marrow stromal cells (BMSCs), takes a role in modulating osteoblast and osteoclast formation. We showed previously that experimentally unilateral anterior crossbites (UACs) elicited OA-like lesions in mice temporomandibular joints (TMJs), displaying as subchondral trabecular bone loss. Herein, we tested the role of BMSC-derived Wnt5a/Ror2 signaling in regulating osteoclast precursor migration and differentiation in this process. The data confirmed the decreased bone mass, increased tartrate-resistant acid phosphatase (TRAP)-positive cell number, and enhanced osteoclast activity in TMJ subchondral trabecular bone of UAC-treated rats. Interestingly, the osteoblast activity in the tissue of TMJ subchondral trabecular bone of these UAC-treated rats was also enhanced, displaying as upregulated expressions of osteoblast markers and increased proliferation, migration, and differentiation capabilities of the locally isolated BMSCs. These BMSCs showed an increased CXCL12 protein expression level and upregulated messenger RNA expressions of Rankl, Wnt5a, and Ror2. Ex vivo data showed that their capacities of inducing migration and differentiation of osteoclast precursors were enhanced, and these enhanced capabilities were restrained after blocking their Ror2 signaling using small interfering RNA (siRNA) assays. Reducing Ror2 expression in the BMSC cell line by siRNA or blocking the downstream signalings with specific inhibitors also demonstrated a suppression of the capacity of the BMSC cell line to promote Wnt5a-dependent migration (including SP600125 and cyclosporine A) and differentiation (cyclosporine A only) of osteoclast precursors. These findings support the idea that Wnt5a/Ror2 signaling in TMJ subchondral BMSCs enhanced by UAC promoted BMSCs to increase Cxcl12 and Rankl expression, in which JNK and/or Ca(2+)/NFAT pathways were involved and therefore were engaged in enhancing the migration and differentiation of osteoclast precursors, leading to increased osteoclast activity and an overall TMJ subchondral trabecular bone loss in the UAC-treated rats.
Project description:Osteoporosis, an imbalance in the bone-forming process mediated by osteoblasts and the bone-resorbing function mediated by osteoclasts, is a bone degenerative disease prevalent among the aged population. Due to deleterious side effects of currently available medications, probiotics as a potential treatment of osteoporosis is an appealing approach. Hence, this study aims to evaluate the beneficial effects of two novel Lactobacilli strain probiotics on bone health in ovariectomized (OVX) induced osteoporotic mice model and its underlying mechanisms. Forty-five 9-week-old Institute of Cancer Research (ICR) mice underwent either a sham-operation (n = 9) or OVX (n = 36). Four days after the operation, OVX mice were further divided into four groups and received either saline alone, Lactobacillus plantarum GKM3, Lactobacillus paracasei GKS6 or alendronate per day for 28 days. After sacrifice by decapitation, right distal femur diaphysis was imaged via micro-computed tomography (MCT) and parameters including bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and bone mineral density (BMD) were measured. Moreover, GKM3 and GKS6 on RANKL-induced osteoclast formation and osteoblast differentiation using in vitro cultures were also investigated. The results showed that both probiotics strains inhibited osteoporosis in the OVX mice model, with L. paracasei GKS6 outperforming L. plantarum GKM3. Besides this, both GKS6 and GKM3 promoted osteoblast differentiation and inhibited RANKL-induced osteoclast differentiation via the Bone Morphogenetic Proteins (BMP) and RANKL pathways, respectively. These findings suggested that both strains of Lactobacilli may be pursued as potential candidates for the treatment and management of osteoporosis, particularly in postmenopausal osteoporosis.
Project description:The bone mineral deficiency in osteoporosis poses a threat to the long-term outcomes of endosseous implants. The inhibitors of cathepsin K (CatK) significantly affect bone turnover, bone mineral density (BMD) and bone strength in the patients with osteoporosis. Therefore, we hypothesised that the application of a CatK inhibitor (CatKI) could increase the osseointegration of endosseous implants under osteoporotic conditions. Odanacatib (ODN), a highly selective CatKI, was chosen as the experimental drug. Sixteen rats were randomised into 4 groups: sham, ovariectomy (OVX) with vehicle, OVX with low-dose ODN (5?mg/kg) and OVX with high-dose ODN (30?mg/kg). Titanium implants were placed into the distal metaphysis of bilateral femurs of each OVX rat. After 8 weeks of gavaging, CatKI treatment increased the removal torque, BMD and bone-to-implant contact (BIC). Moreover, high-dose CatKI exerted a better influence than low-dose CatKI. Furthermore, CatKI treatment not only robustly suppressed CatK gene (CTSK) expression, but also moderately reduced expression of the osteoblast-related genes Runx2, Collagen-1, BSP, Osterix, OPN, SPP1 and ALP. Thus, CatKI could affect the osteoblast-related genes, although the balance of bone turnover was achieved mainly by CatK inhibition. In conclusion, CatKI prevented bone loss and aided endosseous implantation in osteoporotic conditions.