Catalpol attenuates osteoporosis in ovariectomized rats through promoting osteoclast apoptosis via the Sirt6-ERα-FasL axis.

BACKGROUND: Catalpol, a major active component of the Chinese herb Rehmannia glutinosa, possesses various pharmacological benefits, including anti-inflammatory, antidiabetic, and antitumor properties. Recent studies have reported that catalpol can attenuate bone loss and enhance bone formation. Nevertheless, the molecular mechanisms underlying its effects on osteoporosis pathogenesis remain unclear. PURPOSE: We investigated whether catalpol had a protective effect against postmenopausal osteoporosis (PMOP) and explored its exact mechanism of action. METHODS: Seventy-two rats were randomly divided into six groups: sham, model, low-dose catalpol (5 mg/kg/day), medium-dose catalpol (10 mg/kg/day), high-dose catalpol (20 mg/kg/day), and positive control (alendronate, 2.5 mg/kg). In this experiment, a ovariectomy was performed to establish a female rat model of PMOP. After 12 weeks of gavage, micro-computed tomography (micro-CT) and histochemical staining were performed to evaluate bone mass, bone microstructure and histological parameters. Furthermore, RAW 264.7 cells were induced by RANKL to form mature osteoclasts to investigate the effect of catalpol on osteoclast differentiation and apoptosis in vitro. Additionally, the osteoclast apoptosis-related proteins of Sirt6, ERα, FasL, NFATc1, cleaved-caspase 8, cleaved-caspase 3, and Bax were assessed using western blotting. The expressions of NFATc1, Ctsk, Oscar, and Trap were quantified using RT-qPCR. The apoptotic rate of the osteoclasts was determined using flow cytometry. Sirt6 knockdown was performed using siRNA gene silencing in experiments to investigate its role in catalpol-mediated osteoclast apoptosis. The deacetylation of ERα in osteoclasts was tested via co-immunoprecipitation. RESULTS: Catalpol (10 and 20 mg/kg) and alendronate (2.5 mg/kg) could significantly improve bone mineral density (BMD) and microstructure and decrease osteoclast density in ovariectomized (OVX) rats. In addition, catalpol (10 and 20 mg/kg) upregulated the expression of Sirt6, ERα, FasL, cleaved-caspase 8, cleaved-caspase 3, Bax, and downregulated the expression of NFATc1, Ctsk, Oscar, Trap both in vivo and in vitro. Catalpol also promoted ERα deacetylation and stabilized ERα protein to enhance the expression of FasL. In addition, Sirt6 knockdown by siRNA prevented ERα deacetylation and eliminated catalpol-mediated osteoclast apoptosis. CONCLUSIONS: The present study demonstrated that catalpol prevents estrogen deficiency-induced osteoporosis by promoting osteoclast apoptosis via the Sirt6-ERα-FasL axis. These findings revealed a novel molecular mechanism underpinning the impact of catalpol in the progression of osteoporosis and provided novel insights into the treatment of osteoporosis.

Anti-osteoporosis Effect of Fisetin against Ovariectomy Induced Osteoporosis in Rats: In silico, in vitro and in vivo Activity.

Osteoporosis is a bone related disease that is characterised by bone loss that further increases the susceptibility to bone fractures and bone frailty due to disturbances in the micro-architecture of bone tissue. Fisetin (flavonoids) exhibited anti-inflammatory and antioxidative stress effects against various diseases. In this protocol, we make an effort to comfort the anti-osteoporosis effect of fisetin against ovariectomy (OVX) induced osteoporosis. A docking study of fisetin and alendronate on the estrogen (α and ß) and vitamin D receptors was carried out. SaOS-2 (osteoblast like human) cells were used for the estimation of cell proliferation. The OVX induced OVX model was used and three doses of fisetin and alendronate was given to rats till 16 weeks. The hormone levels, bone turnover markers and biochemical parameters were estimated. Fisetin was docked into estrogen (α and ß) and vitamin D receptors, resulting in stable complexes with lower binding scores. Fisetin significantly (p < 0.001) exhibited the induction of cell proliferation against the SaOS-2 cells. OVX induced osteoporosis rats exhibited a suppression of body weight and uterus index, after the Fisetin treatment. Fisetin treatment significantly (p < 0.001) improved the level of bone mineral content (BMC), bone mineral density (BMD) and biochemical parameters such as energy, maximum load, stiffness, young modules, maximum stress and reduced the level of 1,25(OH) 2 D3 and E 2 . Fisetin treatment significantly (p < 0.001) declined the level of phosphorus (P), calcium (Ca) and boosted the level of VitD. Fisetin treatment significantly (p < 0.001) reduced the malonaldehyde (MDA) level and enhanced the glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) level in the bone, intestine and hepatic tissue. Fisetin treatment suppressed the cytokines, RANKL/OPG ratio, receptor activator of nuclear factor-κB ligand (RANKL) and improved the level of osteoprotegerin (OPG). The findings suggest that fisetin could be a beneficial phytoconstituent for the treatment and prevention of postmenopausal osteoporotic complications.

A Novel H2S-releasing Amino-Bisphosphonate which combines bone anti-catabolic and anabolic functions.

Bisphosphonates (BPs) are the first-line treatment of bone loss resulting from various pathological conditions. Due to their high affinity to bone they have been used to develop conjugates with pro-anabolic or anti-catabolic drugs. We recently demontrated that hydrogen sulfide (H2S), promotes osteogenesis and inhibits osteoclast differentiation. Here we developed an innovative molecule, named DM-22, obtained from the combination of alendronate (AL) and the H2S-releasing moiety aryl-isothiocyanate. DM-22 and AL were assayed in vitro in the concentration range 1-33 µM for effects on viability and function of human osteoclasts (h-OCs) and mesenchymal stromal cells (h-MSCs) undergoing osteogenic differentiation. Amperometric measures revealed that DM-22 releases H2S at a slow rate with a thiol-dependent mechanism. DM-22 significantly inhibited h-OCs differentiation and function, maintaining a residual h-OCs viability even at the high dose of 33 µM. Contrary to AL, in h-MSCs DM-22 did not induce cytotoxicity as revealed by LDH assay, significantly stimulated mineralization as measured by Alizarin Red staining and increased mRNA expression of Collagen I as compared to control cultures. In conclusion, DM-22 is a new BP which inhibits h-OCs function and stimulate osteogenic differentiation of h-MSCs, without cytotoxicity. DM-22 is an ideal candidate for a novel family of osteoanabolic drugs.

A scoping review of strategies for the prevention of hip fracture in elderly nursing home residents.

BACKGROUND: Elderly nursing home residents are at increased risk of hip fracture; however, the efficacy of fracture prevention strategies in this population is unclear. OBJECTIVE: We performed a scoping review of randomized controlled trials of interventions tested in the long-term care (LTC) setting, examining hip fracture outcomes. METHODS: We searched for citations in 6 respective electronic searches, supplemented by hand searches. Two reviewers independently reviewed all citations and full-text papers; consensus was achieved on final inclusion. Data was abstracted in duplicate. FINDINGS: We reviewed 22,349 abstracts or citations and 949 full-text papers. Data from 20 trials were included: 7--vitamin D (n = 12,875 participants), 2--sunlight exposure (n = 522), 1--alendronate (n = 327), 1--fluoride (n = 460), 4--exercise or multimodal interventions (n = 8,165), and 5--hip protectors (n = 2,594). Vitamin D, particularly vitamin D(3) > or = 800 IU orally daily, reduced hip fracture risk. Hip protectors reduced hip fractures in included studies, although a recent large study not meeting inclusion criteria was negative. Fluoride and sunlight exposure did not significantly reduce hip fractures. Falls were reduced in three studies of exercise or multimodal interventions, with one study suggesting reduced hip fractures in a secondary analysis. A staff education and risk assessment strategy did not significantly reduce falls or hip fractures. In a study underpowered for fracture outcomes, alendronate did not significantly reduce hip fractures in LTC. CONCLUSIONS: The intervention with the strongest evidence for reduction of hip fractures in LTC is Vitamin D supplementation; more research on other interventions is needed.

Alendronate has an anabolic effect on bone through the differentiation of mesenchymal stem cells.

UNLABELLED: We committed MSCs to differentiate into either osteoblasts or adipocytes and examined the effect of ALN on both adipogenesis and osteoblastogenesis. ALN inhibited adipogenesis while promoting osteoblast differentiation and activity. Our results reveal a new anabolic effect of ALN in differentiating bone marrow cells. INTRODUCTION: Alendronate (ALN) prevents bone loss in postmenopausal patients through the regulation of osteoclastic activity. However, it has also proven to be effective in older adults where the pathophysiological mechanism is the predominance of adipogenesis over osteoblastogenesis. The aim of this study is to determine the in vitro effect of ALN on both osteoblastogenesis and adipogenesis. MATERIALS AND METHODS: Human mesenchymal stem cells (MSCs) were plated at a density of 5 x 10(5) cells/well in 100-cm2 dishes containing MSC growth media. After confluence, cells were committed to differentiate adding either adipogenic or osteogenic media with and without 1,25(OH)2D3 (10(-8) M) and supplemented with ALN at increasing concentrations (10(-9) to 10(-7) M). Untreated differentiating MSCs were used as control. Alkaline phosphatase (ALP), oil red O, and Alizarin red staining were performed at timed intervals (weeks 1 and 2). Additionally, levels of expression of both osteogenesis and adipogenesis transcription factors were measured in protein extracts. Finally, the effect of ALN on PPARgamma2 nuclear activation complex was assessed. RESULTS: We found that ALN has a significant and dose-dependent effect on osteoblastogenesis. This effect was not modified by the presence of 1,25(OH)2D3 in the medium. Furthermore, adipogenic differentiation of MSCs was affected by addition of both ALN and 1,25(OH)2D3 to the media as confirmed by phenotype changes and lower number of lipid droplets. Finally, expression of adipogenic transcription factors and PPARgamma2 activation were reduced in adipose differentiating MSCs treated with either ALN or ALN + 1,25(OH)2D3. CONCLUSIONS: This study shows a potential anabolic effect of ALN in vitro through the stimulation of osteogenic differentiation of MSCs. Additionally, a previously unknown inhibitory effect of ALN on bone marrow adipogenesis was also found.

Synthesis and evaluation of water-soluble polymeric bone-targeted drug delivery systems.

Four polymeric bone-targeting conjugates were synthesized based on poly(ethylene glycol) (PEG, two conjugates) and poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA, two conjugates). The well-known bone-targeting compounds, alendronate and aspartic acid peptide, were used as bone-targeting moieties. Fluorescein isothiocyanate (FITC) was attached to the conjugates as a model drug for detection purposes. The bone-targeting potential of these conjugates was tested in vitro with hydroxyapatite (HA) and in mice. The data obtained indicated that these novel delivery systems could specifically accumulate in the bone tissue.