Therapeutic Agent For Osteoporosis Research Articles

High molecular weight hyaluronic acid alleviates ovariectomy-induced bone loss in mice

BackgroundThe rapid decline in ovarian function associated with menopause promotes osteoclast differentiation and increases bone resorption, disrupting of bone homeostasis and increasing the risk of osteoporosis. Hyaluronic acid (HA) is a polysaccharide ubiquitously present in the connective tissues. Recent reports indicate that high-molecular-weight HA (HMW-HA) promotes osteoblast proliferation, enhances alkaline phosphatase activity and mineral deposition, and promotes the expression of bone differentiation markers, such as Runx2 and osteocalcin. HMW-HA also inhibits the expression of the receptor activator of nuclear factor kappa-B ligand (RANKL) in osteoblasts. These results suggest that HMW-HA may be an effective therapeutic agent against postmenopausal osteoporosis. Therefore, this study aimed to examine whether HMW-HA alleviates ovariectomy (OVX)-induced bone loss in mice.MethodsEight-week-old female C57BL/6 J mice were randomly divided into the following five groups: Group 1: Sham/saline, Group 2: OVX/saline, Group 3: OVX/HMW-HA [15 mg/kg]; Group 4: OVX/HMW-HA [30 mg/kg]; and Group 5: OVX/HMW-HA [60 mg/kg]. Mice were administered HMW-HA or saline subcutaneously starting from 1 week after OVX and changes in bone mass were analyzed at 5 weeks using three-dimensional micro-computed tomography (3D-μCT). In addition, changes in osteoclast parameters were analyzed histologically.ResultsThe reduction in trabecular bone volume and trabecular number was significantly ameliorated in the OVX/HMW-HA group compared with that observed in the OVX/saline group, along with a significant inhibition of the increase in trabecular spacing. In addition, the OVX/HMW-HA group exhibited a significant reduction in osteoclast surface area and number compared with the OVX/saline group, with no significant differences compared with the sham group. In vitro experiments revealed that depletion of HMW-HA from the culture medium by hyaluronidase treatment increased RANKL expression in the bone marrow stromal cell line ST2. These data suggest that HMW-HA alleviates OVX-induced bone loss by downregulating osteoclast formation and/or activity in mice.ConclusionHMW-HA is a potential novel therapeutic agent for osteoporosis.

Strontium-Doped Mesoporous Bioactive Glass-Loading Bisphosphonates Inhibit Osteoclast Differentiation and Prevent Osteoporosis in Ovariectomized Mice

Postmenopausal osteoporosis, a metabolic bone disease associated with aging that affects bones throughout the body, is emerging as an urgent public health concern and imposes a substantial healthcare burden on society. The clinical application of bisphosphonate, the primary treatment for osteoporosis, is limited owing to the drug’s severe complications. Herein, we investigate the synthesis and utilization of strontium-doped mesoporous bioactive glass loaded with alendronate (ALN@Sr-MBG) as a novel therapeutic agent for osteoporosis, to explore its potential as an alternative to alendronate (ALN). Strontium-doped mesoporous bioactive glass (Sr-MBG) was synthesized using the sol–gel method, while ALN@Sr-MBG was obtained via incorporating Sr-MBG into an alendronate saturated solution. The bioactivities of ALN@Sr-MBG, including biotoxicity, inflammation inhibition, and anti-osteoclast differentiation, were investigated in vitro using CCK-8, flow cytometry, tartrate-resistant acid phosphatase (TRAP) staining, and RT-PCR assays. Animal models were established by surgically removing the ovaries from non-pregnant female C57/BL mice, followed by weekly intraperitoneal injections of ALN@Sr-MBG, ALN, or excipients. After 8 weeks, the mice femurs were extracted and analyzed through micro-CT scanning and hematoxylin-eosi, osteoblastic, and osteogenic staining. These in vitro findings demonstrate that ALN@Sr-MBG displays enhanced biological efficacy compared to ALN in terms of inflammation inhibition, osteogenesis promotion, and osteoclastogenesis inhibition. Furthermore, micro-CT analysis revealed that ALN@Sr-MBG significantly augments bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), while reducing trabecular separation (Tb.Sp) and the structural model index (SMI) in mice with ovariectomy-induced osteoporosis. The osteoblast and osteogenic staining results indicate the enhanced bioactivities of ALN@Sr-MBG in promoting bone formation and inhibiting bone resorption compared to ALN. In vitro and in vivo assessments further confirmed that ALN@Sr-MBG exhibits superior anti-osteoporotic bioactivity compared to ALN. This study’s findings confirm the potential of ALN@Sr-MBG as a novel alternative therapy to ALN and a drug candidate for the treatment of osteoporosis.

Morinda officinalis saponins promote osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells via the BMP-SMAD signaling pathway.

Morinda officinalis saponins (MOS), a traditional Chinese medicine extracted from M. officinalis roots, have been used as a health supplement. Existing evidence suggests that extracts from this plant can be used for osteoporosis treatment. However, the molecular mechanisms underlying the anti-osteoporotic effects of M. officinalis remain poorly understood. In this study, we investigated the osteogenesis-promoting effects of MOS on human umbilical cord-derived mesenchymal stem cells (HUC-MSCs). Alkaline phosphatase staining, alizarin red staining, and quantitative reverse transcription-PCR demonstrated that MOS promoted the osteogenic differentiation of HUC-MSCs in a concentration-dependent manner. RNA sequencing results showed that the expression of key osteogenic differentiation-related genes, including BMP4, as well as the activity of transforming growth factor-β and calcium signaling pathways increased following MOS treatment. Furthermore, treatment with the bone morphogenetic protein (BMP) antagonist Noggin reversed the MOS-induced pro-osteogenic differentiation effects and the upregulation of osteoblast-specific markers. Overall, the results indicate that MOS can partially promote osteogenic differentiation of HUC-MSCs by regulating the BMP-SMAD signaling pathway. These findings indicate the potential utility of MOS as a therapeutic agent for osteoporosis, particularly in the context of stem cell therapy.

Potential of 20 unexplored genes to be candidates for osteogenic regulators as novel therapeutic strategies in bone diseases

Background: Bone homeostasis is maintained by a balance between bone formation by osteoblasts and bone resorption by osteoclasts. As a therapeutic agent for osteoporosis, a strategy of suppressing bone resorption by inhibiting the function of osteoclasts is used; however, long-term use causes problems, such as decreased formation of osteoblasts and osteonecrosis of the jaw. Therefore, various growth factors and hormones have been used to promote the proliferation of osteoblasts, but the current situation is that long-term use cannot be applied owing to issues such as carcinogenic risk. To establish an appropriate osteogenesis-promoting strategy, it is essential to ensure the fate of progenitor cells in the mature osteoblasts. Purpose: To investigate potential osteogenic regulators based on published single-cell transcriptome data as an alternative approach to bone diseases treatment. Methods: a systematic review using articles through several databases with descriptions related to various genes were identified based on single-cell transcriptome data as candidates for osteogenic regulators. Results: 20 unexplored genes were identified based on single-cell transcriptome data as candidates for osteogenic regulators. Conclusions: Increasing osteoblast activity through the promotion of osteoblast differentiation shows promise as a novel approach for treating bone-formation-related diseases.

Safranal inhibits estrogen-deficiency osteoporosis by targeting Sirt1 to interfere with NF-κB acetylation

BackgroundOsteoporosis is a prevalent bone metabolic disease in menopause, and long-term medication is accompanied by serious side effects. Estrogen deficiency-mediated hyperactivated osteoclasts is the initiating factor for bone loss, which is regulated by nuclear factor-κB (NF-κB) signaling. Safranal (Saf) is a monoterpene aldehyde produced from Saffron (Crocus sativus L.) and possesses multiple biological properties, particularly the anti-inflammatory property. However, Saf's role in osteoporosis remains unknown. PurposeThis study aims to validate the role of Saf in osteoporosis and explore the potential mechanism. Study DesignThe RANKL-exposed mouse BMM (bone marrow monocytes) and the castration-mediated osteoporosis model were applied to explore the effect and mechanism of Saf in vitro and in vivo. MethodThe effect of Saf on osteoclast formation and function were assessed by TRAcP staining, bone-resorptive experiment, qPCR, immunoblotting and immunofluorescence, etc. Micro-CT, HE, TRAcP and immunohistochemical staining were performed to estimate the effects of Saf administration on OVX-mediated osteoporosis in mice at imaging and histological levels. ResultsSaf concentration-dependently inhibited RANKL-mediated osteoclast differentiation without affecting cellular viability. Meanwhile, Saf-mediated anti-osteolytic capacity and Sirt1 upregulation were also found in ovariectomized mice. Mechanistically, Saf interfered with NF-κB signaling by activating Sirt1 to increase p65 deacetylation and inactivating IKK to decrease IκBα degradation. ConclusionOur results support the potential application of Saf as a therapeutic agent for osteoporosis.

AKT/GSK3β/NFATc1 and ROS signal axes are involved in AZD1390-mediated inhibitory effects on osteoclast and OVX-induced osteoporosis.

As a common disease in modern society, osteoporosis is caused by osteoclast hyperactivation, leading to enhanced bone resorption. Reactive oxygen species (ROS) metobolism and nuclear factor-activated T cells 1 (NFATc1) activities are two crucial processes during osteoclastogenesis. AZD1390 (AZD), an inhibitor of ataxia telangiectasia mutated (ATM), has been reported for antitumor effects, but little is known about how it plays a function in metabolic bone disease. Here, we found that AZD inhibitsthe generation, function and ROS-scavenging enzyme activity of mature osteoclast induced by RANKL stimulation, in a dose-dependent manner.Mechanistic analysis shows thatAZD affects osteoclast function and differentiation by inhibiting RANKL-induced NFATc1 signaling pathway and by increasing ROS-scavenging enzymes production in oxidative stress pathways. Preclinical studies have shown that AZD protects against bone loss in an ovariectomy (OVX) mouse model. Finally, our data confirm that AZD may prevent OVX-induced bone loss by abrogating RANKL-induced AKT/GSK3β/NFATc1 signaling pathways, and by promoting the expression of ROS scavenging enzymes in oxidative stress pathways.Collectively, our research shows that AZD has the potential as a new therapeutic agent for osteoporosis.

Antiosteoporosis Effects, Pharmacokinetics, and Drug Delivery Systems of Icaritin: Advances and Prospects.

Osteoporosis is a systemic skeletal disorder affecting over 200 million people worldwide and contributes dramatically to global healthcare costs. Available anti-osteoporotic drug treatments including hormone replacement therapy, anabolic agents, and bisphosphonates often cause adverse events which limit their long-term use. Therefore, the application of natural products has been proposed as an alternative therapy strategy. Icaritin (ICT) is not only an enzyme-hydrolyzed product of icariin but also an intestinal metabolite of eight major flavonoids of the traditional Chinese medicinal plant Epimedium with extensive pharmacological activities, such as strengthening the kidney and reinforcing the bone. ICT displays several therapeutic effects, including osteoporosis prevention, neuroprotection, antitumor, cardiovascular protection, anti-inflammation, and immune-protective effect. ICT inhibits bone resorption activity of osteoclasts and stimulates osteogenic differentiation and maturation of bone marrow stromal progenitor cells and osteoblasts. As for the mechanisms of effect, ICT regulates relative activities of two transcription factors Runx2 and PPARγ, determines the differentiation of MSCs into osteoblasts, increases mRNA expression of OPG, and inhibits mRNA expression of RANKL. Poor water solubility, high lipophilicity, and unfavorable pharmacokinetic properties of ICT restrict its anti-osteoporotic effects, and novel drug delivery systems are explored to overcome intrinsic limitations of ICT. The paper focuses on osteogenic effects and mechanisms, pharmacokinetics and delivery systems of ICT, and highlights bone-targeting strategies to concentrate ICT on the ideal specific site of bone. ICT is a promising potential novel therapeutic agent for osteoporosis.

Anti-Osteoporotic Effects of n-trans-Hibiscusamide and Its Derivative Alleviate Ovariectomy-Induced Bone Loss in Mice by Regulating RANKL-Induced Signaling.

Osteoporosis is characterized by the deterioration of bone structures and decreased bone mass, leading to an increased risk of fracture. Estrogen deficiency in postmenopausal women and aging are major factors of osteoporosis and are some of the reasons for reduced quality of life. In this study, we investigated the effects of n-trans-hibiscusamide (NHA) and its derivative 4-O-(E)-feruloyl-N-(E)-hibiscusamide (HAD) on receptor activator of nuclear factor kappa-Β (NF-κB) ligand (RANKL)-induced osteoclast differentiation and an ovariectomized osteoporosis mouse model. NHA and HAD significantly inhibited the differentiation of osteoclasts from bone marrow-derived macrophages (BMMs) and the expression of osteoclast differentiation-related genes. At the molecular level, NHA and HAD significantly downregulated the phosphorylation of mitogen-activated protein kinase (MAPK) signaling molecules. However, Akt and NF-κB phosphorylation was inhibited only after NHA or HAD treatment. In the ovariectomy (OVX)-induced osteoporosis model, both NHA and HAD effectively improved trabecular bone structure. C-terminal telopeptide (CTX), a bone resorption marker, and RANKL, an osteoclast stimulation factor, were significantly reduced by NHA and HAD. The tartrate-resistant acid phosphatase (TRAP)-stained area, which indicates the osteoclast area, was also decreased by these compounds. These results show the potential of NHA and HAD as therapeutic agents for osteoporosis.

PTX3 Effects on Osteogenic Differentiation in Osteoporosis: An In Vitro Study.

Pentraxin 3 (PTX3) is a glycoprotein belonging to the humoral arm of innate immunity that participates in the body’s defence mechanisms against infectious diseases. It has recently been defined as a multifunctional protein, given its involvement in numerous physiological and pathological processes, as well as in the pathogenesis of age-related diseases such as osteoporosis. Based on this evidence, the aim of our study was to investigate the possible role of PTX3 in both the osteoblastic differentiation and calcification process: to this end, primary osteoblast cultures from control and osteoporotic patients were incubated with human recombinant PTX3 (hrPTX3) for 72 h. Standard osteinduction treatment, consisting of β-glycerophosphate, dexamethasone and ascorbic acid, was used as control. Our results showed that treatment with hrPTX3, as well as with the osteogenic cocktail, induced cell differentiation towards the osteoblastic lineage. We also observed that the treatment not only promoted an increase in cell proliferation, but also the formation of calcification-like structures, especially in primary cultures from osteoporotic patients. In conclusion, the results reported here suggest the involvement of PTX3 in osteogenic differentiation, highlighting its osteoinductive capacity, like the standard osteoinduction treatment. Therefore, this study opens new and exciting perspectives about the possible role of PTX3 as biomarker and therapeutic agent for osteoporosis.

Fusarium solani G6, a novel vitexin-producing endophytic fungus: characterization, yield improvement and osteoblastic proliferation activity.

The study aimed to characterize a novel vitexin-producing endophytic fungus Fusarium solani G6 from Cajanus cajan, improve its capability for producing vitexin and evaluate its osteoblastic proliferation activity. A total of 153 endophytic fungi, classified into 6 genera, were isolated from C. cajan. Among them, only one strain, endophyte G6 identified as Fusarium solani, was found to producevitexin. After the optimization of fermentation conditions, the highest vitexin yield (18.72mg/L) for the strain was observed in PDB liquid medium containing 20.54g/L of glucose and 8.90g/L of ammonium sulfate, at an initial medium pH of 5.1 and at 28°C for 6days of cultivation. Moreover, the fungal vitexin exhibited notable osteoblastic proliferation stimulating activity. A novel vitexin-producing endophytic fungus F. solani G6 was characterized from C. cajan for the first time. The findings highlighted its potential use for large-scale production of vitexin and might have a promising use as therapeutic agent for osteoporosis.

Vaccine of RANKL mutant conjugated with KLH effectively stabilizing bone metabolism and preventing trabecular microstructural degeneration in osteoporotic rats.

Receptor activator of nuclear factor kappa-B ligand (RANKL) is one of the key factors regulating the maturation of osteoclasts and an important target for osteoporosis treatment. A monoclonal antibody against RANKL showed effective therapeutic activity against osteoporosis by inhibiting bone resorption by osteoclasts. However, being an exogenous protein, its efficacy decreases after long-term use, and its discontinuation increases the risk of vertebral fractures. Here, we aimed to design an active immunotherapeutic agent to induce a T-cell dependent primary response. The agent, a mutant RANKL vaccine (mRv), was produced by cross-linking mutant RANKL, lacking the ability to stimulate osteoclast maturation, with the carrier protein keyhole limpet hemocyanin, a neo-antigen with a large molecular mass. Subcutaneous injection of mRv stimulated rats with ovariectomy-induced osteoporosis to produce high titers of anti-RANKL antibodies. The mutant RANKL vaccine decreased serum CTX-1 and BALP levels and inhibited the microstructural degeneration of trabecular bone in osteoporotic rats. mRv overcame immune system tolerance, stimulated rats to produce therapeutic antibodies, stabilized bone metabolism, and inhibited trabecular microstructural degeneration. These findings confirm the potential of the mutant RANKL vaccine to be developed into an effective preventive and therapeutic agent for osteoporosis.

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL.

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

Zanthoxylum piperitum alleviates the bone loss in osteoporosis via inhibition of RANKL-induced c-fos/NFATc1/NF-κB pathway

BackgroundThe fruit of Zanthoxylum piperitum (ZP) is an herbal medicine as well as a spice agent in Asia to treat carminative, stomachic, anthelmintic and degenerative diseases. Z. piperitum was reported to have anti-oxidant, anti-inflammatory, anti-osteoarthritic and osteosarcoma proliferation-control effects. Purpose and Study designThis study was conducted to determine the anti-osteoporotic effects and mechanisms of action of ZP. MethodsFemale ICR mice underwent ovariectomies (OVX) and were orally administered ZP at 1, 10 and 100 mg/kg for 6 weeks. The femoral and tibial bones were assessed by dual‐energy X‐ray absorptiometry and histology to analyze the bone mineral density (BMD) and the number of osteoclasts. Raw 264.7 cells were stimulated by 100 ng/ml receptor activator of nuclear factor-κB ligand (RANKL) for 7 days in the presence of ZP. RANKL-induced signaling molecules were analyzed in osteoclasts. ResultsThe levels of femoral and tibial BMD were significantly increased by ZP administration. Serum biomarkers such as osteocalcin, calcium, alkaline phosphatase and bone-specific alkaline phosphatase concentrations were markedly recovered to normal levels in ZP-treated osteoporotic mice. In addition, the number of osteoclasts in the head, trochanter and body of the femur was obviously decreased in the ZP treatment groups. Moreover, ZP treated-cells showed a reduction in the number of TRAP-positive multinuclear cells in RANKL-stimulated Raw 264.7 cells. ZP decreased the RANKL-activated NFATc1 and c-fos, transcription factors of osteoclast formation. The nuclear translocation of NF-κB and phosphorylation of ERK42/44 were inhibited by the ZP treatment in RANKL-induced osteoclasts. ConclusionCollectively, ZP exerts its inhibitory effect against bone resorption by regulating RANKL-mediated c-fos/NFATc1/NF-κB in osteoclast. ZP may prove to be a therapeutic agent for osteoporosis.

Discovery and development of berberine derivatives as stimulants of osteoblast differentiation

Berberine is an essential phytochemical for the treatment of various diseases; however, when used to treat osteoporosis, it has minor effect as compared with that of the currently available drugs. This study aimed to find a new compound that would have a better anti-osteoporotic effect than that of berberine. Based on structure and activity relationship study, we identified compound 2c, a berberine derivative, to be the most potent compound to affect osteoblast differentiation. Compound 2c was more effective than berberine and exhibited no toxicity within its effective concentration. Compound 2c increased, in a dose-dependent manner, ALP activity during osteoblast differentiation and enhanced the mRNA expression of osteogenic factors including ALP, Runx2, and Osterix. Furthermore, compound 2c increased the transcriptional activity induced by BMP4 on the ALP and BSP promoter. Taken together, compound 2c shows promise as a therapeutic agent for osteoporosis by promoting osteoblast differentiation.

Anti-osteoporotic effects of Salvia miltiorrhiza Bunge EtOH extract both in ovariectomized and naturally menopausal mouse models

Ethnopharmacological relevanceSalvia miltiorrhiza is a traditional oriental medicine widely used for preventing and treating disorders of the liver, menstrual, and blood circulation systems. Osteoporosis, loss of bone with age and/or estrogen deficiency, is an important causal factor of fracture. S. miltiorrhiza extract has been used to alleviate dysmenorrhea and painful osteoarthritis. Aim of the studyThis study was performed to investigate the anti-osteoporosis activity of the Salvia miltiorrhiza ethanol extract (SME) in osteoporosis-prone conditions: ovariectomized (OVX) and naturally menopaused (NM) ICR mice. Materials and methodsAnti-osteoporotic potentials of SME (50–200 mg/kg) were evaluated based on bone mineral density using microCT analysis, biochemical parameters, and changes in the gene expressions involved in bone resorption. ResultsSME ameliorated the loss of trabecular bone both in OVX and NM mice. SME was effective in correcting aberrant levels of RANKL, osteocalcin, and BALP, which are critically involved in bone resorption. In addition, SME suppressed the expression of TRAF6 and NFATc1, which play a role in osteoclast differentiation. ConclusionsSME suppressed the loss of trabecular bone via suppressing bone resorption and osteoclast differentiation both in OVX and NM mice. SME is likely to be developed as a therapeutic agent for osteoporosis.

Extracellular vesicles from human urine-derived stem cells prevent osteoporosis by transferring CTHRC1 and OPG

Osteoporosis is a debilitating bone disease affecting millions of people. Here, we used human urine-derived stem cells (USCs), which were noninvasively harvested from unlimited and easily available urine, as a “factory” to obtain extracellular vesicles (USC-EVs) and demonstrated that the systemic injection of USC-EVs effectively alleviates bone loss and maintains bone strength in osteoporotic mice by enhancing osteoblastic bone formation and suppressing osteoclastic bone resorption. More importantly, the anti-osteoporotic properties of USC-EVs are not notably disrupted by the age, gender, or health condition (with or without osteoporosis) of the USC donor. Mechanistic studies determined that collagen triple-helix repeat containing 1 (CTHRC1) and osteoprotegerin (OPG) proteins are enriched in USC-EVs and required for USC-EV-induced pro-osteogenic and anti-osteoclastic effects. Our results suggest that autologous USC-EVs represent a promising novel therapeutic agent for osteoporosis by promoting osteogenesis and inhibiting osteoclastogenesis by transferring CTHRC1 and OPG.

Systemic administration of low-dose naltrexone increases bone mass due to blockade of opioid growth factor receptor signaling in mice osteoblasts

AimsOpioid receptor blockers such as naloxone and naltrexone have been suggested to have a bone mass-increasing effect. However, the mechanisms at play have not been clarified. We examined the effects of naltrexone on osteoblasts and determined the expression of opioid growth factor receptor (OGFR) in osteoblasts. Naltrexone blocks the OGFR and other canonical opioid receptors. Thus, we designed experiments to clarify the effects of naltrexone on bone tissue by examining the physiological role of OGFR signaling in osteoblasts and the changes in bone structure after naltrexone systemic administration in mice. Main methodsWe used mouse osteoblast-like cell line MC3T3-E1 for in vitro experiments. We cultured MC3T3-E1 cells in the presence of the OGFR agonist met-enkephalin (met-enk). Then, we measured cell proliferation activity and analyzed the expression levels of cell proliferation-related genes. For our in vivo experiments, we administered naltrexone intraperitoneally to mice daily for 28 days and administered the animals in the control group equivalent volumes of saline. After sacrificing the mice, we performed micro-computed tomography and bone morphology analyses. Key findingsMet-enk suppressed cell proliferation in MC3T3-E1 cells. Moreover, Low dose naltrexone administration significantly increased their femoral bone mass, bone formation ratio, and osteoblast number/bone surface values when comparing the values for the same variables in the control group. SignificanceOur results suggest that naltrexone increases bone mass due to osteoblast number increments caused by the OGFR signaling block. Opioid receptor blockers have potential as therapeutic agents for osteoporosis as well as opioid antagonists.

Osteoporosis as a risk factor for cardiovascular disease.

Osteoporosis(OP)is associated with high risk for cardiovascular disease(CVD). Concomitant occurrence of both OP and CVD seems to result from common pathological mechanisms between bone system and vascular system. It is expected that therapeutic agent for OP may be effective not only in normalization of bone metabolism but also in reducing vascular calcification. I give an outline with the relationship between bone metabolism and vascular calcification including epidemiology, outbreak mechanism, and effect on vascular calcification of OP treatment.

Etidronate attenuates tactile allodynia by spinal ATP release inhibition in mice with partial sciatic nerve ligation.

Etidronate is widely used as a therapeutic agent for osteoporosis. We have recently shown that intrathecal administration of etidronate into mice produces an analgesic effect against the capsaicin-induced nociceptive behavior. However, the effect of etidronate on neuropathic pain at the spinal level remains unknown. Therefore, we examined whether etidronate attenuates pain after partial sciatic nerve ligation (PSNL). We evaluated tactile allodynia 7days after PSNL by measuring paw withdrawal with the von Frey filament test. The mRNA and protein levels of SLC17A9 in the ipsilateral lumbar dorsal spinal cord of PSNL-operated mice were determined using real-time PCR and western blotting, respectively. PSNL-induced tactile allodynia was attenuated by oral and intrathecal administration of etidronate, with maximum efficiency at 90 and 60min after injection, respectively. The anti-allodynic effect of intrathecally administered etidronate was completely inhibited by an intrathecal administration of adenosine triphosphate (ATP). The solute carrier family, SLC17, mediates the transport of pain transmitters, like ATP and glutamate. Indeed, we detected several members of the SLC17 family in the mouse dorsal lumbar spinal cord. Among the detected mRNAs, only Slc17a9, encoding for neuronal vesicular ATP transporter, was significantly increased upon PSNL. SLC17A9 protein levels were also significantly increased. In mice subjected to PSNL, SLC17A9 was present in neurons and microglia, but not in astrocytes of the lumbar superficial dorsal horn. Collectively, our results suggest that etidronate produces its anti-allodynic effects by inhibiting SLC17A9-dependent exocytotic ATP release from the dorsal horn in mice subjected to PSNL.

Antioxidative 1,4-Dihydropyridine Derivatives Modulate Oxidative Stress and Growth of Human Osteoblast-Like Cells In Vitro.

Oxidative stress has been implicated in pathophysiology of different human stress- and age-associated disorders, including osteoporosis for which antioxidants could be considered as therapeutic remedies as was suggested recently. The 1,4-dihydropyridine (DHP) derivatives are known for their pleiotropic activity, with some also acting as antioxidants. To find compounds with potential antioxidative activity, a group of 27 structurally diverse DHPs, as well as one pyridine compound, were studied. A group of 11 DHPs with 10-fold higher antioxidative potential than of uric acid, were further tested in cell model of human osteoblast-like cells. Short-term combined effects of DHPs and 50 µM H2O2 (1-h each), revealed better antioxidative potential of DHPs if administered before a stressor. Indirect 24-h effect of DHPs was evaluated in cells further exposed to mild oxidative stress conditions induced either by H2O2 or tert-butyl hydroperoxide (both 50 µM). Cell growth (viability and proliferation), generation of ROS and intracellular glutathione concentration were evaluated. The promotion of cell growth was highly dependent on the concentrations of DHPs used, type of stressor applied and treatment set-up. Thiocarbatone III-1, E2-134-1 III-4, Carbatone II-1, AV-153 IV-1, and Diethone I could be considered as therapeutic agents for osteoporosis although further research is needed to elucidate their bioactivity mechanisms, in particular in respect to signaling pathways involving 4-hydroxynoneal and related second messengers of free radicals.