Genistein prevents bone resorption diseases by inhibiting bone resorption and stimulating bone formation

Genistein, a soybean-derived isoflavone, has been shown to suppress osteoclastic bone resorption. To clarify the mechanisms underlying this action, we investigated the effects of genistein on the differentiation, cytoskeleton and function in mice osteoclasts in vitro and bone metabolism in ovariectomized rats. Study design: Primary OCs were isolated from 3 week-old mice and induced by 1,25(OH)(2)D(3). Then OCs were exposed to genistein at various concentration of 0 M, 10(-9) M, 10(-8) M, 10(-7) M, 10(-6) M, and 10(-5) M. The number of TRAP+ cells were counted as well as the surface area of bone resorption on bone slice. F-actin change was observed by Confocal. In vivo, forty 12 week-old female SD rats were randomly assigned to four groups: (1) sham operated (Sham); (2) (OVX); (3) ovariectomized and treated with estradiol (OVX-E); (4) ovariectomized and received genistein (OVX-G). After 12 weeks, BMD, body weight, serum level of alkaline phosphatase (ALP), acid phosphatase (ACP), osteocalcin (OC), IL-1beta, TNFalpha, IL-6 and calcitonin (CT) were evaluated. Femur were sectioned. In addition, the serum estradiol, the weight of uteri and histological behavior were also examined to indicate the side effect of genistein to the uteri. Results: In vitro, the number of TRAP+ cells decreased depending on the concentration of genistein as well as the area of bone resorption. F-actin became disorder under Confocal. In vivo, after treated with genistein, BMD and the serum level of ALP, ACP, osteocalcin increased significantly, while the serum level of IL-1beta and TNFalpha decreased. Especially, the increase of ALP and osteocalcin of OVX-G was higher than that of OVX-E. Histologically, the pachy-trabecula were observed as well as the more mineral deposition lines. Additionally, the uterus weight index and the serum estradiol in OVX-G rats were lower significantly than those of OVX-E. The epithelia of uteri gland in OVX-G appeared cubic while those of OVX-E became squamous. Conclusions: Genistein can prevent bone resorption diseases by the promotion of bone formation and the prevention of bone resorption with slight side effect.     

Inhibitory effects of gamma-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones

The effects of mouse recombinant gamma-interferon (gamma-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9-bradykinin and prostaglandin E2 (PGE2) have been studied using cultures of neonatal calvarial bones and analyzing the release of 45Ca from prelabelled bones as a parameter of bone resorption. In addition, the effects of gamma-IFN and indomethacin on formation of PGE2 in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mumol/l) totally abolished bradykinin (1 mumol/l) induced 45Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE2 (1 mumol/l). gamma-IFN (1000 U/ml) almost totally inhibited 45Ca release stimulated by bradykinin (1 mumol/l), but the inhibitory effect could only be partially overcome by PGE2. gamma-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg9-bradykinin (1 mumol/l) on 45Ca release. The stimulatory effects of PGE2 (1 mumol/l) on radioactive calcium mobilization was partially inhibited by gamma-IFN (1000 U/ml), whereas indomethacin (1 mumol/l) was without effect. The inhibitory effect of gamma-IFN on 45Ca release stimulated by bradykinin and PGE2 was dose-dependent with threshold for action at 3-30 U/ml. Comparative dose-response curves showed that gamma-IFN was most potent as inhibitor of bradykinin induced 45Ca release. Bradykinin (1 mumol/l) significantly stimulated PGE2 formation by a mechanism that was completely inhibited by indomethacin (1 mumol/l). gamma-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE2 formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of protein kinase C in bradykinin-induced prostaglandin formation in osteoblasts

Bradykinin (1 μM 5 min) induced translocation of protein kinase C (PKC) to the plasma membrane fraction in osteoblastic MC3T3-E1 cells. Bradykinin also enhanced the binding of phorbol 12,13-dibutyrate (PDBu) to intact cells, a measure of PKC activation. Addition of bradykinin (1 μM) to cells preincubated with [³H]PDBu (10 nM, 20 min) caused an increase in specific PDBu binding that was maximal after 5–10 min. The bradykinin-induced enhancement of PDBu binding was seen at 1 nM and was maximal at 10 nM. The bradykinin B1 receptor agonist des-Arg⁹-bradykinin (1 μM) did not enhance specific PDBu binding to intact MC3T3-E1 cells. PDBu at and above 3 nM stimulated the formation of prostaglandin E2 (PGE₂) in MC3T3-EI cells. This stimulatory effect was seen after 15–20 min incubation. The Ca²⁺ ionophore A23187 at and above 1 μM induced a rapid (within seconds) burst of PGE₂ formation in MC3T3-E1 cells. The effect of PDBu and A23187 on PGE₂ formation was synergistic. The PKC inhibitor staurosporine (200 nM) inhibited basal as well as bradykinin-induced prostaglandin-formation in MC3T3-E1 cells. In conclusion: bradykinin enhances PKC activation in osteoblastic MC3T3-E1 cells. This kinase activation may be involved in bradykinin-induced prostaglandin formation.     

GDF8 inhibits bone formation and promotes bone resorption in mice

Growth Differentiation Factor 8 (GDF8), also called myostatin, is a member of the transforming growth factor (TGF)‐β super‐family. As a negative regulator of skeletal muscle growth, GDF8 is also associated with bone metabolism. However, the function of GDF8 in bone metabolism is not fully understood. Our study aimed to investigate the role of GDF8 in bone metabolism, both in vitro and in vivo. Our results showed that GDF8 had a negative regulatory effect on primary mouse osteoblasts, and promoted receptor activator of nuclear factor κB ligand (RANKL)‐induced osteoclastogenesis in vitro. Intraperitoneal injection of recombinant GDF8 repressed bone formation and accelerated bone resorption in mice. Furthermore, treatment of aged mice with a GDF8 neutralizing antibody stimulated new bone formation and prevented bone resorption. Thus, our study showed that GDF8 plays a significant regulatory role in bone formation and bone resorption, thus providing a potential therapeutic pathway for osteoporosis.     

Cistanche deserticola extract increases bone formation in osteoblasts

Objectives We investigated the effect of Cistanche deserticola Ma. (CD) on bone formation by cultured osteoblasts. Methods The mineralized nodule formation assay was used to examine the in‐vitro effects of CD on bone formation. Alkaline phosphatase (ALP), bone morphogenetic proteins (BMP)‐2 and osteopontin (OPN) mRNA expression was analysed by quantitative real‐time polymerase chain reaction. The mechanism of action of CD extract was investigated using Western blotting. The in‐vivo anti‐osteoporotic effect of CD extract was assessed in ovariectomized mice. Key findings CD extract had no effect on the proliferation, migration or wound healing of cultured osteoblasts, but increased ALP, BMP‐2 and OPN mRNA and bone mineralization. Mitogen‐activated protein kinase (MAPK) or nuclear factor (NF)‐κB inhibitors reduced CD extract‐induced bone formation and ALP, BMP‐2 and OPN expression. However, CD extract did not affect osteoclastogenesis. In addition, CD extract prevented the bone loss induced by ovariectomy in vivo. Conclusions CD may be a novel bone formation agent for the treatment of osteoporosis.     

Effects of methylprednisolone on bone formation and resorption in rats

Excessive glucocorticoids induce osteoporosis. However, there is some controversy regarding the mechanism of action, and even the endpoint result. The present study was carried out to obtain further insight into the action of glucocorticoids on bone formation and resorption in rats. Growing rats were injected subcutaneously with methylprednisolone (mPSL) at doses of 0, 2.5, 5, 10 or 20 mg/kg per day for 4 weeks. Bone mineral density (BMD), enchondral and periosteal bone formation, collagen synthetic activities of osteoblasts, numbers of osteoblasts and osteoclasts, and serum markers to assess bone turnover were determined. Administration of mPSL dose-dependently increased the BMD in the tibial metaphysis, while it dose-dependently decreased the BMD in the diaphysis. Both enchondral and periosteal bone formation were decreased in a dose-dependent fashion. The incorporation and secretion of (3)H-proline by osteoblasts were both decreased in trabecular and cortical bones. The number of osteoclasts, together with the number of osteoblasts, in the tibial metaphysis was drastically decreased. Serum alkaline phosphatase and osteocalcin were decreased at higher doses. These results support the recent notion that glucocorticoids inhibit both bone formation and resorption. In addition, BMD as an endpoint result might differ from site to site in bone due to a different balance between bone formation and resorption.     

Effect of strontium chloride on bone resorption induced by prostaglandin E2 in cultured bone

We examined whether the bone resorption induced by PGE₂ was inhibited by SrCl₂ using⁴⁵Ca-labelled calvaria of CD-strain mice in tissue culture. It was found that Sr salts inhibited physiological bone resorption in a dose-dependent manner (0.1–5.0 mM) and did not act via PGE₂. Accordingly, it was suggested that Sr salts did not inhibit bone resorption induced by exogenous PGE₂.Abbreviations PGE₂ Prostaglandin E₂ - PTH Parathyroid hormone - CT Calcitonin - Vit. D₃ Vitamin D₃ - HSDM₁ Harvard School of Dental Medicine 1     

Regulation of bone resorption and formation by purines and pyrimidines

Growing evidence suggests that extracellular nucleotides, signalling through P2 receptors, might play important roles in the regulation of bone and cartilage metabolism. ATP and other nucleotides can exert impressive stimulatory effects on the formation and activity of osteoclasts (bone-resorbing cells) in addition to inhibiting bone formation by osteoblasts. In this review, the current understanding of the actions of nucleotides on skeletal cells and the probable receptor subtypes involved are discussed.     

Stimulative effects of cadmium on bone resorption in neonatal parietal bone resorption.

Effects of cadmium on bone resorption were investigated using neonatal mouse parietal bone culture system. Cadmium at 0.5 microM and above stimulated hydroxyproline release as well as 45Ca release. As cadmium-stimulated bone resorption was inhibited by calcitonin, bone resorption induced by cadmium is osteoclast-mediated bone resorption. CI-1, collagenase inhibitor, depressed cadmium-stimulated bone resorption in a dose-dependent manner. Osteoblasts are also involved in cadmium-induced bone resorption. Indomethacin-inhibited cadmium-stimulated bone resorption and cadmium-treated bones released prostaglandin E2 to a greater extent than untreated bones. Cadmium-stimulated bone resorption was shown to be dependent on the production of prostaglandin E2. 3-Isobutyl-1-methylxanthine potentiated cadmium-stimulated bone resorption and verapamil depressed it. It is possible that an increase in levels of cAMP and calcium ion in bone cells is involved in cadmium-induced bone resorption. From these results, cadmium was found to stimulate osteoclast-mediated bone resorption which is dependent on prostaglandin E2. Second messengers in cadmium-induced bone resorption may be cAMP and calcium ion.     

Role of carbonic anhydrase in bone resorption induced by prostaglandin E2 in vitro

The possible role of carbonic anhydrase in bone resorption induced by prostaglandin E2 (PGE2) was studied using an in vitro neonatal mouse calvarial culture system. PGE2 (10(-6) M) was effective in stimulating resorption, as assessed by calcium release into culture media. This enhanced resorption was accompanied by significant increases in calvarial carbonic anhydrase activity over control values at 48 and 96 h. At 48 h, bones treated with PGE2 had 20% more carbonic anhydrase activity than controls. By 96 h, treated bones contained 79% more carbonic anhydrase activity than controls. PGE2-induced bone resorption was inhibited by the carbonic anhydrase inhibitor acetazolamide in a dose-dependent fashion from 10(-5) to 10(-4) M, with 77% inhibition observed at 10(-4) M. The acetazolamide analogue CL 13,850 (N-t-butylacetazolamide), which does not inhibit carbonic anhydrase, failed to inhibit PGE2-induced resorption. These results are consistent with the hypothesis that carbonic anhydrase is a necessary component of the osteoclastic bone resorptive mechanism.     

Flufenamic acid inhibits osteoclast formation and bone resorption and act against estrogen-dependent bone loss in mice

Postmenopausal osteoporosis is one of the most common types of osteoporosis resulting from estrogen deficiency in elderly women. Nonsteroidal anti-inflammatory drugs (NSAIDs) are important drugs for pain relief in patients with osteoporosis. In this study, we report for the first time that flufenamic acid, a clinically approved and widely used NSAID, not only has analgesic properties but also shows a significant effect in terms of preventing postmenopausal osteoporosis. Quantitative RT-PCR analysis showed that treatment with flufenamic acid significantly downregulated the genes associated with osteoclast differentiation. Meanwhile, RNA-sequencing and western blot analyses suggested that flufenamic acid could inhibit the bone resorption by suppressing the phosphorylation of MAPK pathways. Moreover, an ovariectomy (OVX)-induced bone-loss mouse model indicated that flufenamic acid might be a potent drug for preventing osteoporotic fractures, as verified by micro-CT scanning and histological analysis. Therefore, this study proposes an attractive and potent drug with analgesic properties for the prevention of postmenopausal osteoporosis.     

Effects of retinoic acid on bone formation and resorption in cultured mouse calvaria

1. The effects of retinoids on bone metabolism were examined in newborn mouse calvaria. 2. Incubation of calvaria with 0.01-1 microM retinoic acid for 4 days decreased their alkaline phosphatase (ALP) activity, mineral content and collagen content in a concentration-dependent fashion. 3. With treatment for 2 days, retinoic acid (1 microM) decreased the ALP activity and collagen content, but not the mineral content. 4. All these inhibitory effects were observed in calvaria from 0-day-old mice, but no inhibition of ALP activity was observed in calvaria from 14-day-old mice. 5. 1-Hydroxyethylidene-1,1-bisphosphonate (HEBP, 1 mM), which inhibits bone resorption, prevented the effect of retinoic acid (1 microM) on the bone mineral content, but not the effects on ALP and collagen (synthesized by osteoblasts). HEBP (1 mM) alone had no effect on the calvarial mineral and collagen contents. 6. These findings indicate that retinoic acid both stimulates bone resorption and inhibits osteoblastic activity by different mechanisms, and that stimulation of bone resorption by retinoic acid is inhibited by HEBP.     

Uncoupling between bone formation and resorption in ovariectomized rats with chronic cadmium exposure

Osteoporosis, osteomalacia, and pathological fractures are characteristic features of Itai-Itai disease. The mechanisms of bone damage caused by cadmium (Cd) exposure have not been fully clarified. We investigated skeletal changes in ovariectomized rats with chronic Cd exposure, using bone histomorphometry and mechanical tests. Female Sprague-Dawley rats at the age of 8 weeks were ovariectomized. Eight weeks after ovariectomy, the rats were divided into two groups: Cd-OVX group (n = 15), ovariectomized rats given cadmium chloride (CdCl(2), 0.18 mg/rat) ip three times a week for 28 weeks; Cont-OVX group (n = 10), ovariectomized rats given distilled water alone for 28 weeks. Cd-OVX rats had a significant increase in serum concentration of intact osteocalcine and showed numerical but not significant increase in urinary excretion of deoxypyridinoline despite a significant decrease in glomerular filtration rate to 40% of the value in Cont-OVX rats. Bone mineral content (BMC) and density were significantly decreased in both the lumbar vertebral body and femur of Cd-OVX rats. Ultimate compressive load in the lumbar body and bending load in the midfemur were significantly lower in Cd-OVX rats than in Cont-OVX rats but the differences were not demonstrated when the values were corrected for BMC. Structural moduli in the lumbar vertebral body and the midfemur were not different between the two groups. Cd-OVX rats showed significant decreases in the trabecular bone volume and trabecular number with increased values in the indices of bone formation and resorption in the lumbar vertebral body cancellous bone in comparison with Cont-OVX rats. In the midfemur, Cd-OVX rats had significantly smaller cortical bone area than Cont-OVX rats but the moment of inertia was identical between the two groups. The indices of bone formation and resorption at endocortical surface of the midfemur were significantly increased in Cd-OVX rats over those in Cont-OVX rats, whereas the indices of bone formation at the periosteal surface were not different between the two groups. These data suggested that chronic Cd exposure exacerbated the uncoupling between bone formation and resorption in ovariectomized rats, which resulted in the osteopenia, structural changes of the bone, and decreased mechanical strength in ovariectomized rats with chronic Cd exposure.     

Tanshinone IIA suppresses inflammatory bone loss by inhibiting the synthesis of prostaglandin E2 in osteoblasts

Tanshinone IIA isolated from Danshen is widely used in Oriental medicine. However, the action of tanshinone IIA in inflammatory bone-resorptive diseases remains unknown. Here we examined the effect of tanshinone IIA in inflammation-mediated osteoclastic bone resorption. Tanshinone IIA inhibited osteoclast differentiation in cocultures of bone marrow cells and calvarial osteoblasts. Tanshinone IIA regulated the expression of receptor activator of NF-kappaB ligand and osteoprotegerin in osteoblasts treated with lipopolysaccharide (LPS). Also, tanshinone IIA inhibited prostaglandin E(2) (PGE(2)) synthesis by inhibiting Cyclooxygenase-2 (COX-2) expression induced by LPS. Furthermore, tanshinone IIA greatly suppressed bone loss in the mouse models of bone loss. Our findings suggest that tanshinone IIA inhibits osteoclast formation by inhibiting COX-2/PGE(2) signaling and by suppressing bone erosion in vivo. These results suggest that tanshinone IIA may be of therapeutic value as an anti-bone-resorptive drug in the treatment of bone-related disease.     

Evidence for pteridine regulation of lead-mediated inhibition of uroporphyrinogen and heme formation in rat bone marrow.

Uroporphyrin I (URO I) accumulation has been reported in the bone marrow of rats exposed to lead, suggesting a sensitivity of uroporphyrinogen III cosynthase (COSYN) to this heavy metal. Furthermore, it has been reported that a polyglutamated folate derivative may serve as a coenzyme for the catalytic action of hepatic uroporphyrinogen III cosynthase. These findings raised the question of whether depletion of polyglutamated folate could enhance the susceptibility of bone marrow COSYN to lead and potentially interfere with the formation of heme. Nitrous oxide, an anesthetic agent capable of causing bone marrow tetrahydrofolate deficiency, depressed total bone marrow polyglutamated folate content by 42% with significant reductions in all three chain lengths (5-7) identified in the bone marrow during an exposure period of 7 days at 4 hr/day. Lead acetate (15 mg/kg) administered by ip injection at Days 0 and 2 during a 7-day exposure to nitrous oxide resulted in an 84% increase of bone marrow URO I content, which was markedly higher than the increases of 22 and 38% seen with sole administration of lead or nitrous oxide, respectively. The combination of agents also produced a 48% rise in COPRO I, a 39 and 43% decrease in COPRO III and protoporphyrin, respectively, and a 42% decline in the activity of microsomal 7-ethoxycoumarin O-deethylase, which is hemoprotein, cytochrome P-450 mediated. Heme oxygenase activity was not altered by nitrous oxide, lead, or their combination. These results suggest that bone marrow folate deficiency may render COSYN more sensitive to lead as characterized by increased uroporphyrin I and coproporphyrin I isomer content, decreased coproporphyrin III and protoporphyrin content, and depressed microsomal hemoprotein, cytochrome P-450-mediated drug-metabolizing capability.     

Triterpenoids from Celastrus orbiculatus Thunb. inhibit RANKL-induced osteoclast formation and bone resorption via c-Fos signaling

Journal of Natural Medicines - Fourteen triterpenes, lup-20(29)-ene-3β,6β-diol (1), betulin (2), lupeol caffeate (3), 3β-caffeoyloxylup-20(29)-en-6α-ol (4),...     

BK1 and BK2 bradykinin receptors in the rat duodenum smooth muscle.

1. The dual action of bradykinin (relaxation and contraction) on the rat duodenum was investigated by studying its effect on adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultured duodenal smooth muscle cells, and the effects of apamin on the isolated muscle responses to agonists and antagonists of BK1 and BK2 receptors. 2. No change was observed in the cyclic AMP content of cultured cells incubated with up to 100 nM bradykinin. 3. Apamin (100-500 nM) inhibited the relaxant component and enhanced the contractile component of the responses to bradykinin and to the BK2-specific analogue [Thi5,8,D-Phe7]-bradykinin. 4. Apamin (100-500 nM) did not affect the contractile response of stretched duodenum preparation to the BK1-specific agonist des-Arg9-bradykinin. 5. The BK2 antagonist, [D-Arg0Hyp3Thi5,8,D-Phe7]-bradykinin, at a concentration which completely inhibited the relaxant response to bradykinin and to [Thi5,8,D-Phe7]-bradykinin, also prevented the contraction in response to either agonist in the presence of apamin. 6. Our results demonstrate two populations of bradykinin receptors in rat duodenum: a BK2 subtype responsible for the biphasic response of the non-stretched duodenum, and a BK1 subtype responsible for the contractile effect on the stretched tissue.     

Zinc-induced hypocalcemia and bone resorption in rats

The changes of calcium levels in serum and in the femur were examined in rats administered oral doses of zinc sulfate (0.1, 1.0, and 10 mg Zn/100 g body weight) for 3 days. All doses of zinc caused significant decreases in calcium levels in serum and in the femoral diaphysis and epiphysis. The decrease in these femoral calcium levels was seen 1 day after administration of zinc (10 mg/100 g). Furthermore, time course studies of the effect of zinc administration showed that, at 1 hr after zinc administration, calcium levels in serum and in femoral epiphysis but not in diaphysis were significantly decreased. In thyroparathyroidectomized rats, however, no significant decrease of the epiphyseal calcium was observed by administration of zinc (10 mg Zn/100 g), but the serum calcium level was significantly lowered. Zinc administration to intact rats caused a significant increase in acid phosphatase activity in the femoral epiphysis but not in the diaphysis. This increase was clearly prevented by thyroparathyroidectomy. Accumulations of zinc in the femoral epiphysis and diaphysis after zinc administration was not significantly altered by thyroparathyroidectomy. These results suggest that zinc-induced hypocalcemia may cause bone resorption which is primarily mediated by the action of the parathyroid hormone and it is related to calcium homeostasis in rats.