RESUMEN
A series of new 6H-benzofuro[3, 2-c]chromenes (BFC, pterocarpans) with structure-activity relationships were investigated for their potential use in osteoporosis treatment. One of the BFCs 3-piperidylethoxypterocarpan 20 promotes osteoblast differentiation and mineralization at a dose as low as 1 pM via activation of ER/P38MAPK/BMP-2 pathway. When evaluated for in-vivo osteogenic activity in female Sprague-Dawley rats, BFC 20 increased bone mineral density and new bone formation, compared with control at 1.0 and 10.0 mg/kg/body weight by oral gavage for 30 days. The compound was devoid of any uterotrophic effect and led to the new bone formation in adult ovariectomized osteopenic rats. BFC 20 compound also inhibited bone resorption by reducing Ovx induced increase in urinary CTx, thus exhibiting both bone anabolic and anti-catabolic action. Finally, BFC 20 treatment to Ovx rats led to improved trabecular microarchitectural restoration and exhibited therapeutic potential as a dual acting anti-osteoporotic agent for the management of osteoporosis.
Asunto(s)
Anabolizantes/uso terapéutico , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Hueso Esponjoso/patología , Ovariectomía , Piperidinas/uso terapéutico , Pterocarpanos/uso terapéutico , Fosfatasa Alcalina/metabolismo , Anabolizantes/síntesis química , Anabolizantes/química , Anabolizantes/farmacología , Animales , Biomarcadores/metabolismo , Densidad Ósea/efectos de los fármacos , Enfermedades Óseas Metabólicas/patología , Proteína Morfogenética Ósea 2/metabolismo , Remodelación Ósea/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Hueso Esponjoso/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Femenino , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Fosforilación/efectos de los fármacos , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/farmacología , Pterocarpanos/síntesis química , Pterocarpanos/química , Pterocarpanos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Estrógenos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
IL-18BP is a natural antagonist of pro-inflammatory IL-18 cytokine linked to autoimmune disorders like rheumatoid arthritis. However, its role in post menopausal osteoporosis is still unknown. In this study, we investigated the role of IL-18BP on murine osteoblasts, its effect on osteoblasts-CD4+ T cells and osteoblasts-CD11b+ macrophage co-culture. mIL-18BPd enhances osteoblast differentiation and inhibits the activation of NLRP3 inflammasome and caspase-1 which process IL-18 to its active form. Using estrogen deficient mice, we also determined the effect of mIL-18BP on various immune and skeletal parameters. Ovariectomized mice treated with mIL-18BPd exhibited decrease in Th17/Treg ratio and pro-inflammatory cytokines. mIL-18BPd treatment restored trabecular microarchitecture, preserved cortical bone parameters likely attributed to an increased number of bone lining cells and reduced osteoclastogenesis. Importantly, these results were corroborated in female osteoporotic subjects where decreased serum IL-18BP levels and enhanced serum IL-18 levels were observed. Our study forms a strong basis for using humanized IL-18BP towards the treatment of postmenopausal osteoporosis.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Osteoporosis Posmenopáusica/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteína Morfogenética Ósea 2/genética , Diferenciación Celular/efectos de los fármacos , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamasomas/metabolismo , Mediadores de Inflamación , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Interleucina-18/metabolismo , Leucocitos Mononucleares , Linfopoyesis/genética , Linfopoyesis/inmunología , Ratones , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoporosis Posmenopáusica/genética , Osteoporosis Posmenopáusica/inmunología , Osteoporosis Posmenopáusica/patología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células Th17/citología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th17/metabolismo , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: Recent studies have shown that immune system plays a major role in pathophysiology of postmenopausal osteoporosis. Previously we have shown that phytoestrogens like daidzein and medicarpin exhibit immunoprotective effects, by virtue of which they alleviate bone loss. With this background, methoxyisoflavones like formononetin (formo) and isoformononetin (isoformo) that have been studied for preventing bone loss in ovariectomized rats were tested for their immunomodulatory effects in estrogen-deficient bone loss mice model. METHODS: Adult Balb/c mice (Nâ=â8/group) were given oral dose of formo and isoformo at 10 mg/kg body weight, post ovariectomy (Ovx) daily for 6 weeks. Animals were autopsied and long bones were harvested to study bone microarchitecture. Peripheral blood mononuclear cells were isolated for fluorescence-activated cell sorting and RNA analysis. Serum was collected for enzyme-linked immunosorbent assay. RESULTS: It was observed that formo and isoformo treatment to Ovx mice led to significant restoration of Ovx-induced deterioration of trabecular microarchitecture. Pro-osteoclastogenic subset Th17 and B cells were decreased in formo/isoformo-treated Ovx mice in comparison with vehicle-treated Ovx group. Formo and isoformo treatment to Ovx mice also led to decreased expression of Th17 diffentiation factors and promoted T-regulatory cell differentiation. Formo was more effective in enhancing the FOXP3 expression compared with isoformo. IL-17A-induced osteoclastogenesis and inhibition of osteoblast apoptosis were also suppressed by formo and isoformo treatment, with formo having a more potent effect. CONCLUSIONS: Our study demonstrates the immunomodulatory activity of methoxyisoflavones, formo, and isoformo, which translate into improved skeletal parameters, thereby preventing Ovx-induced bone loss.
Asunto(s)
Linfocitos B/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Isoflavonas/farmacología , Linfopoyesis/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Fitoestrógenos/farmacología , Células Th17/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Linfocitos B/citología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Menopausia/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Osteoblastos/efectos de los fármacos , Osteoporosis Posmenopáusica/prevención & control , Ovariectomía , Células Th17/citologíaRESUMEN
Activated T cell has a key role in the interaction between bone and immune system. T cells produce proinflammatory cytokines, including receptor activator of NF-κB ligand (RANKL), tumor necrosis factor α (TNF-α), and interleukin 17 (IL-17), all of which augment osteoclastogenesis. RANKL and TNF-α are targeted by inhibitors such as denosumab, a human monoclonal RANKL antibody, and infliximab, which neutralizes TNF-α. IL-17 is also an important mediator of bone loss, and an antibody against IL-17 is undergoing phase II clinical trial for rheumatoid arthritis. Although there are a few studies showing suppression of Th17 cell differentiation and induction of regulatory T cells (Tregs) by infliximab, the effect of denosumab remains poorly understood. In this study, we investigated the effects of anti-TNF-α, anti-RANKL, or anti-IL-17 antibody administration to estrogen-deficient mice on CD4(+) T-cell proliferation, CD28 loss, Th17/Treg balance and B lymphopoesis, and finally, the translation of these immunomodulatory effects on skeletal parameters. Adult Balb/c mice were treated with anti-RANKL/-TNF-α/-IL-17 subcutaneously, twice a week, postovariectomy (Ovx) for 4 weeks. Animals were then autopsied; bone marrow cells were collected for FACS and RNA analysis and serum collected for ELISA. Bones were dissected for static and dynamic histomorphometry studies. We observed that although anti-RANKL and anti-TNF-α therapies had no effect on Ovx-induced CD4(+) T-cell proliferation and B lymphopoesis, anti-IL-17 effectively suppressed both events with concomitant reversal of CD28 loss. Anti-IL-17 antibody reduced proinflammatory cytokine production and induced Tregs. All three antibodies restored trabecular microarchitecture with comparable efficacy; however, cortical bone parameters, bone biomechanical properties, and histomorphometry were best preserved by anti-IL-17 antibody, likely attributable to its inhibitory effect on osteoblast apoptosis and increased number of bone lining cells and Wnt10b expression. Based on the superior immunoprotective effects of anti-IL-17, which appears to translate to a better skeletal preservation, we propose beginning clinical trials using a humanized antibody against IL-17 for treatment of postmenopausal osteoporosis.
Asunto(s)
Anticuerpos/farmacología , Huesos/inmunología , Estrógenos/deficiencia , Interleucina-17/inmunología , Ligando RANK/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Antígenos CD/metabolismo , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/fisiología , Proliferación Celular/efectos de los fármacos , Citocinas/biosíntesis , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Linfopoyesis/efectos de los fármacos , Ratones Endogámicos BALB C , Osteoclastos/efectos de los fármacos , Ovariectomía , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
With a longitudinally designed study, we tested whether an acetone soluble fraction (ASF) from the stem bark of Butea monosperma resulted in maximizing bone gain in rats during growth and maturation and thus protected against osteopenia following ovariectomy (OVx) with concomitant treatment withdrawal. Female rats at weaning were given ASF (100 mg/kg/d) or vehicle for 12 weeks, and baseline skeletal parameters (micro-CT) and total plasma antioxidant status (TAS) were measured. At this stage, one group was OVx and the other group was sham operated. Vehicle group (untreated) after OVx was given E2 or continued with vehicle (OVx control). ASF group after OVx was given vehicle (ASF withdrawn, ASFW). After another 12 weeks, all groups were killed and various skeletal parameters were determined. ASF resulted in substantially better skeletal parameters and higher plasma TAS over control at maturity. Rats treated with ASF before OVx had reduced rates of bone loss compared to OVx control. Twelve weeks after OVx, the ASFW group exhibited better trabecular microarchitectural preservation, bone turnover profiles, increased cortical deposition, and biomechanical strength over the OVx control, and the effects were comparable to OVx + E2 group. ASF supplementation during skeletal growth could maximize bone accrual and could confer increased resistance to post-OVx osteopenia despite treatment withdrawal.
RESUMEN
Th17 cells produce IL-17, and the latter promotes bone loss in collagen-induced arthritis in mice. Blocking IL-17 action in mouse model of rheumatoid arthritis reduces disease symptoms. These observations suggest that Th17 cells may be involved in the pathogenesis of bone loss. However, the role of Th17 cell in estrogen (E2) deficiency-induced bone loss is still not very clear. We investigated the effect of E2 on Th17 differentiation in vivo and IL-17 mediated regulation of osteoclast and osteoblast differentiation. Additionally, effect of IL-17 functional block under E2 deficiency-induced bone loss was studied. In murine bone marrow cells, E2 suppressed IL-17 mediated osteoclast differentiation. IL-17 inhibited formation of mineralized nodules in osteoblasts and this effect was suppressed by E2. E2 treatment to mouse calvarial osteoblasts inhibited the IL-17-induced production of osteoclastogenic cytokines and NF-kB translocation. In ovariectomized mice, there was increase in the number of Th17 cells, transcription factors promoting Th17 cell differentiation and circulating IL-17 levels. These effects were reversed by E2 supplementation. Treatment of neutralizing IL-17 monoclonal antibody to Ovx mice mitigated the E2 deficiency-induced trabecular bone loss and reversed the decreased osteoprotegerin-to-receptor activator of nuclear factor kappa B ligand (RANKL) transcript levels in long bones, increased osteoclast differentiation from the bone marrow precursor cells and decreased osteoblast differentiation from the bone marrow stromal cells. Our findings indicate that E2 deficiency leads to increased differentiation of Th17 cells with attendant up regulation of STAT3, ROR-γt and ROR-α and downregulation of Foxp3 which antagonizes Th17 cell differentiation. Increased IL-17 production in turn induces bone loss by increasing pro-osteoclastogenic cytokines including TNF-α, IL-6 and RANKL from osteoblasts and functional block of IL-17 prevents bone loss. IL-17 thus plays a critical causal role in Ovx-induced bone loss and may be considered a potential therapeutic target in pathogenesis of post menopausal osteoporosis.
Asunto(s)
Diferenciación Celular , Estradiol/deficiencia , Interleucina-17/metabolismo , Osteoporosis/etiología , Linfocitos T Colaboradores-Inductores/citología , Animales , Línea Celular , Ratones , FN-kappa B/metabolismo , Osteoblastos/citología , Osteoclastos/citología , Osteoprotegerina/genética , Ovariectomía , Ligando RANK/genética , ARN Mensajero/genética , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway. On the other hand, formononetin maximally stimulated osteoblast differentiation at 100 nM that involved p38 MAPK pathway but had no effect on osteoblast proliferation. Unlike daidzein, these two compounds neither activated estrogen receptor in osteoblast nor had any effect on osteoclast differentiation. Daily oral administration of each of these compounds at 10.0 mg kg(-1) day(-1) dose to recently weaned female Sprague-Dawley rats for 30 consecutive days, increased bone mineral density at various anatomic positions studied. By dynamic histomorphometry of bone, we observed that rats treated with cladrin exhibited increased mineral apposition and bone formation rates compared with control, while formononetin had no effect. Cladrin had much better plasma bioavailability compared with formononetin. None of these compounds exhibited estrogen agonistic effect in uteri. Our data suggest that cladrin is more potent among the two in promoting parameters of peak bone mass achievement, which could be attributed to its stimulatory effect on osteoblast proliferation and better bioavailability. To the best of our knowledge, this is the first attempt to elucidate structure-activity relationship between the methoxylated forms of daidzein and their osteogenic effects.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Isoflavonas/farmacología , Osteoblastos/fisiología , Animales , Disponibilidad Biológica , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Isoflavonas/sangre , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Medicarpin, a pterocarpan class of naturally occurring benzopyran furanobenzene compound was synthesized in gram scale to investigate its effects on murine bone cells and in ovariectomized (OVx) mice. Medicarpin, at as low as 10(-10)M suppressed osteoclastogenesis in bone marrow cells (BMCs). Medicarpin-induced apoptosis of mature osteoclasts isolated from long bones. Effects of medicarpin in osteoclasts appear to be independent of estrogen receptor (ER) activation as ICI 180,782 failed to abrogate its effects on osteoclasts. In calvarial osteoblasts, medicarpin (10(-10)M) blocked nuclear factor kappaB (NF-kappaB) signaling assessed by tumor necrosis factor alpha (TNFalpha)-stimulated nuclear translocation of p65 subunit of NF-kappaB. Medicarpin also inhibited the expression of TNFalpha in mouse calvarial osteoblasts. This effect was ER dependent as ICI 180,782 reversed the suppressive effect of medicarpin on TNFalpha mRNA levels in osteoblasts. In addition, like 17beta-estradiol, presence of medicarpin inhibited TNFalpha-induced upregulation of interleukin-1, and -6 mRNA levels in osteoblasts. In co-cultures consisting of calvarial osteoblasts and BMCs, presence of medicarpin increased osteoprotegerin (OPG)/receptor activator of NF-kappaB ligand (RANKL) ratio and reduced mRNA levels of osteoclast markers including tartrate-resistant acid phosphatase and RANK. OVx mice administered medicarpin (10.0mgkg(-1)day(-1)) orally for 30days had reduced formation of osteoclasts but increased formation of osteoprogenitor cells in BMCs compared with OVx+vehicle group. Medicarpin treatment to OVx mice maintained parameters of trabecular microarchitecure. Medicarpin exhibited no uterine estrogenicity. Our findings point towards direct and indirect inhibitory effects of medicarpin on osteoclastogenesis in vitro that contribute to its bone sparing effect in OVx mice.