iNOS regulates hematopoietic stem and progenitor cells via mitochondrial signaling and is critical for bone marrow regeneration.

Hematopoietic stem cells (HSCs) replenish blood cells under steady state and on demand, that exhibit therapeutic potential for Bone marrow failures and leukemia. Redox signaling plays key role in immune cells and hematopoiesis. However, the role of reactive nitrogen species in hematopoiesis remains unclear and requires further investigation. We investigated the significance of inducible nitric oxide synthase/nitric oxide (iNOS/NO) signaling in hematopoietic stem and progenitor cells (HSPCs) and hematopoiesis under steady-state and stress conditions. HSCs contain low levels of NO and iNOS under normal conditions, but these increase upon bone marrow stress. iNOS-deficient mice showed subtle changes in peripheral blood cells but significant alterations in HSPCs, including increased HSCs and multipotent progenitors. Surprisingly, iNOS-deficient mice displayed heightened susceptibility and delayed recovery of blood progeny following 5-Fluorouracil (5-FU) induced hematopoietic stress. Loss of quiescence and increased mitochondrial stress, indicated by elevated MitoSOX and MMPhi HSCs, were observed in iNOS-deficient mice. Furthermore, pharmacological approaches to mitigate mitochondrial stress rescued 5-FU-induced HSC death. Conversely, iNOS-NO signaling was required for demand-driven mitochondrial activity and proliferation during hematopoietic recovery, as iNOS-deficient mice and NO signaling inhibitors exhibit reduced mitochondrial activity. In conclusion, our study challenges the conventional view of iNOS-derived NO as a cytotoxic molecule and highlights its intriguing role in HSPCs. Together, our findings provide insights into the crucial role of the iNOS-NO-mitochondrial axis in regulating HSPCs and hematopoiesis.

9-Demethoxy-medicarpin: A potential bone health supplement for the management of protein deficiency-induced bone loss in growing rats.

Human skeleton requires an adequate supply of many different nutritional factors for optimal growth and development. The role of nutrition in bone growth has piqued interest in recent years, especially in relation to maximizing peak bone mass and reducing the risk of osteoporosis. Protein deficiency-induced bone loss was induced in female growing rats. All experimental rodent diets were prepared as per recommendations for growing animals. 9-Demethoxy-medicarpin (DMM) treatment was given to growing Sprague Dawley (SD) rats at 1 mg and 10 mg dose orally for 30 days. Bones were collected for bone mineral density (BMD). Bone marrow cells were isolated from femur for calcium nodule formation. Serum samples were collected for biochemical parameters. We found that DMM treatment speeds up the recovery of musculoskeletal weakness by replenishing nutrients in proven rodent model. DMM supplementation for four weeks showed significantly increased vertebral, femur and tibial BMD compared with the untreated PD group. Albumin levels were significantly enhanced in treatment groups, in which 10 mg dose imparted a better effect. We conclude that DMM treatment led to increased BMD and biochemical parameters in protein deficient condition in growing rats and has potential as a bone growth supplement.

Cladrin alleviates dexamethasone-induced apoptosis of osteoblasts and promotes bone formation through autophagy induction via AMPK/mTOR signaling.

Glucocorticoid-induced osteoporosis (GIOP) is a common clinical consequence that arises due to the extensive usage of glucocorticoids. Cladrin (Clad), a methoxylated isoflavone has been reported to have a bone protecting effect by enhancing osteoblast proliferation and differentiation. However, its consequences on GIOP are not reported yet. This study investigates whether Clad protects against the deleterious effects of Dexamethasone (Dex) on osteoblast and bone. Mice calvarial osteoblasts were treated with Clad and then exposed to Dex to study the effect on osteoblast differentiation, proliferation, and survival. Further, GIOP mice were treated with Clad (5 and 10 mg/kg) doses along with reference standard alendronate (ALN 3 mg/kg) for evaluation of bone protecting effect of Clad. We analyzed bone and vertebral microarchitecture, mechanical strength, and biochemical parameters. We observed that Clad at 10 nM concentration mitigated Dex-induced cytotoxicity and defend osteoblasts against apoptosis. Subsequent results demonstrate that Clad suppressed apoptosis of osteoblast in the presence of Dex by enhancing autophagy in a way that was reliant on the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway. Furthermore, micro-CT scanning, eco MRI results, and serum CTX levels revealed that 12 weeks of Clad treatment prevented bone loss and preserved trabecular bone mass in GIOP animals. We also observed that Clad treated osteoblasts had a lower rate of apoptosis and a greater LC3-II/LC3-I ratio than the Dex group. Our findings show that Clad can protect osteoblasts against glucocorticoids by inducing autophagy via the AMPK/mTOR pathway.

3-PEP promotes bone regeneration by up regulating BCL-2 expression via ERK phosphorylation.

Abstract: Bone healing and regeneration is a complex process that recapitulates embryonic skeletal development and is delayed in diseases like osteoporosis. Bone healing therapies like recombinant bone morphogenetic-2 protein (rhBMP-2) and parathyroid hormone (PTH), an approved bone anabolic therapy reduces fracture risks but are fraught with high cost and several side effects. Thus, there is an unmet need for cost-effective bone healing agents. In this study, we have synthesized 3-piperidinylethoxypterocarpan (3-PEP) which is a hybrid of bone supplement ipriflavone and anti-resorptive drug raloxifene and evaluated its bone regeneration and healing potential. Prior to studies in animal models, the potency of 3-PEP was confirmed in calvarial osteoblast cells. Bromodeoxy uridine cell proliferation and cell viability assay revealed that 3-PEP at 100 pM concentration increased the proliferation and survival of osteoblasts simultaneously inhibiting the apoptosis by involving activation of BCL-2 by phosphorylation at Ser70 site through MEK-ERK pathway. In vivo studies were conducted in estrogen-deficient ovariectomized Balb/c mice and drill hole injury was generated in the mid diaphysis of the femur in all the animals. Treatment with 3-PEP commenced the next day onward and terminated at 7 and 15 days. Micro-CT analysis and calcein labeling of newly generated bone at the drill hole injury site showed that 3-PEP promotes bone healing and new bone formation at a dose of 5 mg/kg at the injury site. These data were also corroborated in non-ovariectomized Balb/c mice cortical defect model. Owing to the side effects associated with rhBMP-2 and PTH, along with the expenses involved, our study proposes an alternative therapeutic option for bone healing.

Discontinuation of PTH therapy amplifies bone loss by increasing oxidative stress: An event ameliorated by sequential IL-17 neutralizing antibody therapy.

Osteoporosis leads to excessive bone resorption which is not accompanied by equal amount of bone formation. PTH (1-34) forms the mainstay of bone anabolic therapy. Intermittent PTH (iPTH) has the ability to reconstruct skeleton, a property not shared by other anti-resorptives. In initial phases of PTH treatment, bone formation exceeds bone resorption. However, gradually this phase is replaced by increased bone resorption. Thus, a replacement post PTH discontinuation is much needed. Studies with bisphosphonates and Denosumab post PTH withdrawal have yielded promising but variable results. Thus, there is scope for trying new combinations. Our previous studies have shown the superior skeletal effects of neutralizing IL17 antibody (NIL17) over anti-RANKL antibody. Thus, here we investigated if sequential treatment of NIL17 after PTH withdrawal (SHIFT) could serve as a promising therapeutic approach for osteoporosis treatment. Our results show that PTH withdrawal (PTH-W) led to mitigation of its anabolic effects as evidenced by reduced BMD, bone trabecular and cortical microarchitectural parameters. In the continuous PTH (PTH-C) and the Shift group, all these parameters were preserved as par with the sham group. Shift therapy also significantly increased PINP levels. Most importantly, serum CTX-I levels and osteoclast numbers, which were elevated in PTH groups were significantly suppressed in NIL17 monotherapy and shift group. Also, expression of FOXO1 and ATF-4, the main regulators of redox balance and function in osteoblasts, were found to be enhanced maximally in the sequential therapy group. Our study thus advocates use of NIL17 as a replacement therapeutic option post PTH discontinuation.

Extract and fraction of Musa paradisiaca flower have osteogenic effect and prevent ovariectomy induced osteopenia.

BACKGROUND: Osteoporosis is an asymptomatic bone disorder leading to altered bone microarchitecture, mineralization and strength. Musa paradisiaca has been reported to have antioxidant and anti-inflammatory effects in various diseases. Its impact on postmenopausal osteoporosis has not been investigated yet. PURPOSE: The intention of the current study was to evaluate the bone regeneration and osteoprotective potential of extract and fraction of M. paradisiaca flower in ovariectomized (Ovx) Sprague Dawley (SD) rats, a model of post-menopausal bone loss. The study also aims to identify osteogenic compounds from active fraction. METHODS: Ethanolic extract (MFE) and butanolic fraction (MFE-Bu) from flower of M. paradisiaca were prepared and their efficacy was tested in rat femur osteotomy model at different doses. Effective dose from both extract (250 mg/kg) and fraction (50 mg/kg) were taken for study in osteopenic bone loss model. PTH was taken as reference standard (20 µg/kg/twice a week). Bones were harvested at autopsy for dynamic and static histomorphometry. Serum was collected for ELISA. Pure compounds were isolated from butanolic fraction (MFE-Bu), and were assessed for their osteogenic effect. RESULTS: MFE and MFE-Bu were observed for their potential in bone healing and prevention of bone loss. Both MFE and MFE-Bu promoted new bone regeneration at injury site as assessed by microCT and calcein dye labeling studies. These also led to restoration of bone microarchitecture deteriorated as a result of osteopenia and improved bone biomechanical properties. Extract as well as the fraction exhibited dual bone anabolic and anti-resorptive properties where they elevated serum procollagen type I N-terminal propeptide (P1NP), a bone formation marker and suppressed serum C-telopeptide of type I collagen (CTX-1), a bone resorption marker. As many as four osteogenic compounds were isolated from MFE-Bu. Oleracein-E was found to be the most potent osteogenic agent based on osteoblast differentiation, mineralization assays, qPCR and protein expression studies. CONCLUSION: Our studies demonstrates that ethanolic extract from the flower of M. paradisiaca and its butanolic fraction exhibit dual osteogenic and anti-resorptive potential, and have an advantage over PTH which though promotes bone formation but is also bone catabolic in nature.

Formononetin, a methoxy isoflavone, enhances bone regeneration in a mouse model of cortical bone defect.

The bone regeneration and healing effect of formononetin was evaluated in a cortical bone defect model that predominantly heals by intramembranous ossification. For this study, female Balb/c mice were ovariectomised (OVx) and a drill-hole injury was generated in the midfemoral bones of all animals. Treatment with formononetin commenced the day after and continued for 21 d. Parathyroid hormone (PTH1-34) was used as a reference standard. Animals were killed at days 10 and 21. Femur bones were collected at the injury site for histomorphometry studies using microcomputed tomography (µCT) and confocal microscopy. RNA and protein were harvested from the region surrounding the drill-hole injury. For immunohistochemistry, 5 µm sections of decalcified femur bone adjoining the drill-hole site were cut. µCT analysis showed that formononetin promoted bone healing at days 10 and 21 and the healing effect observed was significantly better than in Ovx mice and equal to PTH treatment in many aspects. Formononetin also significantly enhanced bone regeneration as assessed by calcein-labelling studies. In addition, formononetin enhanced the expression of osteogenic markers at the injury site in a manner similar to PTH. Formononetin treatment also led to predominant runt-related transcription factor 2 and osteocalcin localisation at the injury site. These results support the potential of formononetin to be a bone-healing agent and are suggestive of its promising role in the fracture-repair process.

Synthesis and biological evaluation of heterocyclic analogues of pregnenolone as novel anti-osteoporotic agents.

The structural modifications of pregnenolone have been described via the introduction of heterocyclic moieties at C-17 position by limiting the acyl group. Novel heterocyclic analogues of pregnenolone have been synthesized by using Friedlander and Claisen-Schmidt reactions, and the synthesized compounds were evaluated for their osteogenic activity. Among the synthesized derivatives, four compounds showed significantly increased ALP activity. Among all four active compounds, the novel compound 3a has shown significant bone matrix mineralization and mRNA expressions of osteogenic marker genes, BMP2, RUNX-2 and OCN at 1pM concentration.