Litsea glutinosa extract promotes fracture healing and prevents bone loss via BMP2/SMAD1 signaling.

Estrogen deficiency is one of the main causes for postmenopausal osteoporosis. Current osteoporotic therapies are of high cost and associated with serious side effects. So there is an urgent need for cost-effective anti-osteoporotic agents. Anti-osteoporotic activity of Litsea glutinosa extract (LGE) is less explored. Moreover, its role in fracture healing and mechanism of action is still unknown. In the present study we explore the osteoprotective potential of LGE in osteoblast cells and fractured and ovariectomized (Ovx) mice models. Alkaline phosphatase (ALP), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and mineralization assays revealed that LGE treatment increased osteoblast cell differentiation, viability and mineralization. LGE treatment at 0.01 µg increased the expression of BMP2, PSMAD, RUNX2 and type 1 col. LGE also mitigated RANKL-induced osteoclastogenesis. Next, drill hole injury Balb/C mice model was treated with LGE for 12 days. Micro-CT analysis and Calcein labeling at the fracture site showed that LGE (20 mg/kg) enhanced new bone formation and bone regeneration, also increased expression of BMP2/SMAD1 signaling genes at fracture site. Ovx mice were treated with LGE for 1 month. µCT analysis indicated that the treatment of LGE at 20 mg/kg dose prevented the alteration in bone microarchitecture and maintained bone mineral density and bone mineral content. Treatment also increased bone strength and restored the bone turnover markers. Furthermore, in bone samples, LGE increased osteogenesis by enhancing the expression of BMP2/SMAD1 signaling components and decreased osteoclast number and surface. We conclude that LGE promotes osteogenesis via modulating the BMP2/SMAD1 signaling pathway. The study advocates the therapeutic potential of LGE in osteoporosis treatment.

Design, synthesis and biological evaluation of novel pyrimidine derivatives as bone anabolic agents promoting osteogenesis via the BMP2/SMAD1 signaling pathway.

Anti-resorptive inhibitors such as bisphosphonates are widely used but they have limited efficacy and serious side effects. Though subcutaneous injection of teriparatide [PTH (1-34)] is an effective anabolic therapy, long-term repeated subcutaneous administration is not recommended. Henceforth, orally bio-available small-molecule-based novel therapeutics are unmet medical needs to improve the treatment. In this study, we designed, synthesized, and carried out a biological evaluation of 31 pyrimidine derivatives as potent bone anabolic agents. A series of in vitro experiments confirmed N-(5-bromo-4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide (18a) as the most efficacious anabolic agent at 1 pM. It promoted osteogenesis by upregulating the expression of osteogenic genes (RUNX2 and type 1 col) via activation of the BMP2/SMAD1 signaling pathway. In vitro osteogenic potential was further validated using an in vivo fracture defect model where compound 18a promoted the bone formation rate at 5 mg kg-1. We also established the structure-activity relationship and pharmacokinetic studies of 18a.

Bone Fracture-healing Properties and UPLC-MS Analysis of an Enriched Flavonoid Fraction from Oxystelma esculentum.

Oxystelma esculentum has been used as a folk medicine to treat jaundice, throat infections, and skin problems. In the current study, the bone fracture-healing properties of a flavonoid-enriched fraction (Oxy50-60F) of O. esculentum were investigated in Swiss mice using a drill-hole injury model. Oxy50-60F (1 mg/kg/day, 5 mg/kg/day, and 10 mg/kg/day) was administered orally (from the next day) after a 0.6 mm drill-hole injury in mice femur mid-diaphysis for 7 days and 14 days. Parathyroid hormone (40 µg/kg; 5 times/week) was given subcutaneously as the positive control. Confocal imaging for bone regeneration, micro-architecture of femur bones, ex vivo mineralization, hematoxyline and eosin staining, measurement of reactive oxygen species, and gene expression of osteogenic and anti-inflammatory genes were studied. Quercetin, kaempferol, and isorhamnetin glycosides were identified in the active fraction using mass spectrometry techniques. Our results confirm that Oxy50-60F treatment promotes fracture healing and callus formation at drill-hole sites and stimulates osteogenic and anti-inflammatory genes. Oxy50-60F administration to fractured mice exhibited significantly better micro-CT parameters in a dose-dependent manner and promoted nodule mineralization at days 7 and 14 post-injury. Oxy50-60F also prevents ROS generation by increasing expression of the SOD2 enzyme. Overall, this study reveals that Oxy50-60F has bone regeneration potential in a cortical bone defect model, which supports its use in delayed-union and non-union fracture cases.

Chebulinic acid alleviates LPS-induced inflammatory bone loss by targeting the crosstalk between reactive oxygen species/NFκB signaling in osteoblast cells.

Chebulinic acid (CA), a plant ellagitannin derived from Triphala, is reported to exhibit both anti-inflammatory & anti-oxidant activity apart from anti-tumour property. However, its role in inflammatory bone loss conditions was unexplored. We hypothesized that CA may prevent the bone loss under inflammatory conditions induced by lipopolysaccharide (LPS) in 10-week-old male C57BL/6J mice. Micro-CT analysis and histomorphometric evaluations were carried out where it was found that CA significantly improved the bone micro-architectures by enhancing trabecular connectivity and strength of the bone. CA also increased the bone regeneration as examined by calcein labelling and ex-vivo mineralisation along with maintaining the bone serum markers. Further, CA ameliorated the reduction in osteoblast cell differentiation, proliferation and viability after LPS stimulation. DCFDA and Mitosox staining revealed that CA presented remarkable protective effects against LPS treatment by attenuating oxidative stress, both at cellular & mitochondrial levels. In addition, CA significantly decreased the production of pro-inflammatory cytokines, and down-regulated the phosphorylation of NFκB and IκBα, indicating that CA could attenuate the inflammatory impairment to primary osteoblast cells by suppressing the NFkB signalling pathway. Taken together, the protective role of CA against LPS-induced bone loss & inhibitory effect on total ROS levels hold promise as a potential novel therapeutic strategy for the inflammatory diseases in bones.

Maintenance of increased bone mass after PTH withdrawal by sequential medicarpin treatment via augmentation of cAMP-PKA pathway.

Osteoporosis is a metabolic bone disorder associated with impaired bone microarchitecture leading to fragility fractures. Long-term usage of parathyroid hormone (PTH) enhances bone resorption and leads to osteosarcoma in rats which limits its exposure to maximum 2 years in human. Notably, the anabolic effects of PTH do not endure in the absence of sustained administration. Studies in our lab identified osteogenic and antiresorptive activity in medicarpin, a phytoestrogen belonging to the pterocarpan class. Considering dual-acting property of medicarpin and limitations of PTH therapy, we envisaged that medicarpin sequential treatment after PTH withdrawal could serve as promising therapeutic approach for osteoporosis treatment. As PTH exerts its bone anabolic effect by increasing osteoblast survival, our study aims to determine whether medicarpin amplifies this effect of PTH. Our results show that PTH withdrawal led to reduced bone mineral density and bone parameters, while sequential treatment of medicarpin after PTH withdrawal significantly enhanced these parameters. Remarkably, these effects were more pronounced than 8-week PTH treatment. Sequential therapy also significantly increased P1NP levels and decreased CTX levels and TRAP positive cells compared to PTH 8W group where CTX levels were quite high due to bone resorptive action of PTH. Protein expression studies revealed that medicarpin along with PTH betters the antiapoptotic potential compared to PTH alone, through augmentation of cyclic adenosine monophosphate-PKA-CREB pathway. These results proclaim that medicarpin sequential treatment prevented the reduction in bone accrual and strength accompanying PTH withdrawal and also aided in antiapoptotic role of PTH. The study points toward the potential use of medicarpin as a replacement therapeutic option postdiscontinuation of PTH.

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.

A machine learning-based approach to determine infection status in recipients of BBV152 (Covaxin) whole-virion inactivated SARS-CoV-2 vaccine for serological surveys.

Data science has been an invaluable part of the COVID-19 pandemic response with multiple applications, ranging from tracking viral evolution to understanding the vaccine effectiveness. Asymptomatic breakthrough infections have been a major problem in assessing vaccine effectiveness in populations globally. Serological discrimination of vaccine response from infection has so far been limited to Spike protein vaccines since whole virion vaccines generate antibodies against all the viral proteins. Here, we show how a statistical and machine learning (ML) based approach can be used to discriminate between SARS-CoV-2 infection and immune response to an inactivated whole virion vaccine (BBV152, Covaxin). For this, we assessed serial data on antibodies against Spike and Nucleocapsid antigens, along with age, sex, number of doses taken, and days since last dose, for 1823 Covaxin recipients. An ensemble ML model, incorporating a consensus clustering approach alongside the support vector machine model, was built on 1063 samples where reliable qualifying data existed, and then applied to the entire dataset. Of 1448 self-reported negative subjects, our ensemble ML model classified 724 to be infected. For method validation, we determined the relative ability of a random subset of samples to neutralize Delta versus wild-type strain using a surrogate neutralization assay. We worked on the premise that antibodies generated by a whole virion vaccine would neutralize wild type more efficiently than delta strain. In 100 of 156 samples, where ML prediction differed from self-reported uninfected status, neutralization against Delta strain was more effective, indicating infection. We found 71.8% subjects predicted to be infected during the surge, which is concordant with the percentage of sequences classified as Delta (75.6%-80.2%) over the same period. Our approach will help in real-world vaccine effectiveness assessments where whole virion vaccines are commonly used.

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.

Synthesis and biological evaluation of substituted amide derivatives of C4-ageratochromene dimer analog.

Substituted amide derivatives of C4-ageratochromene dimer analog (19) were synthesized through structural modification of precocene-I (4a), isolated from the essential oil of Ageratum conyzoides L. The target compounds (18-20, 23I-VI, 24I-VI, and 25I-VI) were evaluated for their bone-forming effect using osteoblast differentiation assay. Seven compounds (23I, 23II, 23IV, 23VI, 24III, 24VI, and 25VI) presented good activity within 1 pM-1 nM concentration. At 1 pM concentration, the most active compound i.e. 23II showed effective mineralization of osteoblast cells along with expression of osteogenic marker genes viz RUNX 2, BMP-2, and type 1 collagen (Type-1 col) without any toxicity towards osteoblast cells. Single crystal X-ray analysis of 18 and 20 revealed that the core nucleus of these molecules bear phenyl rings in a Trans-stilbenoid system and had a good structural correlation with 17ß-estradiol (1) and diethylstilbestrol (DES, 3). In-silico study about 23II showed its structural complementarities with the LBD of estrogen receptor (ER) which indicated possible ER-mediated activity of compounds.