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.

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.

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.

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.