Fasciola helminth defense molecule-1 protects against experimental arthritis by inhibiting osteoclast formation and function without modulating the systemic immune response.

An inverse correlation between helminth infection and the autoimmune disease appears to be contributed by the anti-inflammatory factors produced by these organisms. Suppressing osteoclast function without affecting the systemic immunological response is an emerging therapeutic strategy for rheumatoid arthritis (RA). We observed that a synthetic peptide corresponding to 34 amino acids of C-terminal sequence of Fasciola helminth defense molecule-1 (C-FhHDM-1) inhibited RANKL-induced osteoclast formation and lysosomal acidification with an attendant upregulation of sequestome1/p62, a negative regulator of NF-κB expression. C-FhHDM-1 also suppressed RANKL production from osteoblasts. Macrophages are the major inflammatory cells in the joints of RA and C-FhHDM-1 suppressed ICAM-1 (an inflammatory surrogate) expression in these cells. In a murine model of collagen II-induced arthritis (CIA), C-FhHDM-1 improved clinical score, protected against cartilage destruction, and maintained bone mass and bone architecture of joints compared with the CIA group. C-FhHDM-1 suppressed the CIA-induced expression of TNF, IL-17, and IFN-γ in joints but not their serum levels. The peptide also had no effect on the CIA-induced suppression of T regulatory response. We conclude that C-FhHDM-1 has a joint-specific protective effect in experimental arthritis without mitigating systemic inflammation, and thus could become an adjuvant anti-arthritis therapy to prevent RA-induced osteopenia.

M2 polarization of macrophages by Oncostatin M in hypoxic tumor microenvironment is mediated by mTORC2 and promotes tumor growth and metastasis.

Oncostatin M (OSM), an inflammatory cytokine belonging to the interleukin-6 (IL-6) superfamily, plays a vital role in multitude of physiological and pathological processes. Its role in breast tumor progression and metastasis to distant organs is well documented. Recent reports implicate OSM in macrophage M2 polarization, a key pro-tumoral phenomenon. M2 polarization of macrophages is believed to promote tumor progression by potentiating metastasis and angiogenesis. In the current study, we delineated the mechanism underlying OSM induced macrophage M2 polarization. The findings revealed that OSM skews macrophages towards an M2 polarized phenotype via mTOR signaling complex 2 (mTORC2). mTORC2 relays signals through two effector kinases i.e. PKC-α and Akt. Our results indicated that mTORC2 mediated M2 polarization of macrophages is not dependent on PKC-α and is primarily affected via Akt, particularly Akt1. In vivo studies conducted on 4T1/BALB/c mouse orthotropic model of breast cancer further corroborated these observations wherein i.v. reintroduction of mTORC2 abrogated monocytes into orthotropic mouse model resulted in diminished acquisition of M2 specific attributes by tumor associated macrophages. Metastasis to distant organs like lung, liver and bone was reduced as evident by decrease in formation of focal metastatic lesions in mTORC2 abrogated monocytes mice. Our study pinpoints key role of mTORC2-Akt1 axis in OSM induced macrophage polarization and suggests for possible usage of Oncostatin-M blockade and/or selective mTORC2 inhibition as a potential anti-cancer strategy particularly with reference to metastasis of breast cancer to distant organs such as lung, liver and bone.

Influence of antifertility agents Dutasteride and Nifedipine on CatSper gene level in epididymis during sperm maturation in BALB/c mice.

Calcium (Ca2+) signaling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility and the acrosome reaction are all mediated by increases in intracellular Ca2+ through CatSper (sperm-specific cation channel). The CatSper channel complex contains four pore-forming α subunits (CatSper1-4) and five accessory subunits called ß, δ, ε, γ and ζ. Genetic deletion of any of the four CatSper genes in mice results in loss of hyperactivated motility and male infertility. Despite their vital role in male fertility, almost very little is known about influence of antifertility agents on CatSper gene expression in epididymis and epididymal spermatozoa. Therefore, we performed quantitative real-time qPCR analysis for CatSper expression in the epididymis and epididymal sperm of BALB/c mice after treatment with Dutasteride (DS), a dual 5-α reductase inhibitor and Nifedipine (NF) a calcium channel blocker as positive control. We observed that treatment with antifertility agents Dutasteride and Nifedipine induced significant decreases in the caput and cauda epididymal sperm counts, motility and fertility which could partly be attributed to alteration in the normal morphology of the sperm associated with downregulation/upregulation of CatSper mRNAs in epididymis and epididymal spermatozoa of male BALB/c mice. These can be explained on the basis of interference with mechanisms affecting calcium ion signaling resulting in changes in intracellular calcium required for sperm activity, finally affecting sperm maturation and fertility of male BALB/c mice. These studies provide some novel avenues for developing new male contraceptives in future.

Formononetin, an isoflavone, activates AMP-activated protein kinase/ß-catenin signalling to inhibit adipogenesis and rescues C57BL/6 mice from high-fat diet-induced obesity and bone loss.

Balance between adipocyte and osteoblast differentiation is the key link of disease progression in obesity and osteoporosis. We have previously reported that formononetin (FNT), an isoflavone extracted from Butea monosperma, stimulates osteoblast formation and protects against postmenopausal bone loss. The inverse relationship between osteoblasts and adipocytes prompted us to analyse the effect of FNT on adipogenesis and in vivo bone loss, triggered by high-fat diet (HFD)-induced obesity. The anti-obesity effect and mechanism of action of FNT was determined in 3T3-L1 cells and HFD-induced obese male mice. Our findings show that FNT suppresses the adipogenic differentiation of 3T3-L1 fibroblasts, through down-regulation of key adipogenic markers such as PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα) and sterol regulatory element-binding protein (SREBP) and inhibits intracellular TAG accumulation. Increased intracellular reactive oxygen species levels and AMP-activated protein kinase (AMPK) activation accompanied by stabilisation of ß-catenin were attributed to the anti-adipogenic action of FNT. In vivo, 12 weeks of FNT treatment inhibited the development of obesity in mice by attenuating HFD-induced body weight gain and visceral fat accumulation. The anti-obesity effect of FNT results from increased energy expenditure. FNT also protects against HFD-induced dyslipidaemia and rescues deterioration of trabecular bone volume by increasing bone formation and decreasing bone resorbtion caused by HFD. FNT's rescuing action against obesity-induced osteoporosis commenced at the level of progenitors, as bone marrow progenitor cells, obtained from the HFD mice group supplemented with FNT, showed increased osteogenic and decreased adipogenic potentials. Our findings suggest that FNT inhibits adipogenesis through AMPK/ß-catenin signal transduction pathways and protects against HFD-induced obesity and bone loss.

On-demand release of a selective MMP-13 blocker from an enzyme-responsive injectable hydrogel protects cartilage from degenerative progression in osteoarthritis.

In osteoarthritis (OA), the degradation of cartilage is primarily driven by matrix metalloprotease-13 (MMP-13). Hence, the inhibition of MMP-13 has emerged as an attractive target for OA treatment. Among the various approaches that are being explored for MMP-13 regulation, blocking of the enzyme with specific binding molecules appears to be a more promising strategy for preventing cartilage degeneration. To enhance effectiveness and ensure patient compliance, it is preferable for the binding molecule to exhibit sustained activity when administered directly into the joint. Herein, we present an enzyme-responsive hydrogel that was designed to exhibit on-demand, the sustained release of BI-4394, a potent and highly selective MMP-13 blocker. The stable and compatible hydrogel was prepared using triglycerol monostearate. The efficacy of the hydrogel to prevent cartilage damage was assessed in a rat model of OA induced by anterior cruciate ligament transection (ACLT). The results revealed that in comparison to the rats administrated weekly with intra-articular BI-4394, the hydrogel implanted rats had reduced levels of inflammation and bone erosion. In comparison to untreated control, the cartilage in animals administered with BI-4394/hydrogel exhibited significant levels of collagen-2 and aggrecan along with reduced MMP-13. Overall, this study confirmed the potential of BI-4394 delivery using an enzyme-responsive hydrogel as a promising treatment option to treat the early stages of OA by preventing further cartilage degradation.

Exposure to As, Cd and Pb-mixture impairs myelin and axon development in rat brain, optic nerve and retina.

Arsenic (As), lead (Pb) and cadmium (Cd) are the major metal contaminants of ground water in India. We have reported the toxic effect of their mixture (metal mixture, MM), at human relevant doses, on developing rat astrocytes. Astrocyte damage has been shown to be associated with myelin disintegration in CNS. We, therefore, hypothesized that the MM would perturb myelinating white matter in cerebral cortex, optic nerve (O.N.) and retina. We observed modulation in the levels of myelin and axon proteins, such as myelin basic protein (MBP), proteolipid protein, 2'-, 3'-cyclic-nucleotide-3'-phosphodiesterase, myelin-associated glycoprotein and neurofilament (NF) in the brain of developing rats. Dose and time-dependent synergistic toxic effect was noted. The MBP- and NF-immunolabeling, as well as luxol-fast blue (LFB) staining demonstrated a reduction in the area of intact myelin-fiber, and an increase in vacuolated axons, especially in the corpus-callosum. Transmission electron microscopy (TEM) of O.N. revealed a reduction in myelin thickness and axon-density. The immunolabeling with MBP, NF, and LFB staining in O.N. supported the TEM data. The hematoxylin and eosin staining of retina displayed a decrease in the thickness of nerve-fiber, plexiform-layer, and retinal ganglion cell (RGC) count. Investigating the mechanism revealed a loss in glutamine synthetase activity in the cerebral cortex and O.N., and a fall in the brain derived neurotrophic factor in retina. An enhanced apoptosis in MBP, NF and Brn3b-containing cells justified the diminution in myelinating axons in CNS. Our findings for the first time indicate white matter damage by MM, which may have significance in neurodevelopmental-pediatrics, neurotoxicology and retinal-cell biology.

Caviunin glycoside (CAFG) from Dalbergia sissoo attenuates osteoarthritis by modulating chondrogenic and matrix regulating proteins.

ETHNOPHARMACOLOGICAL RELEVANCE: Dalbergia sissoo DC. (Indian rosewood or Sheesham) is a traditional medicinal plant, reported since time immemorial for its analgesic, anti-nociceptive, anti-inflammatory, and immuno-modulatory properties. D. sissoo DC (DS). is being used traditionally to cure joint inflammation and joint pain. AIM: To study the potential of DS leaves and its derived novel compound CAFG to treat the clinical symptoms of osteoarthritis (OA) and its underlying mechanism. METHODS: The chemical profile of DS extract (DSE) with isoflavonoids and isoflvaonoid glycosides from the DS was established by UHPLC-PDA and UHPLC-MS/MS. Monosodium iodoacetate (MIA) was injected into the knee joint to develop the OA model in rats. DSE was given orally for 28 days daily at 250 and 500 mg.kg-1day-1. For in-vitro experiments, chondrocytes isolated from joint articular cartilage were negatively induced with interleukin-1ß (IL-1ß) and CAFG was given to the cells as a co-treatment. RESULTS: Chondrocytes undergo apoptosis following inflammation and proteoglycan synthesis affected in MIA injected knees. DSE administration prevented these effects as assessed by H&E and Toluidine blue staining. Micro-CT analysis showed that subchondral bone loss was restored. DSE decreased elevated serum levels of cartilage-bone degradation (CTX-I, CTX-II, and COMP), inflammation markers IL-1ß, and matrix-degrading MMP-3 and 13. The effects of IL-1ß on gene expression of chondrocytes were reversed by CAFG treatment at 1 µM. CONCLUSION: Data showed that DSE protected joint cartilage and deterioration in subchondral bone in vivo while in in-vitro, its active ingredient CAFG prevented interleukin-1ß induced effects and inhibited OA. This finding suggest that DSE and CAFG could be used as a possible therapeutic to treat osteoarthritis.

Heartwood extract from Dalbergia sissoo promotes fracture healing and its application in ovariectomy-induced osteoporotic rats.

OBJECTIVES: This study was undertaken to investigate the effects of a heartwood ethanolic extract (HEE) made from the Dalbergia sissoo on facture healing and in the prevention of pathological bone loss resulting from estrogen deficiency in ovariectomized (Ovx) rats. METHODS: Heartwood ethanolic extract (250, 500 and 1000 mg/kg per day) was administered orally immediately next day after drill-hole injury and continued for 2 weeks. Ovx rats received HEE at same doses for 12 weeks and compared with 17-ß estradiol (E2; 100 µg/kg for 5 days/week subcutaneously) group. Confocal imaging for fracture healing, micro-architecture of long bones, biomechanical strength, formation of mineralized nodule by bone marrow osteoprogenitor cells, bone turnover markers and gene expression were studied. One-way ANOVA was used to test significance. KEY FINDINGS: Heartwood ethanolic extract treatment promoted fracture healing, formation of new bone at the drill-hole site and stimulated osteogenic genes at callus region. HEE administration to the Ovx rats exhibited better micro-architectural parameters at various anatomical positions, better bone biomechanical strength and more osteoprogenitor cells in the bone marrow compared with Ovx + vehicle group. HEE exhibited no uterine estrogenicity. CONCLUSIONS: Oral administration of HEE was found to promote fracture healing and exhibited osteoprotective effect by possibly stimulation of osteoblast function.

Odanacatib Restores Trabecular Bone of Skeletally Mature Female Rabbits With Osteopenia but Induces Brittleness of Cortical Bone: A Comparative Study of the Investigational Drug With PTH, Estrogen, and Alendronate.

Cathepsin K (CK), a lysosomal cysteine protease, is highly expressed in mature osteoclasts and degrades type 1 collagen. Odanacatib (ODN) is a selective and reversible CK inhibitor that inhibits bone loss in preclinical and clinical studies. Although an antiresorptive, ODN does not suppress bone formation, which led us to hypothesize that ODN may display restorative effect on the osteopenic bones. In a curative study, skeletally mature New Zealand rabbits were ovarectomized (OVX) and after induction of bone loss were given a steady-state exposure of ODN (9 mM/d) for 14 weeks. Sham-operated and OVX rabbits treated with alendronate (ALD), 17b-estradiol (E2), or parathyroid hormone (PTH) served as various controls. Efficacy was evaluated by assessing bone mineral density (BMD), bone microarchitecture (using micro-computed tomography), fluorescent labeling of bone, and biomechanical strength. Skeletal Ca/P ratio was measured by scanning electron microscopy (SEM) with X-ray microanalysis, crystallinity by X-ray diffraction, and bone mineral density distribution (tissue mineralization) by backscattered SEM. Between the sham and ODN-treated osteopenic groups, lumbar and femur metaphyseal BMD, Ca/P ratio, trabecular microstructure and geometric indices, vertebral compressive strength, trabecular lining cells, cortical parameters (femoral area and thickness and periosteal deposition), and serum P1NP were largely comparable. Skeletal improvements in ALD-treated or E2-treated groups fell significantly short of the sham/ODN/PTH group. However, the ODN group displayed reduced ductility and enhanced brittleness of central femur, which might have been contributed by higher crytallinity and tissue mineralization. Rabbit bone marrow stromal cells expressed CK and when treated with ODN displayed increased formation of mineralized nodules and decreased apoptosis in serum-deficient medium compared with control. In vivo, ODN did not suppress remodeling but inhibited osteoclast activity more than ALD. Taken together, we show that ODN reverses BMD, skeletal architecture, and compressive strength in osteopenic rabbits; however, it increases crystallinity and tissue mineralization, thus leading to increased cortical bone brittleness.

BMP signaling is required for adult skeletal homeostasis and mediates bone anabolic action of parathyroid hormone.

Bmp2 and Bmp4 genes were ablated in adult mice (KO) using a conditional gene knockout technology. Bones were evaluated by microcomputed tomography (µCT), bone strength tester, histomorphometry and serum biochemical markers of bone turnover. Drill-hole was made at femur metaphysis and bone regeneration in the hole site was measured by calcein binding and µCT. Mice were either sham operated (ovary intact) or ovariectomized (OVX), and treated with human parathyroid hormone (PTH), 17ß-estradiol (E2) or vehicle. KO mice displayed trabecular bone loss, diminished osteoid formation and reduced biomechanical strength compared with control (expressing Bmp2 and Bmp4). Both osteoblast and osteoclast functions were impaired in KO mice. Bone histomorphomtery and serum parameters established a low turnover bone loss in KO mice. Bone regeneration at the drill-hole site in KO mice was lower than control. However, deletion of Bmp2 gene alone had no effect on skeleton, an outcome similar to that reported previously for deletion of Bmp4 gene. Both PTH and E2 resulted in skeletal preservation in control-OVX, whereas in KO-OVX, E2 but not PTH was effective which suggested that the skeletal action of PTH required Bmp ligands but E2 did not. To determine cellular effects of Bmp2 and Bmp4, we used bone marrow stromal cells in which PTH but not E2 stimulated both Bmp2 and Bmp4 synthesis leading to increased Smad1/5 phosphorylation. Taken together, we conclude that Bmp2 and Bmp4 are essential for maintaining adult skeletal homeostasis and mediating the anabolic action of PTH.

Pathophysiological Mechanism of Bone Loss in Type 2 Diabetes Involves Inverse Regulation of Osteoblast Function by PGC-1α and Skeletal Muscle Atrogenes: AdipoR1 as a Potential Target for Reversing Diabetes-Induced Osteopenia.

Type 2 diabetes is associated with increased fracture risk and delayed fracture healing; the underlying mechanism, however, remains poorly understood. We systematically investigated skeletal pathology in leptin receptor-deficient diabetic mice on a C57BLKS background (db). Compared with wild type (wt), db mice displayed reduced peak bone mass and age-related trabecular and cortical bone loss. Poor skeletal outcome in db mice contributed high-glucose- and nonesterified fatty acid-induced osteoblast apoptosis that was associated with peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) downregulation and upregulation of skeletal muscle atrogenes in osteoblasts. Osteoblast depletion of the atrogene muscle ring finger protein-1 (MuRF1) protected against gluco- and lipotoxicity-induced apoptosis. Osteoblast-specific PGC-1α upregulation by 6-C-ß-d-glucopyranosyl-(2S,3S)-(+)-5,7,3',4'-tetrahydroxydihydroflavonol (GTDF), an adiponectin receptor 1 (AdipoR1) agonist, as well as metformin in db mice that lacked AdipoR1 expression in muscle but not bone restored osteopenia to wt levels without improving diabetes. Both GTDF and metformin protected against gluco- and lipotoxicity-induced osteoblast apoptosis, and depletion of PGC-1α abolished this protection. Although AdipoR1 but not AdipoR2 depletion abolished protection by GTDF, metformin action was not blocked by AdipoR depletion. We conclude that PGC-1α upregulation in osteoblasts could reverse type 2 diabetes-associated deterioration in skeletal health.

Developmental exposure to As, Cd, and Pb mixture diminishes skeletal growth and causes osteopenia at maturity via osteoblast and chondrocyte malfunctioning in female rats.

We studied the effect of metal mixture (MM), comprising As, Cd, and Pb, in developing female rat skeleton from gestation day 5 until postnatal day 60 (P-60). MM resulted in synergistic inhibition in viability and differentiation of osteoblasts in vitro, likely induced by reactive oxygen species. MM, administered at their most frequently occurring concentrations present in the groundwater of India, i.e., As: 0.38 ppm, Pb: 0.22 ppm, and Cd: 0.098 ppm or 10× of the ratio to developing rats, exhibited a synergistic decrease in ex vivo mineralization of bone marrow stromal (osteoprogenitor) cells. MM group showed a dose-dependent attenuation in weight and axial lengths and shortening of tibias at P-60. Furthermore, the growth plate was shortened, which was associated with shorter proliferative and hypertrophic zones, decreased parathyroid hormone-related protein and Indian hedgehog expression in the chondrocytes, reduced primary and secondary spongiosa, and hypomineralized osteoids-a major characteristic of osteomalacia. In addition, compared with the control, MM-treated rats were clearly osteopenic based on bone mineral density, microarchitecture, biomechanical strength, and particularly the biochemical profile, that suggested high turnover bone loss. Finally, in comparison to the control, the fracture-healing ability of MM group was delayed and accompanied by inferior quality of the healed bone. Together, these data demonstrated that the mixture of As, Cd, and Pb induced synergistic toxicity to developing skeleton, thereby diminishing modeling-directed bone accrual, inducing osteopenia and dampening fracture healing.

Anti-apoptotic role of omega-3-fatty acids in developing brain: perinatal hypothyroid rat cerebellum as apoptotic model.

Inadequate maternal intake of omega-3-fatty acids (omega3 FAs) causes adverse neurodevelopmental outcome in the progeny; however, their molecular mechanism of action is obscure. Since omega3 FAs are known to inhibit neuronal apoptosis during neuro-degeneration, we investigated their possible contribution in regulating neuronal apoptosis during brain development. Using rat model of hypothyroidism-induced neuronal apoptosis, we provide evidence for anti-apoptotic role of omega3 FAs during cerebellar development. omega3 FAs were supplemented as a mixture of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to pregnant and lactating rats, and primary hypothyroidism was induced by administering methimazole. The cerebella from postnatal day 16 (d16) pups were isolated, and studies on apoptosis were conducted. We observed that omega3 FA-supplementation significantly reduced DNA fragmentation and caspase-3 activation in developing cerebellum of hypothyroid pups. The protection provided by omega3 FAs was associated with their ability to prevent increases in the level of pro-apoptotic basal cell lymphoma protein-2 (Bcl-2)-associated X protein (Bax) in the cerebellum during thyroid hormone (TH) deficiency. omega3 FAs increased the levels of anti-apoptotic proteins like Bcl-2 and Bcl-extra large (Bcl-x(L)), known to be repressed in hypothyroidism. omega3 FAs also restored levels of cerebellar phospho (p)-AKT, phospho-extracellular regulated kinase (p-ERK) and phospho-c-Jun N-terminal kinase (p-JNK), which were altered by hypothyroid insults, without interfering with the expression of TH responsive gene, myelin basic protein (mbp). Taken together, these results supplement an insight into the molecular mechanism of action of omega3 FAs in developing brain that involves regulation of apoptotic signaling pathways under stress.