Anirudha Karvande

MicroRNA 874-3p Exerts Skeletal Anabolic Effects Epigenetically during Weaning by Suppressing Hdac1 Expression

Embryonic skeletogenesis and postnatal bone development require the transfer of calcium from the mother to the offspring during pregnancy and lactation. Therefore, bone resorption in the mother becomes elevated during these periods, resulting in significant maternal skeletal loss. There follows an anabolic phase around weaning during which there is a remarkable recovery of the maternal skeleton. However, the mechanism(s) of this anabolic response remain(s) largely unknown. We identified eight differentially expressed miRNAs by array profiling, of which miR-874-3p was highly expressed at weaning, a time when bone loss was noted to recover. We report that this weaning-associated miRNA is an anabolic target. Therefore, an agomir of miR-874-3p induced osteoblast differentiation and mineralization. These actions were mediated through the inhibition of Hdac1 expression and enhanced Runx2 transcriptional activation. When injected in vivo, the agomir significantly increased osteoblastogenesis and mineralization, reversed bone loss caused by ovariectomy, and increased bone strength. We speculate that elevated miR-874-3p expression during weaning enhances bone formation and that this miRNA may become a therapeutic target for conditions of bone loss.

A neoflavonoid dalsissooal isolated from heartwood of Dalbergia sissoo Roxb. has bone forming effects in mice model for osteoporosis.

Dalbergia sissoo Roxb. is a well known medicinal plant of India, enriched with various flavonoids used for treating multiple diseases. Earlier, we have shown that extract of Dalbergia sissoo Roxb. leaves mitigate ovariectomy induced bone loss and pure compounds (neoflavonoids) isolated from it, promote osteoblastogenesis in primary calvarial osteoblasts cells in vitro. Here, we hypothesize that dalsissooal (DSL), a novel neoflavonoid isolated from the heartwood of Dalbergia sissoo Roxb. is an important constituent of the extract that imparts bone forming effects. Treatment with DSL enhanced trabecular bone micro-architecture parameters, biomechanical strength, increased bone formation rate and mineral apposition rate in OVx mice comparable to 17β-estradiol. It increased bone formation by enhancing osteoblast gene expression and reduced bone turnover by decreasing osteoclastic gene expressions. Interestingly, we observed that DSL has no uterine estrogenic effects. At cellular levels, DSL promoted differentiation of bone marrow cells as well as calvaria osteoblast cells towards osteoblast lineage by enhancing differentiation and mineralizing ability to form mineralizing nodules via stimulating BMP-2 and RunX-2 expressions. Overall, our data suggest that oral supplementation of a novel neoflavonoid dalsissooal isolated from heartwood of Dalbergia sissoo Roxb. exhibited bone anabolic action by improving structural property of bone, promoting new bone formation and reducing bone turnover rate in post-menopausal model for osteoporosis with no uterine hyperplasia.

Evaluation of anti-osteoporotic activity of butanolic fraction from Passiflora foetida in ovariectomy-induced bone loss in mice

OBJECTIVE In this study, we have evaluated the skeletal effects of butanolic fraction (BF) from Passiflora foetida in an estrogen deficient mice bone loss model. STUDY DESIGN Skeletal effect of BF was studied in ovariectomized (OVx) female Balb/c mice. BF (50 and 100mg/kg/day dose orally) was given for 8 weeks. Micro-architecture of long bones, biomechanical strength, formations of mineralized nodule by bone marrow osteoprogenitor cells, osteoid formation and bone turnover markers were studied. One way ANOVA was used to test the significance of effects of Passiflora foetida. RESULTS OVx mice treated with BF represented with better micro-architectural parameters at various anatomical positions, better bone biomechanical strength and more osteoprogenitor cells in the bone marrow compared with OVx group. BF did not exhibit uterine estrogenicity. CONCLUSION Oral administration of BF at both the doses (50 and 100mg/kg/day) derived from Passiflora Foetida, was found to afford anti-osteoporotic effect under estrogen deficiency by likely stimulation of osteoblast function and inhibition of osteoclast function.

Quercetin-loaded solid lipid nanoparticles improve osteoprotective activity in an ovariectomized rat model: a preventive strategy for post-menopausal osteoporosis

A formulation of quercetin-based solid lipid nanoparticles (QSLNs) was developed to increase the bioavailability of quercetin, with an aim to evaluate its effects on bone health in comparison to free quercetin (Q). The QSLNs were prepared by emulsification solvent evaporation followed by a cold homogenization method. The QSLNs were spherical when observed under atomic force microscopy, with an average diameter of 172.9 ± 12.65 nm. This formulation was pharmaceutically characterized and then evaluated for osteoprotective activity in ovariectomized (OVx) rats. A single oral dose of QSLNs (5 mg kg−1 d−1) significantly increased the bioavailability compared to free quercetin. The oral administration of QSLNs to ovariectomized rats increased serum quercetin levels by 3.5-fold compared to free quercetin. After 12 weeks of treatment, the bone mineral density of the femur, tibia and lumbar spine L-5 measured by micro-computed tomography (μCT) was restored in the QSLNs group, and was equivalent to the sham control group compared to in the OVx group, but the QSLNs had no effect on adipogenesis and uterine weight in OVx rats. μCT analysis showed that the QSLNs group had an improved trabecular microarchitecture in the distal femoral, proximal tibial and lumbar spine cancellous bones. The developed quercetin formulation based on solid lipid nanoparticles inhibited bone loss in osteopenic rats. Q and QSLNs inhibited the receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast cells differentiation and the expression of osteoclast-specific genes in in vitro experiments using bone marrow cells treated with RANKL and M-CSF. The data from this study suggest that, overall, QSLNs treatment recovers bone loss (femur, BV/TV; ∼21%, p < 0.01) more so than in the Q treatment group, while both inhibit bone loss without showing any hyperplasic effect on the uteri in OVx rats.

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

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.

Dietary flavonoid kaempferol inhibits glucocorticoid-induced bone loss by promoting osteoblast survival

OBJECTIVE Kaempferol, a dietary flavonoid found in fruits and vegetables, has been reported to reverse osteopenic condition in ovariectomized rats. Because kaempferol is endowed with osteogenic activity, the aim of this study was to determine whether it has a beneficial effect on glucocorticoid (GC)-induced bone loss. METHODS Adult female rats were divided into four groups as control (vehicle; distilled water), methylprednisolone (MP; 5 mg•kg•d, subcutaneously), MP + kaempferol (5 mg•kg•d, oral), and MP + human parathyroid 1-34 (30 µg/kg, 5 times/wk, subcutaneously) and treated for 4 wk. To study the antagonizing effect of kaempferol on GC-induced inhibition of fracture healing, drill-hole injury was performed on control and GC-treated rats. An oral dose of kaempferol was given for 14 d to observe the effect on callus formation at the site of injury. After treatment, bones were collected for further analysis. RESULTS GC was associated with a decreased bone mineral density and impaired bone microarchitecture parameters. Consumption of kaempferol induced bone-sparing effects in GC-induced osteopenic condition. Additionally, improved callus formation at site of drill injury in femur diaphysis was observed with kaempferol consumption in animals on GC. Consistent with the in vivo data, kaempferol elicited a higher expression of osteogenic markers in vitro and antagonized the apoptotic effect of dexamethasone on calvarial osteoblasts. CONCLUSION These results suggested that kaempferol reduced GC-induced bone loss and enhanced bone regeneration at fractured site, thus emphasizing the positive role of flavonoids on bone health.

Glucose dependent miR-451a expression contributes to parathyroid hormone mediated osteoblast differentiation.

Parathyroid hormone (PTH; amino acid 1-34, known as teriparatide) has reported promoting differentiation and glucose uptake in osteoblasts. However, how PTH regulates glucose metabolism to facilitate osteoblast differentiation is not understood. Here, we report that PTH promotes glucose dependent miR-451a expression which stimulates osteoblast differentiation. In addition to glucose uptake, PTH suppresses AMPK phosphorylation via PI3K-mTOR-AKT axis thereby preventing phosphorylation and inactivation of octamer-binding transcription factor 1 (OCT-1) which has been reported to act on the promoter region of miR-451a. Modulation of AMPK activity controls miR-451a levels in differentiating osteoblasts. Moreover, pharmacological inhibition of PI3K-mTOR-AKT axis suppressed miR-451a via increased AMPK activity. We report that this glucose regulated miRNA is an anabolic target and transfection of miR-451a mimic induces osteoblast differentiation and mineralization in vitro. These actions were mediated through the suppression of Odd-skipped related 1 (Osr1) and activation of Runx2 transcription. When injected in vivo, the miR-451a mimic significantly increased osteoblastogenesis, mineralization, reversed ovariectomy induced bone loss and improved bone strength. Together, these findings suggest that enhanced osteoblast differentiation associated with bone formation in case of PTH therapy is also a consequence of elevated miR-451a levels via glucose regulation. Consequently, this miRNA has the potential to be a therapeutic target for conditions of bone loss.