Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone.

Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic ß subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and TNFα) and expression of skeletal osteoclastogenic genes. It also increased new bone formation and expression of osteogenic genes in the femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid hormone and alendronate (FDA approved drugs). WFA promoted the process of cortical bone regeneration at drill-holes site in the femur mid-diaphysis region and cortical gap was bridged with woven bone within 11 days of both estrogen sufficient and deficient (ovariectomized, Ovx) mice. Together our data suggest that WFA stimulates bone formation by abrogating proteasomal machinery and provides knowledge base for its clinical evaluation as a bone anabolic agent.

Isoformononetin, a methoxydaidzein present in medicinal plants, reverses bone loss in osteopenic rats and exerts bone anabolic action by preventing osteoblast apoptosis.

PURPOSE: Daidzein (Daid) has been implicated in bone health for its estrogen-'like' effects but low bioavailability, unfavorable metabolism and uterine estrogenicity impede its clinical potential. This study was aimed at assessing isoformononetin (Isoformo), a naturally occurring methoxydaidzein, for bone anabolic effect by overcoming the pitfalls associated with Daid. METHODS: Sprague-Dawley ovariectomized (OVx) rats with established osteopenia were administered Isoformo, 17ß-oestradiol (E2) or human parathyroid hormone. Efficacy was evaluated by bone microarchitecture using microcomputed tomography and determination of new bone formation by fluorescent labeling of bone. Osteoblast apoptosis was measured by co-labeling of bone sections with Runx-2 and TUNEL. Biochemical markers of bone metabolism were measured by ELISA. Plasma and bone marrow levels of Isoformo and Daid were determined by LC-MS-MS. Rat bone marrow stromal cells were harvested to study osteoblastic differentiation by Isoformo and Daid. New born rat pups were injected with Isoformo and Daid to study the effect of the compounds on the expression of osteogenic genes in the calvaria by real time PCR. RESULTS: In osteopenic rats, Isoformo treatment restored trabecular microarchitecture, increased new bone formation, increased the serum osteogenic marker (procollagen N-terminal propeptide), decreased resorptive marker (urinary C-terminal teleopeptide of type I collagen) and diminished osteoblast apoptosis in bone. At the most effective osteogenic dose of Isoformo, plasma and bone marrow levels were comprised of ~90% Isoformo and the rest, Daid. Isoformo at the concentration reaching the bone marrow achieved out of its most effective oral dosing induced stromal cell mineralization and osteogenic gene expression in the calvaria of neonatal rats. Isoformo exhibited uterine safety. CONCLUSIONS: Our study demonstrates that Isoformo reverses established osteopenia in adult OVx rats likely via its pro-survival effect on osteoblasts. Given its bone anabolic and anti-catabolic effects accompanied with safety at uterine level we propose its potential in the management of postmenopausal osteoporosis.