Baicalein alleviates osteoarthritis by protecting subchondral bone, inhibiting angiogenesis and synovial proliferation.

Osteoarthritis (OA) is one of the most frequent chronic joint diseases with the increasing life expectancy. The main characteristics of the disease are loss of articular cartilage, subchondral bone sclerosis and synovium inflammation. Physical measures, drug therapy and surgery are the mainstay of treatments for OA, whereas drug therapies are mainly limited to analgesics, glucocorticoids, hyaluronic acids and some alternative therapies because of single therapeutic target of OA joints. Baicalein, a traditional Chinese medicine extracted from Scutellaria baicalensis Georgi, has been widely used in anti-inflammatory therapies. Previous studies revealed that baicalein could alleviate cartilage degeneration effectively by acting on articular chondrocytes. However, the mechanisms involved in baicalein-mediated protection of the OA are not completely understood in consideration of integrality of arthrosis. In this study, we found that intra-articular injection of baicalein ameliorated subchondral bone remodelling. Further studies showed that baicalein could decrease the number of differentiated osteoblasts by inhibiting pre-osteoblasts proliferation and promoting pre-osteoblasts apoptosis. In addition, baicalein impaired angiogenesis of endothelial cells and inhibited proliferation of synovial cells. Taken together, these results implicated that baicalein might be an effective medicine for treating OA by regulating multiple targets.

The effects of tranylcypromine on osteoclastogenesis in vitro and in vivo.

Identification of anti-osteoclastogenic agents is important for the treatment of bone loss diseases that feature excessive osteoclast (OC) activity and bone resorption. Tranylcypromine (TCP), an irreversible inhibitor of monoamine oxidase (MAO), has been used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders. TCP has been discovered to exert anabolic effect on osteoblasts, and MAO-A has also been verified as an important mediator in prostate cancer cells to accelerate osteoclastogenesis. In current study, we were focused on TCP and MAO-A effects on osteoclastogenesis. As illustrated by tartrate-resistant acid phosphatase staining, TCP was capable of inhibiting osteoclastogenesis induced by receptor activators of the NF-κB ligand (RANKL) in bone marrow-derived macrophage cells without any cytotoxicity. It was also shown to effectively suppress bone resorption of OCs. The subsequent study revealed that TCP inhibited osteoclastogenesis-related genes in a time-dependent manner through protein kinase B (AKT)-mediated mechanism followed by the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)-c-fos pathway. And TCP could overcome the osteoclastogenic effects of AKT activator SC79. In addition, our results indicated that the expression and catalytic activity of MAO-A were up-regulated by RANKL stimulation and down-regulated by TCP in vitro and in vivo. Furthermore, the effects of MAO-A knockdown on OC differentiation indicated that MAO-A played an important role in osteoclastogenesis in vitro and might contribute to the inhibitory effects of TCP. And AKT, NFATc1, and c-fos were involved in the MAO-A pathway. Notably, our in vivo study reflected that TCPs were capable of restoring the bone loss in LPS-induced calvaria osteolysis and estrogen deficiency-induced osteoporosis models. Thus, our current work provided a potential option for the treatment of bone loss diseases and highlighted the important role of MAO-A in osteoclastogenesis as well.-Liu, Z., Yang, K., Yan, X., Wang, T., Jiang, T., Zhou, Q., Qi, J., Qian, N., Zhou, H., Chen, B., Huang, P., Guo, L., Zhang, X., Xu, X., Jiang, M., Deng, L. The effects of tranylcypromine on osteoclastogenesis in vitro and in vivo.

Development of Small-Molecules Targeting Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-Receptor Activator of Nuclear Factor-κB (RANK) Protein-Protein Interaction by Structure-Based Virtual Screening and Hit Optimization.

Targeting RANKL/RANK offers the possibility of developing novel therapeutic approaches to treat bone metabolic diseases. Multiple efforts have been made to inhibit RANKL. For example, marketed monoclonal antibody drug Denosumab could inhibit the maturation of osteoclasts by binding to RANKL. This study is an original approach aimed at discovering small-molecule inhibitors impeding RANKL/RANK protein interaction. We identified compound 34 as a potent and selective RANKL/RANK inhibitor by performing structure-based virtual screening and hit optimization. Disruption of the RANKL/RANK interaction by 34 effectively inhibits RANKL-induced osteoclastogenesis and bone resorption. The expression of osteoclast marker genes was also suppressed by treatment of 34. Furthermore, 34 markedly blocked the NFATc1/c-fos pathway. Thus, our current work demonstrates that the chemical tractability of the difficult PPI (RANKL/RANK) target by a small-molecule compound 34 offers a potential lead compound to facilitate the development of new medications for bone-related diseases.

Small molecule nAS-E targeting cAMP response element binding protein (CREB) and CREB-binding protein interaction inhibits breast cancer bone metastasis.

Bone is the most common metastatic site for breast cancer. The excessive osteoclast activity in the metastatic bone lesions often produces osteolysis. The cyclic-AMP (cAMP)-response element binding protein (CREB) serves a variety of biological functions including the transformation and immortalization of breast cancer cells. In addition, evidence has shown that CREB plays a key role in osteoclastgenesis and bone resorption. Small organic molecules with good pharmacokinetic properties and specificity, targeting CREB-CBP (CREB-binding protein) interaction to inhibit CREB-mediated gene transcription have attracted more considerations as cancer therapeutics. We recently identified naphthol AS-E (nAS-E) as a cell-permeable inhibitor of CREB-mediated gene transcription through inhibiting CREB-CBP interaction. In this study, we tested the effect of nAS-E on breast cancer cell proliferation, survival, migration as well as osteoclast formation and bone resorption in vitro for the first time. Our results demonstrated that nAS-E inhibited breast cancer cell proliferation, migration, survival and suppressed osteoclast differentiation as well as bone resorption through inhibiting CREB-CBP interaction. In addition, the in vivo effect of nAS-E in protecting against breast cancer-induced osteolysis was evaluated. Our results indicated that nAS-E could reverse bone loss induced by MDA-MB-231 tumour. These results suggest that small molecules targeting CREB-CBP interaction to inhibit CREB-mediated gene transcription might be a potential approach for the treatment of breast cancer bone metastasis.

SF-deferoxamine, a bone-seeking angiogenic drug, prevents bone loss in estrogen-deficient mice.

Deferoxamine (DFO) possesses a good chelating capability and is therefore used for the clinical treatment of ion deposition diseases. Increasing evidence shows that DFO can inhibit the activity of proline hydroxylase (PHD) by chelating iron, resulting in hypoxia-induced factor (HIF) signaling activation and angiogenesis promotion. However, clinical evidence indicates that a high concentration of DFO could be biotoxic due to its enrichment in related organs. Thus, we established a new compound by conjugating DFO with the bone-seeking agent iminodiacetic acid (IDA); the new agent is called SF-DFO, and we verified its promotion of HIF activation and tube formation in vivo. After confirming the bone-seeking property of SF-DFO in the femur and vertebra of both male and female mice and comparing it to that of DFO, we analyzed the protective effect of DFO and SF-DFO in an ovariectomized (OVX) mouse model. The serum CTX-I level revealed no influence of DFO and SF-DFO on osteoclast activity, but the blood vessels and osteoblasts in the metaphysis were more abundant after SF-DFO treatment, which resulted in a greater protective effect against trabecular bone loss compared to the DFO group. Additionally, the cortical parameters and bone strength performance were identical between the DFO and SF-DFO groups. However, the diffuse inflammatory response in the liver and spleen that occurred after DFO injection was not observed in the SF-DFO group. Thus, with reduced biotoxicity and an equivalent bone-seeking capability, SF-DFO may be a better choice for the prevention of vascular degradation-induced osteoporosis.

A Novel Rhein Derivative Modulates Bone Formation and Resorption and Ameliorates Estrogen-Dependent Bone Loss.

Osteoporosis, an osteolytic disease that affects millions of people worldwide, features a bone remodeling imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Identifying dual target-directed agents that inhibit excessive bone resorption and increase bone formation is considered an efficient strategy for developing new osteoporosis treatments. Rhein, a natural anthraquinone, can be isolated from various Asian herbal medicines. Rhein and its derivatives have been reported to have various beneficial pharmacological effects, especially their bone-targeting ability and anti-osteoclastogenesis activity. Moreover, hydrogen sulfide (H2 S) was reported to prevent ovariectomy- (OVX-) induced bone loss by enhancing bone formation, and sulfur replacement therapy has been considered a novel and plausible therapeutic option. Based on this information, we synthesized a rhein-derived thioamide (RT) and investigated its effects on bone resorption and bone formation in vitro and in vivo. It has been found that the RT-inhibited receptor activator of the nuclear factor-κB (NF-κB) ligand- (RANKL-) induced osteoclastogenesis and bone resorption in a dose-dependent manner. The expression of osteoclast marker genes was also suppressed by RT treatment. Furthermore, exploration of signal transduction pathways indicated that RT markedly blocked RANKL-induced osteoclastogenesis by attenuating MAPK pathways. However, RT treatment in an osteoblastic cell line, MC3TE-E1, indicated that RT led to an increase in the deposition of minerals and the expression of osteoblast marker genes, as demonstrated by Alizarin Red staining and alkaline phosphatase activity. Importantly, an OVX mouse model showed that RT could attenuate the bone loss in estrogen deficiency-induced osteoporosis in vivo with a smart H2 S-releasing property and that there was a considerable improvement in the biomechanical properties of bone. Accordingly, our current work highlights the dual regulation of bone remodeling by the rhein-derived molecule RT. This may be a highly promising approach for a new type of anti-osteoporosis agent. © 2018 American Society for Bone and Mineral Research.

Vascularized 3D printed scaffolds for promoting bone regeneration.

3D printed scaffolds hold promising perspective for bone tissue regeneration. Inspired by process of bone development stage, 3D printed scaffolds with rapid internal vascularization ability and robust osteoinduction bioactivity will be an ideal bone substitute for clinical use. Here, we fabricated a 3D printed biodegradable scaffold that can control release deferoxamine, via surface aminolysis and layer-by-layer assembly technique, which is essential for angiogenesis and osteogenesis and match to bone development and reconstruction. Our in vitro studies show that the scaffold significantly accelerates the vascular pattern formation of human umbilical endothelial cells, boosts the mineralized matrix production, and the expression of osteogenesis-related genes during osteogenic differentiation of mesenchymal stem cells. In vivo results show that deferoxamine promotes the vascular ingrowth and enhances the bone regeneration at the defect site in a rat large bone defect model. Moreover, this 3D-printed scaffold has excellent biocompatibility that is suitable for mesenchymal stem cells grow and differentiate and possess the appropriate mechanical property that is similar to natural cancellous bone. In summary, this 3D-printed scaffold holds huge potential for clinical translation in the treatment of segmental bone defect, due to its flexibility, economical friendly and practicality.

Estrogen inhibits osteoclasts formation and bone resorption via microRNA-27a targeting PPARγ and APC.

Inhibition of osteoclasts formation and bone resorption by estrogen is very important in the etiology of postmenopausal osteoporosis. The mechanisms of this process are still not fully understood. Recent studies implicated an important role of microRNAs in estrogen-mediated responses in various cellular processes, including cell differentiation and proliferation. Thus, we hypothesized that these regulatory molecules might be implicated in the process of estrogen-decreased osteoclasts formation and bone resorption. Western blot, quantitative real-time polymerase chain reaction, tartrate-resistant acid phosphatase staining, pit formation assay and luciferase assay were used to investigate the role of microRNAs in estrogen-inhibited osteoclast differentiation and bone resorption. We found that estrogen could directly suppress receptor activator of nuclear factor B ligand/macrophage colony-stimulating factor-induced differentiation of bone marrow-derived macrophages into osteoclasts in the absence of stromal cell. MicroRNA-27a was significantly increased during the process of estrogen-decreased osteoclast differentiation. Overexpressing of microRNA-27a remarkably enhanced the inhibitory effect of estrogen on osteoclast differentiation and bone resorption, whereas which were alleviated by microRNA-27a depletion. Mechanistic studies showed that microRNA-27a inhibited peroxisome proliferator-activated receptor gamma (PPARγ) and adenomatous polyposis coli (APC) expression in osteoclasts through a microRNA-27a binding site within the 3'-untranslational region of PPARγ and APC. PPARγ and APC respectively contributed to microRNA-27a-decreased osteoclast differentiation and bone resorption. Taken together, these results showed that microRNA-27a may play a significant role in the process of estrogen-inhibited osteoclast differentiation and function.

The inhibition of RANKL expression in fibroblasts attenuate CoCr particles induced aseptic prosthesis loosening via the MyD88-independent TLR signaling pathway.

Periprosthetic osteolysis and aseptic loosening are mainly caused by wear particles (Ps) that are generated from friction interfaces. However, the mechanisms underlying the development of aseptic loosening remain unclear. Therefore, we aimed toclarify how the myeloid differentiation factor 88 (MyD88)-independent Toll-like receptor (TLR) signaling pathway mediates cobalt and chromium (CoCr)-Ps-induced osteolysis. We quantified the expression levels of TLRs, MyD88, RANKL, and inflammatory factors in patients experiencing aseptic loosening after primary total hip arthroplasty (THA) with metal-on-metal (MoM) bearings and hip osteoarthritis (hOA). We observed the in vitro and in vivo levels of RANKL, TLRs, and MyD88 in fibroblasts challenged with CoCr Ps by applying shMyD88 interference lentivirus vectors to block the MyD88-independent TLR pathway. The levels of TLRs, MyD88, RANKL, and inflammatory factors in the revision THA (rTHA) with MoM group were higher than those in the hOA group. Our data collectively revealed that inhibiting MyD88 expression could reduce osteoclastogenesis in vitro and CoCr-Ps-induced osteolysis in vivo. Our findings suggested that osteoclastogenesis is promoted by the CoCr-Ps-induced expression of RANKL in fibroblasts and that MyD88 is a potential target in the treatment of wear Ps-induced osteolysis.

The prevention of latanoprost on osteoclastgenesis in vitro and lipopolysaccharide-induced murine calvaria osteolysis in vivo.

Identification of agents that inhibit osteoclast formation and function is important for the treatment of osteolytic diseases which feature excessive osteoclast formation and bone resorption. Latanoprost (LTP), an analog of prostaglandin F2α, is a medication which works to lower pressure inside the eyes. Prostaglandin F2α was reported to regulate bone metabolism, however, the effect of LTP in osteoclastogenesis is still unknown. Here, we found that LTP suppressed RANKL-induced osteoclastogenesis in a dose-dependent manner as illustrated by TRAP activity and TRAP staining. In addition, the osteoclast function was also reduced by LTP treatment, as indicated in less osteoclastic resorption pit areas. Furthermore, LTP inhibited the mRNA expressions of osteoclast marker genes such as TRAP and cathepsin K. In order to illustrate its molecular mechanism, we examined the changing of mRNA and protein levels of NFATc1 and c-fos by LTP treatment, as well as the phosphorylation of ERK, AKT, JNK, and p38. The results suggested that LTP inhibited RANKL-induced osteoclastgenesis and function by inhibiting ERK, AKT, JNK, and p38 cascade, following by the c-fos/NFATc1 pathway. In agreement with in vitro results, using an in vivo lipopolysaccharide-induced murine calvaria osteolysis mouse model, we found that administration of LTP was able to reverse the lipopolysaccharide-induced bone loss. Together, these data demonstrated that LTP attenuated the bone loss in lipopolysaccharide-induced murine calvaria osteolysis mice through inhibiting osteoclast formation and function. Our study thus provided the evidences that LTP was a potential treatment option against osteolytic bone diseases.

Desferrioxamine reduces ultrahigh-molecular-weight polyethylene-induced osteolysis by restraining inflammatory osteoclastogenesis via heme oxygenase-1.

As wear particles-induced osteolysis still remains the leading cause of early implant loosening in endoprosthetic surgery, and promotion of osteoclastogenesis by wear particles has been confirmed to be responsible for osteolysis. Therapeutic agents targeting osteoclasts formation are considered for the treatment of wear particles-induced osteolysis. In the present study, we demonstrated for the first time that desferrioxamine (DFO), a powerful iron chelator, could significantly alleviate osteolysis in an ultrahigh-molecular-weight polyethylene (UHMWPE) particles-induced mice calvaria osteolysis model. Furthermore, DFO attenuated calvaria osteolysis by restraining enhanced inflammatory osteoclastogenesis induced by UHMWPE particles. Consistent with the in vivo results, we found DFO was also able to inhibit osteoclastogenesis in a dose-dependent manner in vitro, as evidenced by reduction of osteoclasts formation and suppression of osteoclast specific genes expression. In addition, DFO dampened osteoclasts differentiation and formation at early stage but not at late stage. Mechanistically, the reduction of osteoclastogenesis by DFO was due to increased heme oxygenase-1 (HO-1) expression, as decreased osteoclasts formation induced by DFO was significantly restored after HO-1 was silenced by siRNA, while HO-1 agonist COPP treatment enhanced DFO-induced osteoclastogenesis inhibition. In addition, blocking of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway promoted DFO-induced HO-1 expression, implicating that p38 signaling pathway was involved in DFO-mediated HO-1 expression. Taken together, our results suggested that DFO inhibited UHMWPE particles-induced osteolysis by restraining inflammatory osteoclastogenesis through upregulation of HO-1 via p38MAPK pathway. Thus, DFO might be used as an innovative and safe therapeutic alternative for treating wear particles-induced aseptic loosening.

Synthesis and biological evaluation of rhein amides as inhibitors of osteoclast differentiation and bone resorption.

Approaches of targeting excessive activation and differentiation of osteoclasts were considered as an effective treatment option for osteoporosis or osteopenia. In the present work, a series of rhein derivatives were synthesized and employed for their cytotoxicity screening against bone marrow-derived macrophages cells (BMMs) and their inhibition effects on osteoclasts activation and differentiation in vitro using an MTT assay and a TRAP activity assay respectively. Two rhein derivatives d6 and d11 inhibited BMMs activation and differentiation with 98% and 85% inhibitory activity respectively, without showing any cytotoxicity on BMMs. Subsequently, the most potent compound d6 was further validated for its inhibitory effects on the formation of TRAP-positive multinucleated cells and bone resorption as evaluated by TRAP staining and bone resorption assay. The regulation by d6 of osteoclast marker genes assay revealed that treatment of BMMs with M-CSF and RANKL resulted in the stimulation of mRNA expressions of NFATc1, c-fos, TRAP, MMP-9 and cathepsin K which were highly related with osteoclast activation and differentiation, while d6 decreased mRNA expressions of these genes. It was indicated that d6 might regulate osteoclasts activity through RANKL/RANK/NFATc1 pathway. Thus our current work is expected to provide a highly promising approach for the development of a new type of anti-osteoporosis agent.

Activation of HIFa pathway in mature osteoblasts disrupts the integrity of the osteocyte/canalicular network.

The hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, are the central mediators of the homeostatic response that enables cells to survive and differentiate in low-oxygen conditions. Previous studies indicated that disruption of the von Hippel-Lindau gene (Vhl) coincides with the activation of HIFα signaling. Here we show that inactivation of Vhl in mature osteoblasts/osteocytes induces their apoptosis and disrupts the cell/canalicular network. VHL-deficient (ΔVHL) mice exhibited a significantly increased cortical bone area resulting from enhanced proliferation and osteogenic differentiation of the bone marrow stromal cells (BMSCs) by inducing the expression of ß-catenin in the BMSC. Our data suggest that the VHL/HIFα pathway in mature osteoblasts/osteocytes plays a critical role in the bone cell/canalicular network and that the changes of osteocyte morphology/function and cell/canalicular network may unleash the bone formation, The underlying mechanism of which was the accumulation of ß-catenin in the osteoblasts/osteoprogenitors of the bone marrow.

MicroRNA-17/20a inhibits glucocorticoid-induced osteoclast differentiation and function through targeting RANKL expression in osteoblast cells.

Glucocorticoids act on the osteoblasts to up-regulate the expression of RANKL, which is very important in the etiology of glucocorticoid-induced osteoclast differentiation and bone resorption. The mechanisms of this process are still not completely understood. Recent studies have shown that glucocorticoids mediate osteoblast function by decreasing the expression of microRNA-17-92a cluster. Coincidentally, we found that the microRNA-17/20a (microRNA-17, microRNA-20a) seed sequences were also complementary to a sequence conserved in the 3'- untranslated region of RANKL mRNA. Therefore, we hypothesized that glucocorticoids might promote osteoblast-derived RANKL expression by down-regulating microRNA-17/20a, which favors differentiation and function of the osteoclasts. In the present study, Western blot analysis showed that microRNA-17/20a markedly lowered the levels of RANKL protein and attenuated dexamethasone-induced RANKL expression in the osteoblasts. The post-transcriptional repression of RANKL by microRNA-17/20a was further confirmed by the luciferase reporter assay. Furthermore, we found that dexamethasone-induced osteoclast differentiation and function were significantly attenuated in co-culture with osteoblast over-expressed microRNA-17/20a and osteoclast progenitors. These results showed that microRNA-17/20a may play a significant role in glucocorticoid-induced osteoclast differentiation and function by targeting the RANKL expression in osteoblast cells.

Bone selective protective effect of a novel bone-seeking estrogen on trabecular bone in ovariectomized rats.

The drawbacks of estrogen restrict the clinical use of hormone replacement therapy, and it would be most helpful to explore new estrogenic substances that could prevent bone loss and be free from any adverse effects. We synthesized a new compound named bone-seeking estrogen (SE2) by combining 17ß-estradiol (E2) with iminodiacetic acid through the Mannich reaction. E2 and SE2 were labeled with isotope (3)H, and the tissue distribution tests of E2-(3)H and SE2-(3)H were analyzed by the radioactivity. The specific nuclear binding of E2 and SE2 in osteoblasts was measured. SE2 exhibited significantly greater affinity for bone but lower affinity for ovary and uterus than did E2, and SE2 maintained a high affinity for the estrogen receptor alpha similar to that of E2. SE2 administration did not induce uterine hypertrophy. Body weight increase was significantly suppressed by treatment with E2 but not by SE2 after ovariectomy (OVX). SE2 decreased bone turnover as E2 after OVX detected by serum biochemical markers. Bone histology and micro-CT analysis revealed that SE2 administration, similar to E2, could improve bone mass and trabecular architecture after OVX. Biomechanical analyses showed that SE2 treatment effectively increased mechanical properties after OVX. The results suggested that SE2 was effective in preventing OVX-induced bone loss and exhibited few side effects on body weight and uterine hypertrophy, which was beneficial in reducing the adverse effects caused by E2. SE2 may be a better choice than E2 for the prevention of postmenopausal osteoporosis.

MicroRNA-17-92a upregulation by estrogen leads to Bim targeting and inhibition of osteoblast apoptosis.

Anti-apoptotic effects of estrogen on osteoblasts are very important in the etiology of estrogen protection of the adult skeleton against bone loss. The mechanisms of this process are still not fully understood. Recent studies implicated an important role of microRNAs in estrogen-mediated responses in various cellular processes, including cell apoptosis and proliferation. Therefore, we hypothesized that these regulatory molecules might be involved with estrogen in protecting osteoblasts from apoptosis. Western blotting, quantitative real-time PCR, flow cytometry and luciferase assays were employed to investigate the role of microRNAs in this process. The microRNA cluster miR-17-92a, a post-transcriptional regulator, was significantly reduced during dexamethasone, etoposide and tumor necrosis factor alpha (TNF-α)-induced osteoblasts apoptosis. The repression of miR-17-92a was significantly attenuated by estrogen. To delineate the role of miR-17-92a in apoptosis, we silenced and overexpressed miR-17-92a in osteoblasts. We found that miR-17-92a depletion significantly enhanced dexamethasone-induced apoptosis and overexpressing miR-17-92a remarkably increased the anti-apoptotic effects of estrogen on osteoblasts. Mechanistic studies showed that miR-17-92a inhibited Bim expression through a microRNA-17-92a-binding site within the 3'-untranslated region of Bim. The post-transcriptional repression of Bim was further confirmed by a luciferase reporter assay. These results showed that miR-17-92a, plays a significant role in the process of estrogen protection of osteoblasts against apoptosis, by regulating Bim expression.