Bone Protective Effects Research Articles

A Network Pharmacology-Based Study on Vital Pharmacological Pathways and Targets of Eucommiae Cortex Acting on Osteoporosis

Background Eucommiae Cortex is a Chinese herbal medicine with bone protective effects and treats osteoporosis. This study aimed to explore the pharmacological mechanisms of this complex mixture. Methods The active compounds and disease targets involved in the study were obtained from publicly available websites and databases. Core target genes were identified by protein-protein interaction (PPI) network and topology analysis and mapped to critical components by a “component-target” regulatory network. Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis and Gene Ontology (GO)analysis were performed to analyze the biological processes of target genes. Moreover, we carried out molecular docking, cell experiments, and quantitative real-time PCR to propel the research forward. Results Eucommiae Cortex contained 28 active ingredients and 85 potential targets for antiosteoporosis. PPI networks and topology analysis screened 17 core target genes. The therapeutic mechanism involves a series of biological reactions, including host-virus response, chemical stress, oxidative stress, and cell cycle control. The KEGG enrichment illustrated that the MAPK signaling pathway might play a significant role. The final experiment detected ten genes (EGF, AKT1, JUN, MAPK8, MAPK1, CASP3, FOS, VEGFA, EGFR, and MYC) and three compounds (quercetin, kaempferol, and beta- carotene) in the MAPK pathway. Firstly, the CCK-8 and ALP activity test results showed that three compounds could enhance the proliferation and differentiation of osteoblast-like MC3T3-E1 cells. Secondly, molecular docking confirmed the favorable binding potential. Subsequently, we observed that adding 1∗10−6 mol/L quercetin, 1∗10−5 mol/L kaempferol, and 1∗10−5 mol/L beta-carotene activated the ERK/JNK cascades and the heterodimer complex AP-1(Fos/Jun) in the MAPK pathway. Conclusion MAPK pathway might provide an essential mechanism for the antiosteoporosis effect of Eucommiae Cortex. Eucommiae Cortex and its active ingredients have the potential to treat osteoporosis.

Identification of PDXDC1 as a novel pleiotropic susceptibility locus shared between lumbar spine bone mineral density and birth weight

An increasing number of epidemiological studies have suggested that birth weight (BW) may be a determinant of bone health later in life, although the underlying genetic mechanism remains unclear. Here, we applied a pleiotropic conditional false discovery rate (cFDR) approach to the genome-wide association study (GWAS) summary statistics for lumbar spine bone mineral density (LS BMD) and BW, aiming to identify novel susceptibility variants shared between these two traits. We detected 5 novel potential pleiotropic loci which are located at or near 7 different genes (NTAN1, PDXDC1, CACNA1G, JAG1, FAT1P1, CCDC170, ESR1), among which PDXDC1 and FAT1P1 have not previously been linked to these phenotypes. To partially validate the findings, we demonstrated that the expression of PDXDC1 was dramatically reduced in ovariectomized (OVX) mice in comparison with sham-operated (SHAM) mice in both the growth plate and trabecula bone. Furthermore, immunohistochemistry assay with serial sections showed that both osteoclasts and osteoblasts express PDXDC1, supporting its potential role in bone metabolism. In conclusion, our study provides insights into some shared genetic mechanisms for BMD and BW as well as a novel potential therapeutic target for the prevention of OP in the early stages of the disease development.Key messagesWe investigated pleiotropy-informed enrichment between LS BMD and BW.We identified genetic variants related to both LS BMD and BW by utilizing a cFDR approach.PDXDC1 is a novel pleiotropic gene which may be related to both LS BMD and BW.Elevated expression of PDXDC1 is related to higher BMD and lower ratio n-6/n-3 PUFA indicating a bone protective effect of PDXDC1.

Asiatic Acid Attenuates Osteoporotic Bone Loss in Ovariectomized Mice Through Inhibiting NF-kappaB/MAPK/ Protein Kinase B Signaling Pathway.

Osteoporosis is a condition associated with osteolytic bone disease that is primarily characterized by inordinate osteoclast activation. Protein kinase B (Akt) pathways activated by receptor activator of nuclear factor kappa-B ligand (RANKL) are essential for osteoclastogenesis. Asiatic acid (AA) is a natural pentacyclic triterpenoid compound extracted from a traditional Chinese herb that exhibits a wide range of biological activities. AA has been found to alleviate the hypertrophic and fibrotic phenotype of chondrocytes via the Akt signaling pathway. In this study, we investigated whether AA alleviated bone loss by inhibiting the Akt signaling pathway during osteoclastogenesis and its effect on osteoblasts. The effect of AA cytotoxicity on mouse bone marrow-derived macrophages/monocytes (BMMs) was evaluated in vitro using a Cell Counting Kit-8 assay. The effects of AA on osteoclast differentiation and function were detected using tartrate-resistant acid phosphatase (TRAP) staining and a pit formation assay. A Western blot and qRT-PCR were conducted to evaluate the expression of osteoclast-specific genes and protein signaling molecules. In addition, alkaline phosphatase and alizarin red staining were performed to assess osteoblast differentiation and mineralization. The bone protective effect of AA was investigated in vivo using ovariectomized mice. we found that AA could dose-dependently inhibit RANKL-induced osteoclastogenesis. Moreover, the pit formation assay revealed that osteoclast function was suppressed by treatment with AA. Moreover, the expression of osteoclast-specific genes was found to be substantially decreased during osteoclastogenesis. Analysis of the molecular mechanisms showed that AA could inhibit NF-kappaB/MAPK/Akt signaling pathway, as well as the downstream factors of NFATc1 in the osteoclast signaling pathway activated by RANKL. However, AA did not significantly promote osteoblast differentiation and mineralization. The in vivo experiments suggested that AA could alleviate ovariectomy-induced bone loss in ovariectomized mice. Our results demonstrate that AA can inhibit osteoclastogenesis and prevent ovariectomy-induced bone loss by inhibiting the NF-kappaB/MAPK/Akt signaling pathway. The discovery of the new molecular mechanism that AA inhibits osteoclastogenesis provides essential evidence to support the use of AA as a potential drug for the treatment of osteoclast-related diseases.

GIPR rs10423928 and bone mineral density in postmenopausal women in Shanghai.

We demonstrated previously that there is a correlation between glucagon-like peptide-1 (GLP-1) single-nucleotide polymorphism (SNP) and bone mineral density in postmenopausal women. Both GLP-1 and glucose-dependent insulinotropic peptide are incretins. The glucose-dependent insulinotropic peptide receptor (GIPR) SNP rs10423928 has been extensively studied. However, it is not clear whether GIPR gene mutations affect bone metabolism. The aim of this study was to investigate the association between rs10423928 and bone mineral density in postmenopausal women in Shanghai. rs10423928 was detected in 884 postmenopausal women in Shanghai, and the correlation between the GIPR SNP and bone mineral density was assessed. The dominant T/T genotype of rs10423928 was found to be related to the bone mineral density of the femoral neck (P = 0.035). Overall, our findings indicate that the dominant T/T genotype of rs10423928 in postmenopausal women is significantly associated with a higher bone mineral density and that the T/T genotype exerts a bone-protective effect.

Sheep bone collagen peptide ameliorates osteoporosis by regulating RANK/RANKL/OPG signal pathway

Sheep bone collagen peptide (SBCP) has attracted attention due to its potential effects on bone health. This study aims to explore the bone protective effect of SBCP in the presence of estrogen deficiency. Ovariectomized (OVX) female rats were given different dose rates (0, 0.68, 2.05, 3.40 g/kg BW) of SBCP for 8 consecutive weeks. At the end of the treatment, the serological indexes and tibial mineral content were measured. The microstructure and morphological changes of femur bones were observed by scanning electron microscopy (SEM) and hematoxylin and eosin (H & E) staining. Femur bones were harvested for determination of expression level of RANK, RANKL, OPG by molecular biological techniques. 3.40 g/kg BW SBCP treatment decreased the PINP and β-CTx levels but increased the tibial calcium and phosphorus contents significantly in estrogen deficient rats. Expression of RANK and RANKL decreased however expression of OPG increased in the bone of estrogen deficient rats which received 3.40 g/kg BW SBCP with greater effects than SBCP-M and SBCP-L treatments. SBCP helps to overcome the adverse effect of estrogen deficiency the bone and thus this nutriment could potentially be used for the treatment and prevention of osteoporosis in postmenopausal women.

The Neuropeptide VIP Limits Human Osteoclastogenesis: Clinical Associations with Bone Metabolism Markers in Patients with Early Arthritis.

We aimed to evaluate the direct action of VIP on crucial molecules involved in human osteoclast differentiation and function. We also investigated the relationship between VIP serum levels and bone remodeling mediators in early arthritis patients. The expression of VIP receptors and osteoclast gene markers in monocytes and in vitro differentiated osteoclasts was studied by real-time PCR. NFATc1 activity was measured using a TransAM® kit. Osteoclastogenesis was confirmed by quantification of tartrate-resistant acid phosphatase positive multinucleated cells. OsteoAssay® Surface Multiple Well Plate was used to evaluate bone-resorbing activity. The ring-shaped actin cytoskeleton and the VPAC1 and VPAC2 expression were analyzed by immunofluorescence. We described the presence of VIP receptors in monocytes and mature osteoclasts. Osteoclasts that formed in the presence of VIP showed a decreased expression of osteoclast differentiation gene markers and proteolytic enzymes involved in bone resorption. VIP reduced the resorption activity and decreased both β3 integrin expression and actin ring formation. Elevated serum VIP levels in early arthritis patients were associated with lower BMD loss and higher serum OPG concentration. These results demonstrate that VIP exerts an anti-osteoclastogenic action impairing both differentiation and resorption activity mainly through the negative regulation of NFATc1, evidencing its bone-protective effects in humans.

A Review on the Ethnomedicinal Usage, Phytochemistry, and Pharmacological Properties of Gentianeae (Gentianaceae) in Tibetan Medicine.

Gentianaceae is a large plant family and is distributed worldwide. As the largest tribe in Gentianaceae, Gentianeae contains 939–968 species, and the Qinghai-Tibet Plateau and adjacent areas are the main centers of diversity for Gentianeae. Species from the Gentianeae are widely used in traditional Tibetan medicine. In this review, a systematic and constructive overview of the information on botany, ethnomedicinal usage, phytochemistry, and pharmacological properties of Gentianeae in Tibetan medicine is provided. The results of this study are based on a literature search, including electronic databases, books, websites, papers, and conference proceedings. Botanical studies showed that Gentianeae includes the subtribe Gentianeae and Swertiinae, and several new genera and taxa have been identified. Approximately 83 species from Gentianeae were used in Tibetan medicine, among which Gentiana and Swertia constituted the largest number of species with 42 and 24 species, respectively. The species from Gentianeae are mainly used as Bangjian (སྤང་རྒྱན།), Jieji (ཀྱི་ལྕེ།), Dida (ཏིག་ཏ།), and Ganggaqiong (གང་གྰཆུང་།) in Tibetan medicine with different clinical applications. More than 240 formulas were found containing Gentianeae species with different attending functions. Phytochemical studies showed that the main active components of Gentianeae species are iridoids, xanthones, flavonoids, and triterpenoids. The bioactivities of plants from Gentianeae include hepatic protection, upper respiratory tract protection, joint and bone protection, glucose regulation, antibacterial, antioxidant, anticancer, and antiviral effects. This review will provide a reference for future research on natural resource protection, plant-based drug development, and further clinical investigation.

Effects of 12 Months Consumption of 100 g Dried Plum (Prunes) on Bone Biomarkers, Density, and Strength in Men.

Several male animal studies have demonstrated bone-protective effects of dried plum; however, no human male study has evaluated the effect of dried plum on bone health. We conducted a randomized controlled clinical study to test if daily inclusion of 100 g of dried plum in the diet positively influenced bone mineral density (BMD), bone strength, and bone biomarkers in men. Sixty-six men were randomly assigned to one of two daily treatment groups: (1) control (0 g dried plum) or (2) 100 g dried plum. Blood samples were collected at baseline and after 3, 6, and 12 months to assess bone biomarkers. Bone was measured at baseline and after 6 and 12 months via dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. Tartrate-resistant acid phosphatase-5b (TRAP5b) and C-terminal collagen cross-link (CTX) levels decreased significantly in the dried plum group at 3-, 6-, and 12-month intervals compared with baseline. No changes were observed in the control group for TRAP5b and CTX levels. Bone-specific alkaline phosphatase levels decreased significantly after 6 and 12 months in the control and dried plum groups. BMD for total body, spine (L1-L4), hip, and ulna did not change in the control and dried plum groups from baseline to 6 or 12 months. In the proximal tibia, endosteal circumferences increased significantly within the dried plum group during the course of treatment. The results suggest that daily consumption of 100 g dried plum for 12 months has modest bone-protective effects in men. ClinicalTrials.gov identifier: NCT04720833.

An emerging role of Prevotella histicola on estrogen deficiency–induced bone loss through the gut microbiota–bone axis in postmenopausal women and in ovariectomized mice

The gut microbiota (GM)-bone axis has emerged as a crucial mediator of bone homeostasis. Estrogen deficiency-induced bone loss is closely associated with an altered GM. However, the underlying mechanisms remain unclear. We sought to explore the putative effects of GM on estrogen deficiency-induced bone loss and determine a potential mechanism. Fecal samples collected from postmenopausal women with osteoporosis (PMO) and with normal bone mass (PMN) were examined by 16S ribosomal RNA (rRNA) gene sequencing and analysis. Prevotella histicola, a typical species of Prevotella, was orally given to female C57BL6/J mice after ovariectomy [ovariectomized (OVX)]. The primary outcomes were changes in bone microstructures as measured by micro-computed tomography scanning and bone histomorphometry analysis. Secondary outcomes included changes in osteoclast activity, the expression of osteoclastogenic cytokines, and gut permeability, which were measured by ELISA, qRT-PCR, western blot, and immunofluorescence. As demonstrated through 16S rRNA gene sequencing and analysis, the GM in the PMO group featured a significantly decreased proportion of the genus Prevotella in comparison with that in the PMN group (∼60%, P<0.05). In animal experiments, P. histicola-treated OVX mice maintained a relatively higher bone volume than OVX controls. Mechanistically, the protective effects of P. histicola on bone mass were found to be associated with its modulation of gut permeability as well as its inhibitory effects on osteoclast activity which function by attenuating osteoclastogenic cytokine expression. The GM diversity and composition between the PMN and PMO groups were significantly different. In particular, the proportion of the genus Prevotella was notably higher in the PMN group, demonstrating its potential bone-protective effects on osteoporosis. Further animal study using osteoporotic mice showed P. histicola could prevent estrogen deficiency-induced bone loss through the GM-bone axis. Thus, P. histicola may serve as a therapeutic agent or target for osteoporosis treatment.

Magnesium Picolinate Improves Bone Formation by Regulation of RANK/RANKL/OPG and BMP-2/Runx2 Signaling Pathways in High-Fat Fed Rats.

Magnesium (Mg) deficiency may affect bone metabolism by increasing osteoclasts, decreasing osteoblasts, promoting inflammation/oxidative stress, and result in subsequent bone loss. The objective of the present study was to identify the molecular mechanism underlying the bone protective effect of different forms of Mg (inorganic magnesium oxide (MgO) versus organic magnesium picolinate (MgPic) compound) in rats fed with a high-fat diet (HFD). Forty-two Wistar albino male rats were divided into six group (n = 7): (i) control, (ii) MgO, (iii) MgPic, (iv) HFD, (v) HFD + MgO, and (vi) HFD + MgPic. Bone mineral density (BMD) increased in the Mg supplemented groups, especially MgPic, as compared with the HFD group (p < 0.001). As compared with the HFD + MgO group, the HFD + MgPic group had higher bone P (p < 0.05) and Mg levels (p < 0.001). In addition, as compared to MgO, MgPic improved bone formation by increasing the levels of osteogenetic proteins (COL1A1 (p < 0.001), BMP2 (p < 0.001), Runx2 (p < 0.001), OPG (p < 0.05), and OCN (p < 0.001), IGF-1 (p < 0.001)), while prevented bone resorption by reducing the levels of RANK and RANKL (p < 0.001). In conclusion, the present data showed that the MgPic could increase osteogenic protein levels in bone more effectively than MgO, prevent bone loss, and contribute to bone formation in HFD rats.

Sema3A accelerates bone formation during distraction osteogenesis in mice

ABSTRACT Distraction osteogenesis (DO) is a bone regeneration technique used to treat maxillofacial disorders, fracture nonunion, and large bone defects. It is well known for its amazing regenerative potential, but an extended consolidation period limits its clinical use. The interaction between the nervous system and bone regeneration has attracted great attention in recent years. Sema3A is a key axonal chemorepellent which has been proved to have bone-protective effects. In this article, we try to improve DO by local administration of Sema3A and explore the possible mechanisms. Forty wildtype, male, adult mice were divided into two groups after tibia osteotomy surgery. Sema3A or Saline was daily injected transcutaneous into the center of the distraction zone during the consolidation period. Micro-CT images were taken at 4, 6,8 and 10 weeks post-surgery; vascular density and biomechanical testing were performed at 10 weeks post-surgery. We also set up in vitro vessel growth assay to evaluate the effect of Sema3A on angiogenesis. Compared with the Saline group, Sema3A treatment significantly accelerated bone regeneration, improved angiogenesis and callus’ biomechanical strength. At 10 weeks post-surgery, compared with the Saline group, the BV/TV, BMD, TMD increased by about 23%, 22%, 18% respectively, vascular density increased by about 49% in the Sema3A group. Histological images and western-blot showed decreased expression of VEGF-A and increased expression of Ang-1 at 4 weeks post-surgery in the Sema3A group. In vitro, Sema3A suppressed VEGF-induced angiogenesis but had little effect on Ang-induced angiogenesis. Conclusion: Sema3A could accelerate bone regeneration and improve angiogenesis during DO.

Modulation of NFκB signalling pathway by tocotrienol: A systematic review.

Tocotrienols have been reported to exert anticancer, anti-inflammatory, antioxidant, cardio-protective and boneprotective effects through modulation of NFκB signalling pathway. The objective of this systematic review is to evaluate available literature showing the effect of tocotrienols on NFκB signalling pathway and identify the potential mechanisms involved. A comprehensive search was conducted using PubMed and SCOPUS databases using the keywords "tocotrienol" and "NFκB" or "nuclear factor kappa b". Main inclusion criteria were English language original articles showing the effect of tocotrienol on NFκB signalling pathway. Fifty-nine articles were selected from the total of 117 articles initially retrieved from the literature search. Modulation of regulatory proteins and genes such as inhibition of farnesyl prenyl transferase were found to be the mechanisms underlying the tocotrienol-induced suppression of NFκB activation.

Prenylated Isoflavonoids-Rich Extract of Erythrinae Cortex Exerted Bone Protective Effects by Modulating Gut Microbial Compositions and Metabolites in Ovariectomized Rats.

Flavonoids, found in a wide variety of foods and plants, are considered to play an important role in the prevention and treatment of osteoporosis. Our previous studies demonstrated that Erythrina cortex extract (EC) rich in prenylated isoflavonoids exerted bone protective effects in ovariectomized (OVX) rats. The present study aimed to investigate the interactions of gut microbiota with the EC extract to explore the underlying mechanisms involved in its beneficial effects on bone. Sprague-Dawley female rats of 3-months-old were ovariectomized and treated with EC extract for 12 weeks. EC extract reversed ovariectomy-induced deterioration of bone mineral density and bone microarchitecture as well as downregulated cathepsin K (Ctsk) and upregulated runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) in the tibia of OVX rats. Its protective effects on bone were correlated with changes in microbial richness and the restorations of several genera. EC increased the serum circulating levels of acetate and propionate in OVX rats. We conclude that the bone protective effects of EC extract were associated with the changes in microbial compositions and serum short chain fatty acids (SCFAs) in OVX rats.

Co-Treatment of Purified Annatto Oil (Bixa orellana L.) and Its Granules (Chronic®) Improves the Blood Lipid Profile and Bone Protective Effects of Testosterone in the Orchiectomy-Induced Osteoporosis in Wistar Rats.

This study aimed to evaluate and compare the effects of co-treatment with purified annatto oil (PAO) or its granules (GRA, Chronic®) with that of testosterone on the orchiectomy-induced osteoporosis in Wistar rats. After surgery, rats were treated from day 7 until day 45 with testosterone only (TES, 7 mg/kg, IM) or TES + PAO or GRA (200 mg/kg, p.o.). The following parameters were evaluated: food/water intake, weight, HDL, LDL, glucose, triglycerides (TG), total cholesterol (TC), alkaline phosphatase levels, blood phosphorus and calcium contents, femur weight, structure (through scanning electron microscopy), and calcium content (through atomic absorption spectrophotometry). Our results show that orchiectomy could significantly change the blood lipid profile and decrease bone integrity parameters. Testosterone reposition alone could improve some endpoints, including LDL, TC, bone weight, and bone calcium concentration. However, other parameters were not significantly improved. Co-treatment with PAO or GRA improved the blood lipid profile and bone integrity more significantly and improved some endpoints not affected by testosterone reposition alone (such as TG levels and trabeculae sizes). The results suggest that co-treatment with annatto products improved the blood lipid profile and the anti-osteoporosis effects of testosterone. Overall, GRA had better results than PAO.

Marantodes pumilum Var Alata (Kacip Fatimah) ameliorates derangement in RANK/RANKL/OPG pathway and reduces inflammation and oxidative stress in the bone of estrogen-deficient female rats with type-2 diabetes

BackgroundM. pumilum has been claimed to protect the bone against the adverse effect of estrogen deficiency. Additionally, it also exhibits anti-diabetic activity. In view of these, this study aims to identify the mechanisms underlying the bone protective effect of M. pumilum in the presence of both estrogen deficiency and diabetes mellitus (DM). MethodsOvariectomized, diabetic female rats were given M. pumilum leave aqueous extract (MPLA) (50 and 100 mg/kg/day), estrogen, glibenclamide and estrogen plus glibenclamide for 28 consecutive days. At the end of the treatment, fasting blood glucose (FBG), serum insulin, Ca2+, PO43− and bone alkaline phosphatase (BALP) levels were measured. Rats were sacrificed and femur bones were harvested for determination of expression level and distribution of RANK, RANKL, OPG and oxidative stress and inflammatory proteins by molecular biological techniques. Results100 mg/kg/day MPLA treatment decreased the FBG and BALP levels but increased the serum insulin, Ca2+ and PO43− levels in estrogen deficient, diabetic rats. Expression and distribution of RANKL, NF-κB p65, IKKβ, IL-6, IL-1β and Keap-1 decreased however expression and distribution of RANK, OPG, BMP-2, Type-1 collagen, Runx2, TRAF6, Nrf2, NQO-1, HO-1, SOD and CAT increased in the bone of estrogen deficient, diabetic rats which received 100 mg/kg/day MPLA with greater effects than estrogen-only, glibenclamide-only and estrogen plus glibenclamide treatments. ConclusionMPLA helps to overcome the adverse effect of estrogen deficiency and DM on the bone and thus this herb could potentially be used for the treatment and prevention of osteoporosis in postmenopausal women with diabetes.

The Novel Subtype-Selective Histone Deacetylase (HDAC) Inhibitor, Chidamide, Exerts Dual Anti-Myeloma and Bone Protective Effect in Vitro and In Vivo

Background and Purpose: Despite the advance in multiple myeloma treatment, drug resistance is still inevitable and the prognosis of most patients remains poor. Chidamide, a new subtype-selective histone deacetylase (HDAC) inhibitor, which is produced independently in China, is designed to inhibit the activity of HDAC 1, 2, 3 and 10. In the present work, we investigated the effect of chidamide on multiple myeloma and its associated osteolytic bone disease with different models.Experiment Design: In vitro cytotoxicity of chidamide was tested on myeloma cell lines (MM.1s, CAG, ARP-1, 8226, U266, H929, LP-1, ANBL-6, OPM2) with CCK-8 and flow cytometry. Synergistic combinations with standard drugs in 8226 and CAG cell lines were analyzed by compusyn software. The effect on osteoclast formation was assessed in osteoclastic cultures. The mechanisms of chidamide were explored by western blot and immunofluorescence. Murine models were employed for in vivo works, IVIS in vivo imaging system, ELISA (human Ig lambda, CTX-I, PINP) and micro-CT were used to surveillance the tumor burden and examine the bone destruction.Results: The cytotoxic effect of chidamide on myeloma cells is due to induction of cell apoptosis and cell cycle disruption (G1 phase arrest) in a dose dependent manner (IC50 varies from 2 to 5 uM in different cell lines) which mediated by increasing Cleaved Caspase-3, Caspase-7, Caspase-8, Caspase-9, p21 and p27, decreasing C-Myc, Mcl-1, Bcl-xL, CDK4 and CDK6. SensitiveSOCS3/JAK2/STAT3 cell signaling was involved in regulating these proteins by increasing the expression of SOCS3, reducing the expression of phospho-JAK2 and phospho-STAT3, thus inhibiting the proliferation and inducing cell cycle disruption. After treating with chidamide, the expression levels of HDAC1, 2, 3, 10 were not changed significantly, which indicated chidamide suppressed the function of these targets while not affected their expression. Notably, chidamide exerted synergistic anti-myeloma effect with dexamethasone, carfilzomib or pomalidomide (combination index < 1). Also, chidamide showed effective cytotoxicity in the co-culture of myeloma cells with bone marrow stromal cells and osteoclasts. Importantly, chidamide suppressed in vitro osteoclast formation and resorption, inhibited the expression of the key molecules (Cathepsin K, c-fos, NFATc1) and de-phosphorylation of p-AKT and p-JNK, and disrupted the F-acting ring formation. Finally, chidamide significantly reduced the tumor volume in the subcutaneous xenografts models, also the level of monoclonal protein Ig lambda secreted by myeloma cells decreased in disseminated myeloma murine models after treatment. Moreover, chidamide not only prevented tumor-associated bone loss in the disseminated murine model which was partially related with reducing the tumor burden but also had a direct bone protective effect on RANKL-induced rapid bone loss in non-tumor bearing mouse model with serum PINP (bone formation) and trabecular numbers increased while serum CTX-I (bone resorption) and trabecular space diminished compared with the vehicle group.Conclusion: Our work demonstrates dual anti-myeloma and bone protective effect of chidamide in vitro and in vivo, and shows its synergistic anti-myeloma effect with dexamethasone, carfilzomib and pomalidomide. These findings strongly suggest the potential clinical use of this drug in multiple myeloma in the near future. DisclosuresNo relevant conflicts of interest to declare.

Final results of the randomized phase 2 LEO trial and bone protective effects of everolimus for premenopausal hormone receptor-positive, HER2-negative metastatic breast cancer.

The phase 2 LEO study showed that everolimus (EVE) plus letrozole (LET) with ovarian suppression increased progression-free survival (PFS) in tamoxifen-exposed premenopausal women with hormone receptor-positive, HER2-negative metastatic breast cancer with visceral metastases. Here we report final survival outcomes from the LEO study, and the results of exploratory analyses of bone turnover marker changes and bone-specific progressive disease. Patients who were exposed to or progressed on tamoxifen as adjuvant/palliative treatments were randomly assigned (2:1) to the EVE (leuprorelin + LET + EVE, n=92) or LET (leuprorelin + LET, n=45) arm. In a median 51-months of follow-up, the median PFS was 17.5 and 13.8months in the EVE and LET arms, respectively (P=.245). Patients in the EVE arm with baseline visceral (median PFS 16.4 vs 9.5months, P=.040) and bone (median PFS 17.1 vs 10.9, P=.003) metastases had greater PFS compared to the LET arm. No differences in overall survival (OS) were observed (median OS, 48.3 vs 50.8months, P=.948). The 1-year cumulative incidences of bone-specific disease progression were 6.0% and 23.4% in the EVE and LET arms, respectively (hazard ratio 0.26, P < .001). Bone turnover markers at 6 and 12 weeks after treatment decreased in the EVE arm but were increased or stationary in the LET arm. Skeletal-related events occurred in 6.5% and 11.1% of patients in the EVE and LET arms, respectively. EVE + LET with ovarian suppression prolonged PFS in patients with baseline visceral or bone metastases and offered bone-protective effects in the overall study population. However, these clinical benefits did not translate into an OS benefit.

103P Long-term results and bone-protective effect of everolimus added to letrozole and ovarian function suppression for premenopausal women with hormone receptor-positive, HER2-negative metastatic breast cancer: An updated analysis of the LEO study

In phase II LEO study, everolimus (EVE) plus letrozole (LET) with ovarian suppression resulted in longer progression-free survival (PFS) in tamoxifen-exposed premenopausal women with hormone receptor-positive, HER2-negative metastatic breast cancer with visceral metastases. Here, we report an updated survival for the LEO study, along with the results of exploratory analyses on bone turnover marker changes and bone-specific progressive disease.

A Biochemical study on the effect of some treatments on bone resorption and formation in osteoporotic rats: Thesis Abstract

Thesis Abstract Osteoporosis thins bones, weakening them and making them more susceptible to fractures. It may be related to withdrawal of sex hormones by ageing. Objective: the present study was conducted to evaluate the bone protective effects of Raloxifene, Risedronate, and their combination on osteoporotic male and female rats. Methods: Osteoporosis was induced by orchidectomy (ORX) of 35 male Wistar rats and ovariectomy (OVX) of 50 female Wistar rats. Four weeks post surgery, each of ORX and OVX rats were randomized into 4 groups: control, Raloxifene (RAL), Risedronate (RIS) and Raloxifene+Risedronate (RAL+RIS). RAL dose was 3 mg/kg given orally 3 times/week. RIS dose was 5µg/kg given by s.c. injection, twice weekly. After 6 weeks of treatment, alkaline (ALP) and acid phosphatase (ACP) activities, bone mineral density (BMD), histochemical localization of ALP, gene expression of osteoprotegerin (OPG) and bone morphogenetic protein-2(BMP-2) were examined. Results: The OVX and ORX rats showed a significant increase in plasma ALP and ACP activities and histochemical activity of ALP in ORX rats and OVX rats as compared with sham, and attenuated by treatment with RAL and RIS. BMD of femur didn't change after gonadectomy. In OVX-RAL and ORX-RIS, BMD was improved compared to untreated rats. Gene expression of OPG and BMP-2 was down regulated in OVX rats versus sham, but was up-regulated by RAL and RIS treatments. In ORX rats, OPG gene expression was up-regulated versus sham, but was down-regulated by RIS treatment. Conclusions: Both RAL and RIS reduced bone turnover and maintained BMD in osteoporotic rats.

Protective effects of berberine on senile osteoporosis in mice.

The incidence of osteoporosis is positively correlated with age. Berberine has been reported to treat osteoporosis due to its beneficial actions on bone formation. However, the direct effects of berberine on senile osteoporosis remain unclear. The present study investigated the protective effects of berberine on senile osteoporosis in mice and preliminarily evaluated its potential mechanism. 20-month-old male C57BL/6J mice were used as senile osteoporosis mouse model and treated with strontium ranelate (SR) or berberine or solvent control by daily gavage for 2months. Thereafter, bone mass and microstructure parameters were assessed. Histological staining was performed to identify the osteogenic, adipogenic and osteoclastic activity of bone tissue. Moreover, role of cAMP/PKA/CREB signaling pathway in berberine affecting bone marrow mesenchymal stem cells (BMSCs) differentiation was clarified by enzyme-linked immunosorbent assay and western blot analysis. The results showed that the SR-treated group displayed a high trabecular bone mass phenotype. For mice administrated with berberine, cancellous bone mass was upregulated in a dose-dependent manner, as indicated by gradually increased bone mass, trabecular bone volume fraction and trabecular number. Furthermore, berberine promotes osteogenic and inhibits adipogenic differentiation of BMSCs via cAMP/PKA/CREB signaling. Also, bone resorption effect becomes more obvious with increasing dose of berberine in vitro. The present results suggest that berberine exerts potent bone protective effects by promoting bone formation, inhibiting marrow fat accumulation and bone resorption. This effect may be achieved through cAMP/PKA/CREB signaling pathway.