Lingzhi and San-Miao-San with hyaluronic acid gel mitigate cartilage degeneration in anterior cruciate ligament transection induced osteoarthritis.

OBJECTIVE: To investigate the mitigate efficacy of Chinese medicine Lingzhi (LZ) and San-Miao-San (SMS) combined with hyaluronic acid (HA)-gel in attenuating cartilage degeneration in traumatic osteoarthritis (OA). METHODS: The standardized surgery of anterior cruciate ligament transection (ACLT) was made from the medial compartment of right hind limbs of 8-week-old female SD rats and resulted in a traumatic OA. Rats (n â€‹= â€‹5/group) were treated once intra-articular injection of 50 â€‹µl HA-gel, 50 â€‹µl HA-gel+50 â€‹µg LZ-SMS, 50 â€‹µl of saline+50 â€‹µg LZ-SMS and null (ACLT group) respectively, except sham group. Limbs were harvested for µCT scan and histopathological staining 3-month post-treatment. Inflammatory cytokines from plasma and synovial fluid were detected using Immunology Multiplex Assay kit. The putative targets of active compounds in LZ-SMS and known therapeutic targets for OA were combined to construct protein-protein interaction network. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was adopted to predict the potential targets and signaling pathway of LZ-SMS in OA through the tool of DAVID Bioinformatics. RESULTS: In vivo, HA-gel â€‹+ â€‹LZ-SMS treatment resulted in a higher volume ratio of hyaline cartilage (HC)/calcified cartilage (CC) and HC/Sum (total volume of cartilage), compared to ACLT and HA-gel groups. In addition, histological results showed the elevated cartilage matrix, chondrogenic and osteoblastic signals in HA-gel â€‹+ â€‹LZ-SMS treatment. Treatment also significantly altered subchondral bone (SCB) structure including an increase in BV/TV, Tb.Th, BMD, Conn.Dn, Tb.N, and DA, as well as a significant decrease in Tb.Sp and Po(tot), which implied a protective effect on maintaining the stabilization of tibial SCB microstructure. Furthermore, there was also a down-regulated inflammatory cytokines and upregulated anti-inflammatory cytokine IL-10 in HA+LZ-SMS group. Finally, 64 shared targets from 37 active compounds in LZ-SMS related to the core genes for the development of OA. LZ-SMS has a putative role in regulating inflammatory circumstance through influencing the MAPK signaling pathway. CONCLUSION: Our study elucidated a protective effect of HA-gel â€‹+ â€‹LZ-SMS in mitigating cartilage degradation and putative interaction with targets and signaling pathway for the development of traumatic OA. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Our results provide a biological rationale for the use of LZ-SMS as a potential candidate for OA treatment.

Formononetin, a methoxy isoflavone, enhances bone regeneration in a mouse model of cortical bone defect.

The bone regeneration and healing effect of formononetin was evaluated in a cortical bone defect model that predominantly heals by intramembranous ossification. For this study, female Balb/c mice were ovariectomised (OVx) and a drill-hole injury was generated in the midfemoral bones of all animals. Treatment with formononetin commenced the day after and continued for 21 d. Parathyroid hormone (PTH1-34) was used as a reference standard. Animals were killed at days 10 and 21. Femur bones were collected at the injury site for histomorphometry studies using microcomputed tomography (µCT) and confocal microscopy. RNA and protein were harvested from the region surrounding the drill-hole injury. For immunohistochemistry, 5 µm sections of decalcified femur bone adjoining the drill-hole site were cut. µCT analysis showed that formononetin promoted bone healing at days 10 and 21 and the healing effect observed was significantly better than in Ovx mice and equal to PTH treatment in many aspects. Formononetin also significantly enhanced bone regeneration as assessed by calcein-labelling studies. In addition, formononetin enhanced the expression of osteogenic markers at the injury site in a manner similar to PTH. Formononetin treatment also led to predominant runt-related transcription factor 2 and osteocalcin localisation at the injury site. These results support the potential of formononetin to be a bone-healing agent and are suggestive of its promising role in the fracture-repair process.

A comparative study of the bone metabolic response to dried plum supplementation and PTH treatment in adult, osteopenic ovariectomized rat.

Dried plum has been reported to have potent effects on bone in osteopenic animal models, but the mechanisms through which bone metabolism is altered in vivo remain unclear. To address this issue, a study comparing the metabolic response of dried plum to the anabolic agent, parathyroid hormone (PTH), was undertaken. Six month-old female Sprague Dawley rats (n=84) were sham-operated (SHAM) or ovariectomized (OVX) and maintained on a control diet for 6wks until osteopenia was confirmed. Treatments were initiated consisting of a control diet (AIN-93M) supplemented with dried plum (0, 5, 15 or 25%; w/w) or a positive control group receiving PTH. At the end of 6wks of treatment, whole body and femoral bone mineral density (BMD) were restored by the two higher doses of dried plum to the level of the SHAM group. Trabecular bone volume and cortical thickness were also improved with these two doses of dried plum. Dried plum suppressed the OVX-induced increase in bone turnover as indicated by systemic biomarkers of bone metabolism, N-terminal procollagen type 1 (P1NP) and deoxypyridinoline (DPD). Dynamic bone histomorphometric analysis of the tibial metaphysis revealed that dried plum restored the OVX-induced increase in cancellous bone formation rate (BFR) and mineralizing surface (MS/BS) to the SHAM group, but some doses of dried plum increased endocortical mineral apposition rate (MAR). As expected, PTH significantly increased endocortical MAR and BFR, periosteal BFR, and trabecular MAR and BFR beyond that of the OVX and maintained the accelerated rate of bone resorption associated with OVX. Dried plum up-regulated bone morphogenetic protein 4 (Bmp4) and insulin-like growth factor 1 (Igf1) while down-regulating nuclear factor T cell activator 1 (Nfatc1). These findings demonstrate that in the adult osteopenic OVX animal, the effects of dried plum differ from that of PTH in that dried plum primarily suppressed bone turnover with the exception of the indices of bone formation at the endocortical surface.

Comparison of chemical compositions and osteoprotective effects of different sections of velvet antler.

ETHNOPHARMACOLOGICAL RELEVANCE: Velvet antlers (VA) have been claimed for centuries to have numerous medical benefits including strengthen bones. To investigate and compare the anti-osteoporotic activities from different sections of VA. MATERIALS AND METHODS: Fresh VA prepared from farmed sika deers (Cervus nippon) was divided into upper (VAU), middle (VAM), and basal (VAB) sections. The chemical constituents and anti-osteoporotic effect of different sections from VA were evaluated using ovariectomized rats. RESULTS: Levels of water-soluble extracts, diluted alcoholic extract, amino acids, testosterone, insulin-like growth factor (IGF)-1 and testosterone plus estradiol significantly differed among the different sections. Levels of these constituents were significantly higher in the upper section than in the basal section. Moreover, levels of testosterone and IGF-1 of the VAM were also significantly higher than those of the VAB. Calcium level increased downward from the tip with statistical significance. The strength of vertebrae increased in all VA-treated groups compared to the control, but only treatment with VAU and VAM increased the strength of the femur and the microarchitecure of the trabecular bone. Alkaline phosphatase levels of VAU- and VAM-treated groups significantly decreased, but osteocalcin did not significantly change. Moreover, VAU and VAM dose-dependently increased proliferation and mineralization of MC3T3-E1 cells. CONCLUSION: Our study provides strong evidence for the regional differences in the effectiveness of velvet antler in treating osteoporosis. However, further studies are needed to elucidate the bioactive chemical constituents associated with the anti-osteoporotic effects of velvet antler.

Effects of unfractionated heparin on renal osteodystrophy and vascular calcification in chronic kidney disease rats.

Unfractionated heparin (UFH) is the most widely used anticoagulant in hemodialysis for chronic kidney disease (CKD) patients. Many studies have verified that UFH can induce bone loss in subjects with normal bone, but few have focused on its effect on renal osteodystrophy. We therefore investigated this issue in adenine-induced CKD rats. As CKD also impairs mineral metabolism systemically, we also studied the impacts of UFH on serum markers of CKD-mineral and bone disorder (CKD-MBD) and vascular calcification. We administered low and high doses of UFH (1U/g and 2U/g body weight, respectively) to CKD rats and compared them with CKD controls. At sacrifice, the serum markers of CKD-MBD did not significantly differ among the two UFH CKD groups and the CKD control group. The mean bone mineral densities (BMDs) of the total femur and a region of interest (ROI) constituted of trabecular and cortical bone were lower in the high-dose UFH (H-UFH) CKD group than in the CKD control group (P<0.05 and P<0.01, respectively). The BMD of the femoral ROI constituted of cortical bone did not differ between the H-UFH CKD group and the CKD control group. Histomorphometrical changes in the CKD rats indicated secondary hyperparathyroidism, and the femoral trabecular bone volume, but not cortical bone volume, significantly decreased with increasing UFH dose. The same decreasing trend was found in osteoblast parameters, and an increasing trend was found in osteoclast parameters; however, most differences were not significant. Moreover, no distinct statistical differences were found in the comparison of vascular calcium or phosphorus content among the CKD control group and the two UFH CKD groups. Therefore, we concluded that UFH could induce bone loss in CKD rats with secondary hyperparathyroidism, mainly by reducing the trabecular volume and had little effect on cortical bone volume. The underlying mechanism might involve inhibition of osteoblast activity and promotion of osteoclast activity by UFH. We did not find any effect of UFH on vascular calcification in CKD rats with secondary hyperparathyroidism.

A novel flavonoid C-glucoside from Ulmus wallichiana preserves bone mineral density, microarchitecture and biomechanical properties in the presence of glucocorticoid by promoting osteoblast survival: a comparative study with human parathyroid hormone.

PURPOSE: 6-C-ß-D-glucopyranosyl-(2S,3S)-(+)-5,7,3',4'-tetrahydroxydihydroflavonol (GTDF) is a novel compound isolated from Ulmus wallichiana, reported to have bone anabolic action in ovariectomized rats. Here, we studied the effect of GTDF in glucocorticoid (GC)-induced bone loss and its mode of action. METHODS: Osteoblasts were cultured from rat calvaria or bone marrow to study apoptosis and differentiation by dexamethasone (Dex), methylprednisolone (MP), GTDF, quercetin and rutin. Female Sprague Dawley rats were treated with Dex or MP with or without GTDF or PTH. Efficacy was evaluated by bone microarchitecture using microcomputed tomography, determination of new bone formation by fluorescent labeling of bone and osteoblast apoptosis by co-labeling bone sections with Runx-2 and TUNEL. Serum osteocalcin was determined by ELISA. RESULTS: GTDF preserved trabecular and cortical bones in the presence of Dex and MP and mitigated the MP-mediated suppression of serum osteocalcin. Co-administration of GTDF to MP rats increased mineral apposition, bone formation rates, bone biomechanical strength, reduced osteoblast apoptosis and increased osteogenic differentiation of bone marrow stromal cells compared to MP group, suggesting in vivo osteogenic effect of GTDF. These effects of GTDF were to a great extent comparable to PTH. GTDF prevented GC-induced osteoblast apoptosis by inhibiting p53 expression and acetylation, and activation of AKT but did not influence transactivation of GC receptor (GR). CONCLUSIONS: GTDF protects against GC-induced bone loss by promoting osteoblast survival through p53 inhibition and activation of AKT pathways but not as a GR antagonist. GTDF has the potential in the management of GC-induced osteopenia.

Energy-restricted diet benefits body composition but degrades bone integrity in middle-aged obese female rats.

This study investigates the effects of a restricted diet (RD) on body composition and musculoskeletal health along with endocrines and molecular mechanism in established mature obese rats. Twenty female rats were fed with a high-fat diet (HFD) ad libitum for 4 months and then assigned to either HFD or RD group for another 4 months. Another 10 rats were on a low-fat diet for 8 months. Outcome measures included body composition, bone mineral density, microarchitecrure, and strength; serum leptin, adiponectin, insulin-like growth factor I, and liver glutathione peroxidase activity; and protein expression and spleen tumor necrosis factor α messenger RNA expression. We hypothesized that mature obese rats on a 35% energy restriction diet for 4 months would improve body composition but degrade microstructural and mechanical properties of long bones, and such changes in musculoskeletal integrity are related to the modulation of obesity-related endocrines and proinflammation. Relative to HFD, RD benefited body composition (decreased body weight and %fat mass and increased %fat-free mass); decreased insulin-like growth factor I and leptin; elevated adiponectin, glutathione peroxidase activity and protein expression and tumor necrosis factor α messenger RNA expression; and suppressed bone formation and increased bone resorption, resulting in decreased trabecular and cortical bone volume, bone mineral density, and bone strength. Relative to low-fat diet, RD had a similar effect on body composition and serum markers but increased bone turnover rate and decreased bone mineral density and strength. Our data suggest that long-term RD has a negative impact on bone remodeling in obese female rats, probably through modification of endocrines and elevation of proinflammation.