Glucocorticoid (GC)-induced osteoporosis (GIO) is characterized by impaired bone formation, which can be alleviated by tanshinol, an aqueous polyphenol isolated from Salvia miltiorrhiza Bunge. In this study we investigated the molecular mechanisms underlying GC-induced modulation of osteogenesis as well as the possibility of using tanshinol to interfere with GIO. Female SD rats aged 4 months were orally administered distilled water (Con), prednisone (GC, 5 mg·kg·d), GC plus tanshinol (Tan, 16 mg·kg·d) or GC plus resveratrol (Res, 5 mg·kg·d) for 14 weeks. After the rats were sacrificed, samples of bone tissues were collected. The changes in bone formation were assessed using Micro-CT, histomorphometry, and biomechanical assays. Expression of Kruppel-like factor 15 (KLF15), peroxisome proliferator-activated receptor γ 2 (PPARγ 2) and other signaling proteins in skeletal tissue was measured with Western blotting and quantitative RT-PCR. GC treatment markedly increased the expression of KLF15, PPARγ2, C/EBPα and aP2, which were related to adipogenesis, upregulated FoxO3a pathway proteins (FoxO3a and Gadd45a), and suppressed the canonical Wnt signaling (β-catenin and Axin2), which was required for osteogenesis. Thus, GC significantly decreased bone mass and bone quality. Co-treatment with Tan or Res effectively counteracted GC-impaired bone formation, suppressed GC-induced adipogenesis, and restored abnormal expression of the signaling molecules in GIO rats. We conclude that tanshinol counteracts GC-decreased bone formation by inhibiting marrow adiposity via the KLF15/PPARγ2/FoxO3a/Wnt pathway.

Numerous studies have demonstrated that rheumatoid arthritis (RA) is often associated with bone loss; however, few experiments have focused on cancellous and cortical bone changes in rats during the process of arthritis. We have investigated bone changes in rats with collagen-induced arthritis (CIA) and have explored the characteristics of how RA induces osteoporosis by means of bone histomorphometry, bone biomechanics studies, bone mineral density studies, micro computer tomography, enzyme-linked immunosorbant assay, immunohistochemistry, and Western blot analysis. Bone mineral density of the femur and lumbar vertebrae and biomechanical properties of the femur were decreased in CIA rats. Trabecular bone volume of the tibia and lumbar vertebrae was decreased whereas bone resorption was increased in CIA rats. Bone formation of the tibial shaft in periosteal surfaces was decreased in CIA rats. Furthermore, the trabecular bone loss in CIA rats was severer at 16 weeks than at 8 weeks, as was cortical bone loss. The serum level of tumor necrosis factor α in CIA rats was increased, and the expression of dickkopf 1 and that of receptor activator of nuclear factor κB (RANKL) ligand (RANKL) in the ankle joints were also increased, but the expression of osteoprotegerin (OPG) was decreased. We conclude that CIA rats developed systemic osteoporosis, and that osteoporosis became more serious with CIA development. The mechanism may be related to the increase of bone resorption in cancellous bone cause by upregulation of the expression of DKK-1 and regulation of the RANKL/RANK/OPG signaling pathway, and the decrease of bone formation in cortical bone caused by an increase in the expression of DKK-1.

Tanshinol is a major water-soluble active component of . In this study, we aimed to investigate whether tanshinol has potential therapeutic effects against glucocorticoid-induced osteoporosis and glucocorticoid-induced myopathy. Ninety-six female Sprague-Dawley rats were randomly assigned to five groups: a control group, a model group, and three model groups treated with 25 or 50 mg/kg of tanshinol, or calcitriol. All model groups received prednisone acetate for 90 days to induce glucocorticoid-induced osteoporosis. Afterwards, all animals underwent a surgical procedure to induce bone defects at the right proximal tibia. Prednisone treatment was stopped after surgery, but tanshinol or calcitriol treatment was continued to the endpoint. At the experimental endpoint, compared to the model group, 25 mg/kg tanshinol could significantly reverse glucocorticoid-induced loss of bone mineral density by 12.5 %, while enhancing mechanical bone strength, causing a significant 11 % increase in trabecular number, and reducing trabecular separation by 28 %. In addition, tanshinol improved the bone microarchitecture and prevented glucocorticoid-induced bone loss by promoting bone formation and inhibiting bone resorption. Moreover, results of bone defect repair and muscle weight measurements revealed that tanshinol accelerated the bone fracture healing process and attenuated muscle atrophy caused by glucocorticoid. Furthermore, qRT-PCR analysis showed a 1-fold upregulation in mRNA levels of transforming growth factor beta and roughly 6-fold increases in vascular endothelial growth factor mRNA expression in calluses from the tanshinol groups. Tanshinol also preserved muscular ubiquitin mRNA levels from glucocorticoid-induced elevation. These findings demonstrate the potential benefits of tanshinol against glucocorticoid-induced osteoporosis and glucocorticoid-induced myopathy, which warrants further investigation in future studies.

Osteoporosis (OP) is a systemic skeletal disorder, manifesting with a reduction in bone mass and deterioration of the microarchitecture. Mesenchymal stem cells (MSCs) have an innate ability to differentiate into several cell types, including osteoblasts (OB). Ginsenoside Rb1 (GRb1) is an ethanol extract from ginseng and contains a highly concentrated form of ginsenoside. GRb1 shows extensive beneficial health effects such as anti-oxidative and anti-inflammatory functions, modulating the immune system and inhibiting osteoclastogenesis. We hypothesized that GRb1 can promote MSC differentiation into OBs and inhibit bone loss. In the present study, we aimed to address two questions: (1) Will GRb1 have a positive effect on osteogenic differentiation of MSCs? and (2) Will GRb1 halt bone loss in ovariectomized (OVX) rats? We investigated the effects of GRb1 on viability and osteogenic differentiation of rat mesenchymal stem cells (rMSCs). Our results showed that GRb1 at concentrations of 10−8 M and 10−6 M can increase alkaline phosphatase activity, mineralization and the expression of osteogenic related proteins, such as osteopontin and osteoprotegerin, while incubating rMSCs with osteogenic induction medium and GRb1. Adding GRb1 into the medium can prevent rMSCs from Oxidative damage at the concentration of 25μM H2O2. Furthermore, 40 4-month-old rats were assigned to 5 groups(8 rats per group): the basal group, the sham group, the OVX group, the high dose of GRb1 group (6 mg/kg/day) and the low dose of GRb1 group (3 mg/kg/day). Rats recrived treatment 3days after surgery and last for 14 weeks. Examinations included serum analysis, mechanical testing, Masson-Goldner trichrome staining and bone histomorphometry analysis. The results showed that OVX can lead to dyslipidemia and excessive oxidative stress, whereas GRb1 cannot significantly halt dyslipidemia and excessive oxidative stress in OVX rats. In addition, the bone density of the lumbar vertebra and femur were decreased significantly in the OVX rats, and GRb1 could not inhibit bone loss. Bone histomorphometry analysis showed that the number and width of bone trabecula of the tibia were reduced in OVX rats, and GRb1 could not prevent their occurrence. A bone biomechanics assay showed that GRb1 cannot improve the ability of bone structure to resist fracture of the femur in OVX rats. The current study demonstrated that GRb1 has an obvious effect on osteogenic differentiation in rMSCs but no obvious effect on bone loss in OVX rats. These findings indicate GRb1 has a positive effect on rMSCs but does not have an effect on bone loss in OVX rats at the concentration we used.

Glucocorticoids (GCs) are often prescribed to treat rheumatoid arthritis (RA) in the long term, but there is still controversy in the administration of GCs, mainly because of the adverse reactions such as osteoporosis. Numerous studies have demonstrated that osteoporosis could be induced by GCs in normal rats. However, few experiments have focused on whether osteoporosis could be induced or aggravated by GCs in collagen induced arthritis (CIA) rats. We have investigated bone changes in CIA rats treated with prednisone at 4.5 mg/kg/day for 30 and 90 days by bone histomorphometry, bone mineral density (BMD), micro-CT, biomechanical test, and enzyme-linked immunosorbant assay. We found that high bone turnover osteoporosis was shown in CIA rats. Prednisone treatment for 30 and 90 days improved articular structure and decelerated the degeneration of the femur in CIA rats, but did not improve BMD and bone biomechanics. We conclude that osteoporosis was not aggravated in CIA rats treated with prednisone for 30 and 90 days. On the contrary, prednisone treatment for 30 and 90 days could prevent bone loss of the femur in CIA rats. There was a negative effect on bone metabolism in CIA rats treated with prednisone for 90 days.

Rheumatoid arthritis (RA) is closely associated with periarticular osteopenia and leads to a high risk of generalized osteoporosis. Although glucocorticoid (GC) treatment ameliorates joint degradation and manages inflammation in RA, GC application may induce further bone quality deterioration in RA patients. Current treatments for RA lack relevant strategies for the prevention and treatment of osteopenia in RA. In this study, we aimed to investigate whether salvianolate treatment ameliorated osteopenia in prednisone-treated RA rats. Lewis rats with collagen-induced arthritis (CIA) were administered prednisone (PDN) or PDN plus salvianolate (PDN+Sal) treatment for 90 days. The effects of Sal were investigated in PDN-treated CIA rats. To further evaluate the effects of Sal under inflammatory conditions, we investigated the effects of Sal treatment on the TNF-α-induced inflammatory response in MC3T3-E1 osteoblasts. Bone histomorphometry, bone mineral density (BMD), bone biomechanical properties, micro-computed tomography (micro-CT), immunohistochemistry, RT-PCR and western blot analyses were performed to evaluate the effects of Sal. The results demonstrated that RA induced bone loss and bone quality deterioration, with high bone turnover in CIA rats. PDN+Sal treatment significantly increased BMD and trabecular/cortical bone mass, suppressed inflammation, and improved bone biomechanical properties compared to CIA control and PDN treatment. PDN+Sal treatment significantly suppressed bone resorption and the RANKL and RANKL/OPG ratios compared to PDN. PDN+Sal and PDN treatment significantly inhibited TNF-α by 82 and 83%, respectively, and both suppressed inflammation in CIA rats. However, there was no significant difference between PDN+Sal and PDN treatment alone in regard to bone formation parameters or the management of inflammation and arthropathy. Sal significantly increased Osterix, OPN, and Col1a1 while decreasing RANKL, TRAF6, and TRAIL gene in TNF-α-induced MC3T3-E1 osteoblasts. Sal significantly increased Osterix, OPN and RUNX2 while decreasing NF-κB, TRAF6 and IL-1β protein in TNF-α-induced MC3T3-E1 osteoblasts. The results suggested that salvianolate treatment ameliorated osteopenia and improved bone quality in prednisone-treated RA rats, and the potential mechanism may be related to the regulation of the RANKL/RANK/OPG signaling pathway, TRAIL-TRAF6-NFκB signal axis, and downregulation of inflammatory cytokines. Salvianolate could be used as a promising supplemental therapeutic strategy to ameliorate osteopenia and improve bone quality in GC-treated RA patients.

Background: MicroRNAs (miRs) hold critical implications in the modulation of osteogenesis. This work was designed to unravel the underlying regulatory mechanism of miR-22 during osteoblast differentiation.Methods: Synthetic miR-22 mimics or inhibitors were transfected into preosteoblast MC3T3-E1 cells to regulate miR-22 expression. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and flow cytometry analyses were employed to assess cell proliferation and apoptosis. A quantitative real-time polymerase chain reaction and western blot assays were applied to measure mRNA and protein expression. Alkaline phosphatase activity and alizarin red staining were tested to further analyze cell differentiation. In silico analysis and luciferase reporter assays were utilized to identify the direct binding between miR-22 and its potential target.Results: MTT and flow cytometry analyses showed that miR-22 repressed MC3T3-E1 cell viability and promoted cell apoptosis. By detecting osteogenicspecific molecule expression, alkaline phosphatase activity and alizarin red staining, miR-22 was observed to suppress osteogenic differentiation of MC3T3-E1 cells. In silico analysis and luciferase reporter assays confirmed that ESR1 is a direct target gene of miR-22. In addition, miR-22 expression affected the phosphorylation of p38 mitogen-activated protein kinase and Jun N-terminal kinase expression in MC3T3-E1 cells. Conclusions:The findings of the present study highlight the functional significance of miR-22 in osteoblast differentiation and suggest its role as a possible therapeutic target in metabolic bone disorders. K E Y W O R D SESR1, miR-22, osteoblast differentiation, p38 MAPK/JNK signaling pathway Chen and Zhang contributed equally to this work.

The aim of the present study was to investigate the effect of salvianolic acid B (Sal B) and danshensu (DSU) on the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) and the mechanisms of the effects. The osteogenic differentiation of MSCs in culture was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin (OCN) production, nitric oxide (NO) production and the mRNA expression levels of osteoprotegerin (OPG) and its ligand by MSCs. MSCs were successfully induced to differentiate into osteoblasts and adipocytes. Sal B and DSU increased the ALP activity and the production of OCN in the absence of an ossification inducer. The increase in ALP activity was more pronounced when induction was combined with the osteogenic inducer, Sal B, which enhanced the expression of OPG; however, Sal B reduced the expression of receptor activator of nuclear factor-κB ligand (RANKL) by MSCs. Sal B reversed the inhibitory effect of N-nitro L-arginine methylester on the MSCs and increased ALP activity, OCN content and the OPG/RANKL ratio. Based on these results, it was concluded that Sal B increases the osteogenic differentiation of MSCs, most likely by regulating the nitric oxide pathway.