This study aims to investigate the pharmacological effects and mechanisms of Yougui Yin in treating glucocorticoid-induced osteonecrosis. A rat model of glucocorticoid-associated osteonecrosis of the femoral head(GA-ONFH) was established by intramuscular injection of dexamethasone at 20 mg·kg~(-1) every other day for 8 weeks. Rats were randomly allocated into control, model, and low-and high-dose(1.5 and 3.0 g·kg~(-1), respectively) Yougui Yin groups. After modeling, rats in Yougui Yin groups were administrated with Yougui Yin via gavage, which was followed by femoral specimen collection. Hematoxylin-eosin staining was employed to observe femoral head repair, and immunofluorescence was employed to assess adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs) within the femoral head. Cell experiments were carried out with dexamethasone(1 μmol·L~(-1))-treated BMSCs to evaluate the effects of Yougui Yin-medicated serum on adipogenic differentiation. Animal experiments demonstrated that compared with the model group, Yougui Yin at both high and low doses significantly improved bone mineral density(BMD), bone volume/total volume(BV/TV) ratio, and trabecular thickness(Tb.Th) in the femoral head. Additionally, Yougui Yin alleviated necrosis-like changes and adipocyte infiltration and significantly reduced the expression level of peroxisome proliferator-activated receptor γ(PPARγ) in the femoral head, thereby suppressing the adipogenic differentiation of BMSCs in GA-ONFH rats. The cell experiments revealed that Yougui Yin-medicated serum markedly inhibited dexamethasone-induced adipogenic differentiation of BMSCs and down-regulated the level of PPARγ. The overexpression of PPARγ attenuated the inhibitory effect of Yougui Yin-medicated serum on the adipogenic differentiation of BMSCs, indicating the critical role of PPARγ in Yougui Yin-mediated suppression of adipogenic differentiation of BMSCs. In conclusion, Yougui Yin exerts therapeutic effects on glucocorticoid-induced osteonecrosis by down-regulating PPARγ expression and inhibiting adipogenic differentiation of BMSCs.
Animals ; Mesenchymal Stem Cells/metabolism* ; PPAR gamma/genetics* ; Rats ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Glucocorticoids/adverse effects* ; Rats, Sprague-Dawley ; Adipogenesis/drug effects* ; Osteonecrosis/genetics* ; Cell Differentiation/drug effects* ; Bone Marrow Cells/metabolism* ; Femur Head Necrosis/chemically induced* ; Humans

PURPOSE: To explore the value of transplanting peripheral blood-derived mesenchymal stem cells from allogenic rabbits (rPBMSCs) to treat osteonecrosis of the femoral head (ONFH). MATERIALS AND METHODS: rPBMSCs were separated/cultured from peripheral blood after granulocyte colony-stimulating factor mobilization. Afterwards, mobilized rPBMSCs from a second passage labeled with PKH26 were transplanted into rabbit ONFH models, which were established by liquid nitrogen freezing, to observe the effect of rPBMSCs on ONFH repair. Then, the mRNA expressions of BMP-2 and PPAR-γ in the femoral head were assessed by RT-PCR. RESULTS: After mobilization, the cultured rPBMSCs expressed mesenchymal markers of CD90, CD44, CD29, and CD105, but failed to express CD45, CD14, and CD34. The colony forming efficiency of mobilized rPBMSCs ranged from 2.8 to 10.8 per million peripheral mononuclear cells. After local transplantation, survival of the engrafted cells reached at least 8 weeks. Therein, BMP-2 was up-regulated, while PPAR-γ mRNA was down-regulated. Additionally, bone density and bone trabeculae tended to increase gradually. CONCLUSION: We confirmed that local transplantation of rPBMSCs benefits ONFH treatment and that the beneficial effects are related to the up-regulation of BMP-2 expression and the down-regulation of PPAR-γ expression.
Animals ; Blood Cells/*cytology ; Bone Morphogenetic Protein 2/genetics ; *Cell- and Tissue-Based Therapy ; Femur Head Necrosis/metabolism/*pathology/*therapy ; Gene Expression Regulation ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology ; Osteonecrosis/*pathology/*therapy ; PPAR gamma/genetics ; Rabbits ; Transplantation, Homologous

BACKGROUNDAppropriate expression and regulation of the transcriptome, which mainly comprise of mRNAs and lncRNAs, are important for all biological and cellular processes including the physiological activities of bone microvascular endothelial cells (BMECs). Through an intricate intracellular signaling systems, the transcriptome regulates the pharmacological response of the cells. Although studies have elucidated the impact of glucocorticoids (GCs) cell-specific gene expression signatures, it remains necessary to comprehensively characterize the impact of lncRNAs to transcriptional changes.

METHODSBMECs were divided into two groups. One was treated with GCs and the other left untreated as a paired control. Differential expression was analyzed with GeneSpring software V12.0 (Agilent, Santa Clara, CA, USA) and hierarchical clustering was conducted using Cluster 3.0 software. The Gene Ontology (GO) analysis was performed with Molecular Annotation System provided by CapitalBio Corporation.

RESULTSOur results highlight the involvement of genes implicated in development, differentiation and apoptosis following GC stimulation. Elucidation of differential gene expression emphasizes the importance of regulatory gene networks induced by GCs. We identified 73 up-regulated and 166 down-regulated long noncoding RNAs, the expression of 107 of which significantly correlated with 172 mRNAs induced by hydrocortisone.

CONCLUSIONSTranscriptome analysis of BMECs from human samples was performed to identify specific gene networks induced by GCs. Our results identified complex RNA crosstalk underlying the pathogenesis of steroid-induced necrosis of femoral head.

Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Femur Head ; cytology ; Gene Expression Profiling ; Glucocorticoids ; pharmacology ; Humans ; Oligonucleotide Array Sequence Analysis ; Osteonecrosis ; genetics ; RNA, Messenger ; genetics ; RNA, Untranslated ; genetics ; Transcriptome ; drug effects ; genetics

Osteonecrosis of the femoral head is frequently observed in patients treated with excessive corticosteroids. However, the pathogenesis of corticosteroid-induced osteonecrosis remains unclear. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signaling pathway in steroid-induced femoral head osteonecrosis in rats. Male Sprague-Dawley rats were injected intramuscularly with 20 mg/kg methylprednisolone (MP) for 8 weeks, twice per week. The animals were sacrificed at 2, 4 and 8 weeks after the last MP injection, respectively, and then allocated to the 2-, 4- and 8-week model groups (n=24 each). Rats in the control group (n=12) were not given any treatment. Histopathological analysis was performed and the concentration of tartrate-resistant acid phosphatase (TRAP) in plasma was determined. The activation of osteoclasts in the femoral head was assessed by TRAP staining. The expression of TLR4, MyD88, TRAF6 and NF-κB p65 that are involved in TLR4 signaling, and MCP-1 production were detected by using real-time PCR (RT-PCR) and Western blotting. The results showed that the osteonecrosis in the femoral head was clearly observed and the concentration of TRAP in the plasma was increased in the model rats. The femoral head tissues in MP-treated rats were positive for TRAP and the intensity of TRAP staining was greater in MP-treated rats than in control rats. As compared with the control group, the mRNA expression of TLR4 signaling-related factors was enhanced significantly at 4 and 8 weeks, and the protein levels of these factors increased significantly with time. It was concluded that MP could induce the femoral head osteonecrosis in rats, which was associated with osteoclast activation via the TLR4 signaling pathway. These findings suggest that TLR4 signaling pathway plays a pivotal role in the pathogenesis of steroid-induced osteonecrosis.
Acid Phosphatase ; metabolism ; Animals ; Blotting, Western ; Chemokine CCL2 ; genetics ; metabolism ; Femur Head ; metabolism ; pathology ; Gene Expression ; Immunohistochemistry ; Isoenzymes ; metabolism ; Male ; Methylprednisolone ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; Osteonecrosis ; chemically induced ; genetics ; metabolism ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; TNF Receptor-Associated Factor 6 ; genetics ; metabolism ; Tartrate-Resistant Acid Phosphatase ; Time Factors ; Toll-Like Receptor 4 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; metabolism

MicroRNAs (miRNAs) have recently been recognized to have a role in human orthopedic disorders. The objective of our study was to explore the expression profile and biological function of miRNA-17-5p (miR-17-5p), which is well known to be related to cancer cell proliferation and invasion, in osteoblastic differentiation and in cell proliferation. The expression levels of miR-17-5p in the femoral head mesenchymal stem cells of 20 patients with non-traumatic osteonecrosis (ON) and 10 patients with osteoarthritis (OA) were examined by quantitative reverse transcription-PCR (qRT-PCR). Furthermore, the interaction between miR-17-5p and SMAD7 was observed. We found that in non-traumatic ON samples the level of mature miR-17-5p was significantly lower than that of OA samples (P=0.0002). By targeting SMAD7, miR-17-5p promoted nuclear translocation of beta-catenin, enhanced expression of COL1A1 and finally facilitated the proliferation and differentiation of HMSC-bm cells. We also demonstrated that restoring expression of SMAD7 in HMSC-bm cells partially reversed the function of miR-17-5p. Together, our data suggested a theory that dysfunction of a network containing miR-17-5p, SMAD7 and beta-catenin could contribute to ON pathogenesis. The present study prompts the potential clinical value of miR-17-5p in non-traumatic ON.
Adult ; Base Sequence ; Bone Morphogenetic Protein 2/metabolism ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Female ; *Gene Expression Regulation ; Humans ; Male ; MicroRNAs/genetics/*metabolism ; Middle Aged ; Osteoarthritis/genetics/metabolism/pathology ; Osteoblasts/*cytology/metabolism/*pathology ; Osteogenesis ; Osteonecrosis/*genetics/metabolism/pathology ; Signal Transduction ; Smad7 Protein/*genetics/metabolism ; beta Catenin/metabolism

BACKGROUNDIn steroid-induced osteonecrosis, hypertrophy and hyperplasia of marrow fat cells and lipid deposition of osteocytes can be found in the femoral head. However, the precise reason is not clear yet. The aim of this study was to observe the effect of dexamethasone (Dex) on differentiation of marrow stromal cells (MSCs), and to investigate the pathobiological mechanism of steroid-induced osteonecrosis.

METHODSMSCs in cultures were treated with increasing concentrations of Dex (0, 10(-9), 10(-8), 10(-7), and 10(-6) mol/L) continuously for 21 days. The cells, which were exposed to 0 mol/L (control) or 10(-7) mol/L Dex for 4 - 21 days, were then cultured for 21 days without Dex. MSCs were stained with Sudan III. Number of adipocytes was counted under a light microscope. The activity of alkaline phosphatase (ALP) of MSCs treated with 0, 10(-8), 10(-7), and 10(-6) mol/L Dex for 12 days, and that treated with 0 mol/L and 10(-7) mol/L Dex for 8, 10, or 12 days were determined. The levels of triglycerides, osteocalcin and cell proliferation of MSCs treated with 0 mol/L and 10(-7) mol/L Dex were detected. The mRNA expression levels of adipose-specific 422 (aP2) gene and osteogenic gene type I collagen in MSCs treated with 0 mol/L and 10(-7) mol/L Dex for 6 days were analyzed by whole-cell dot-blot hybridization. Statistical analysis was performed using Student's t test and analysis of variance. P values less than 0.05 were considered significant statistically.

RESULTSThe number of adipocytes in cultures increased with the duration of MSCs' exposure to Dex and the concentration of Dex. The level of ALP activity in the MSCs decreased with concentration of Dex. In the control group, it was 8.69 times of that in the 10(-7) mol/L Dex group on day 12 (t = 20.51, P < 0.001). The level of triglycerides in 10(-7) mol/L Dex group was 3.40 times of that in the control (t = 11.00, P < 0.001). The levels of cell proliferation and osteocalcin in the control were 1.54 and 2.42 times of that in the 10(-7) mol/L Dex group respectively. As compared to the control, the mRNA expression of adipose-specific 422 (aP2) gene in 10(-7) mol/L Dex group was significantly increased (t = 36.48, P < 0.001), and that of osteogenic gene type I collagen was decreased (t = 42.07, P < 0.001).

CONCLUSIONSDex can directly induce the differentiation of MSCs into a large number of adipocytes and inhibit their osteogenic differentiation, which provide a novel explanation for the pathologic changes of steroid-induced osteonecrosis.

Adipogenesis ; drug effects ; Alkaline Phosphatase ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dexamethasone ; toxicity ; Female ; Mice ; Osteocalcin ; genetics ; Osteonecrosis ; chemically induced ; RNA, Messenger ; analysis ; Radioimmunoassay ; Stromal Cells ; cytology