Baicalin attenuates dexamethasone-induced apoptosis of bone marrow mesenchymal stem cells by activating the hedgehog signaling pathway.

BACKGROUND: Perturbations in bone marrow mesenchymal stem cell (BMSC) differentiation play an important role in steroid-induced osteonecrosis of the femoral head (SONFH). At present, studies on SONFH concentrate upon the balance within BMSC osteogenic and adipogenic differentiation. However, BMSC apoptosis as well as proliferation are important prerequisites in their differentiation. The hedgehog (HH) signaling pathway regulates bone cell apoptosis. Baicalin (BA), a well-known compound in traditional Chinese medicine, can affect the proliferation and apoptosis of numerous cell types via HH signaling. However, the potential role and mechanisms of BA on BMSCs are unclear. Thus, we aimed to explore the role of BA in dexamethasone (Dex)-induced BMSC apoptosis in this study. METHODS: Primary BMSCs were treated with 10 -6 mol/L Dex alone or with 5.0 µmol/L, 10.0 µmol/L, or 50.0 µmol/L BA for 24 hours followed by co-treatment with 5.0 µmol/L, 10.0 µmol/L, or 50.0 µmol/L BA and 10 -6 mol/L Dex. Cell viability was assayed through the Cell Counting Kit-8 (CCK-8). Cell apoptosis was evaluated using Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining followed by flow cytometry. The imaging and counting, respectively, of Hochest 33342/PI-stained cells were used to assess the morphological characteristics and proportion of apoptotic cells. To quantify the apoptosis-related proteins (e.g., apoptosis regulator BAX [Bax], B-cell lymphoma 2 [Bcl-2], caspase-3, and cleaved caspase-3) and HH signaling pathway proteins, western blotting was used. A HH-signaling pathway inhibitor was used to demonstrate that BA exerts its anti-apoptotic effects via the HH signaling pathway. RESULTS: The results of CCK-8, Hoechst 33342/PI-staining, and flow cytometry showed that BA did not significantly promote cell proliferation (CCK-8: 0 µmol/L, 100%; 2.5 µmol/L, 98.58%; 5.0 µmol/L, 95.18%; 10.0 µmol/L, 98.11%; 50.0 µmol/L, 99.38%, F   =  2.33, P   >  0.05), but it did attenuate the effect of Dex on apoptosis (Hoechst 33342/PI-staining: Dex+ 50.0 µmol/L BA, 12.27% vs. Dex, 39.27%, t  = 20.62; flow cytometry: Dex + 50.0 µmol/L BA, 12.68% vs. Dex, 37.43%, t  = 11.56; Both P  < 0.05). The results of western blotting analysis showed that BA reversed Dex-induced apoptosis by activating the HH signaling pathway, which down-regulated the expression of Bax, cleaved-caspase 3, and suppressor of fused (SUFU) while up-regulating Bcl-2, sonic hedgehog (SHH), and zinc finger protein GLI-1 (GLI-1) expression (Bax/Bcl-2: Dex+ 50.0 µmol/L BA, 1.09 vs. Dex, 2.76, t  = 35.12; cleaved caspase-3/caspase-3: Dex + 50.0 µmol/L BA, 0.38 vs . Dex, 0.73, t  = 10.62; SHH: Dex + 50.0 µmol/L BA, 0.50 vs . Dex, 0.12, t  = 34.01; SUFU: Dex+ 50.0 µmol/L BA, 0.75 vs . Dex, 1.19, t  = 10.78; GLI-1: Dex+ 50.0 µmol/L BA, 0.40 vs . Dex, 0.11, t  = 30.68. All P  < 0.05). CONCLUSIONS: BA antagonizes Dex-induced apoptosis of human BMSCs by activating the HH signaling pathway. It is a potential candidate for preventing SONFH.

Icariin stimulates osteogenesis and suppresses adipogenesis of human bone mesenchymal stem cells via miR-23a-mediated activation of the Wnt/ß-catenin signaling pathway.

BACKGROUND: Icariin (ICA) is a bioactive compound isolated from epimedium-derived flavonoids that modulates bone mesenchymal stem cell osteogenesis and adipogenesis. However, its precise mechanism in this process is unknown. PURPOSE: The purpose of this study was to elucidate the role of ICA on human bone mesenchymal stem cell (hBMSC) osteogenesis and adipogenesis by focusing on miR-23a mediated activation of the Wnt/ß-catenin signaling pathway. METHODS: After ICA treatment, hBMSC osteogenesis and adipogenesis were evaluated using alkaline phosphatase staining, an alkaline phosphatase activity assay, Oil Red O staining, and cellular triglyceride levels. Moreover, the mRNA and protein expression levels of osteogenic and adipogenic markers as well as key factors of the Wnt/ß-catenin signaling pathway were measured using quantitative reverse transcription polymerase chain reaction and western blotting. Lithium chloride, an activator of the Wnt/ß-catenin signaling pathway, was used as a positive control. Finally, to investigate the role of miR-23a in ICA-induced activation of the Wnt/ß-catenin signaling pathway, hBMSCs were transfected with miR-23a mimics or a miR-23a inhibitor. RESULTS: ICA significantly promoted hBMSC osteogenic differentiation by upregulating alkaline phosphatase activity and the expression of bone sialoprotein II (BSPII) and runt-related transcription factor-2 (Runx-2). In contrast, ICA inhibited hBMSC adipogenic differentiation by reducing lipid droplet formation and cellular triglyceride levels as well as by downregulating the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT enhancer-binding protein-α (C/EBP-α). ICA mediated its effects on hBMSCs by activating the Wnt/ß-catenin signaling pathway. It did so by upregulating ß-catenin, low density lipoprotein receptor-related protein 5 (LRP5), and T cell factor 1 (TCF1). Notably, the up-regulation of these proteins was blocked by Dickkopf-related protein 1 (DKK1). Critically, the effects of ICA on hBMSCs were similar to that of the positive control, lithium chloride. Notably, ICA-induced activation of the Wnt/ß-catenin signaling pathway was significantly attenuated following miR-23a up-regulation. Conversely, miR-23a downregulation affected hBMSCs in the same manner as ICA; i.e., it activated the Wnt/ß-catenin signaling pathway. CONCLUSION: ICA promotes and inhibits, respectively, hBMSC osteogenesis and adipogenesis via miR-23a-mediated activation of the Wnt/ß-catenin signaling pathway.

Epimedium-derived flavonoids modulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats via Wnt/ß-catenin signal pathway activation.

OBJECTIVE: To observe the function of wnt/ß-catenin signal pathway on the process that epimedium-derived flavonoids (EFs) regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, and to provide an experimental evidence for the mechanism of EFs on treating postmenopausal osteoporosis. METHODS: Bone marrow stromal cells from ovariectomized rats were separated and cultivated in the condition of osteoinductive medium or liquid medium for 15 days. Low- (1 µg/mL), medium- (10 µg/mL) and high- (100 µg/mL) dose EFs were administrated correspondingly. Alkaline phosphatase (ALP) staining, ALP activity determination, oil red O staining and realtime polymerese chain reaction (RT-PCR) were used to determine the effect of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats. Moreover, in order to explore the mechanism of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, Dickkopf-related protein 1 (DKK1) was used in the medium group. Enzymelinked immunosorbent assay (ELISA) and RT-PCR were used to determine mRNA levels of ß-catenin, low density lipoprotein receptor-related protein 5 (LRP5) and T cell factor (TCF) protein, known as wnt/ß-catenin signal pathway related factors. RESULTS: EFs increased mRNA expression levels of ALP and early osteoblast differentiation factors, such as runt-related transcription factor 2 (Runx2), osteocalcin and collagen I, and decreased mRNA expression levels of fat generation factors, such as peroxisome proliferator activated receptor gamma 2 (PPARγ-2) and CCAAT enhancer-binding protein-α (C/EBPα) in a dose-dependent manner. While osteoblast differentiation factors were down-regulated, fat generation factors were up-regulated when DKK1 was applied. Also EFs up-regulated mRNA expression levels of ß-catenin, LRP5 and TCF protein which could be blocked by DKK1. CONCLUSION: EFs regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats by activating wnt/ß-catenin signal pathway, which may be an important molecular mechanism of EFs on treating postmenopausal osteoporosis.

[The expression and significance of Wnt/beta-catenin signal pathway protein in the effect of bushen huoxue granule containing serum on the osteoblast].

OBJECTIVE: To investigate the effect of Wnt/beta-catenin signal pathway protein in the effect of Bushen Huoxue Granule (BHG) containing serum on the osteoblast, and to provide necessary experimental reliance for its action of mechanism in treatment of osteoporosis. METHODS: The osteoblast from cranial bones of neonates rat were isolated and cultured in vitro, which was divided into the blank control group and the BHG containing serum group. The alkaline phosphatase (ALP) activities of the osteoblast in each group were quantitatively detected and the ALP staining was performed six days later. The alizarin red staining was performed eighteen days later. At the same time, levels of Wnt/beta-catenin signal pathway protein--beta-catenin, low density lipoprotein correlated protein 5 (LRP 5), and T cell factor (TCF) of osteoblasts in each group were detected by ELISA. RESULTS: BHG containing serum could significantly increase the expression of ALP and promote the formation of mineralizing nodus in the osteoblasts. At the same time it also markedly up-regulated the expressions of p-catenin, LRP 5, and TCF in this process. CONCLUSION: BHG containing serum could markedly increase ossify activities and mineralization of osteoblast. This action was closely correlated with Wnt/p-catenin signal pathway. So it indicated that Wnt/beta-catenin signal pathway played a very important role in the treatment of the osteoporosis by BHG.