Background Dysfunctional osteogenesis of bone marrow mesenchymal stem cells (BMSCs) plays an important role in osteoporosis occurrence and development. However, the molecular mechanisms of osteogenic differentiation remain unclear. This study explored whether microfibrillar-associated protein 5 (MFAP5) regulated BMSCs osteogenic differentiation. Methods We used shRNA or cDNA to knock down or overexpress MFAP5 in C3H10 and MC3T3-E1 cells. AR-S- and ALP-staining were performed to quantify cellular osteogenic differentiation. The mRNA levels of the classical osteogenic differentiation biomarkers Runx2, Col1α1, and OCN were quantified by qRT-PCR. Finally, we employed Western blotting to measure the levels of Wnt/β-catenin and AMPK signaling proteins. Results At days 0, 3, 7, and 14 after osteogenic induction, AR-S- and ALP-staining was lighter in MFAP5 knockdown compared to control cells, as were the levels of Runx2, Col1α1 and OCN. During osteogenesis, the levels of β-catenin, p-GSK-3β, AMPK, and p-AMPK were upregulated, while that of GSK-3β was downregulated, indicating that Wnt/β-catenin and AMPK signaling were activated. The relevant molecules were expressed at lower levels in the knockdown than control group; the opposite was seen for overexpressing cell lines. Conclusions MFAP5 regulates osteogenesis via Wnt/β‑catenin- and AMPK-signaling; MFAP5 may serve as a therapeutic target in patients with osteoporosis.
Background Increased adipogenesis and reduced osteogenesis of bone marrow stromal cells (BMSCs) are key features of glucocorticoid-induced osteoporosis (GIOP). However, the mechanism of controlling the differentiation balance of bone marrow stromal cells (BMSCs) is still unclear. Recent years, studies on the relationship between cholesterol metabolism and bone metabolism have been increasing. Various cholesterol metabolism-related molecules have been proved to be involved in BMSCs differentiation. Cholesterol-25-Hydroxylase (CH25H) is a multi-transmembrane endoplasmic reticulum related enzyme which participated in various metabolic process, including immunity, stem cell differentiation and inflammation. Here we discovered and proved its role in regulating stem cell differentiation and explored the mechanism. Methods RNAi sequences or cDNA were designed to knockdown or overexpress CH25H in cells. Alkaline phosphatase (ALP), alizarin red S (AR-S) staining and Oil Red O staining were used to identify osteogenic or adipogenic differentiation ability of different cells. Meanwhile, biomarkers of osteogenesis or adipogenesis were tested by quantitative real time polymerase chain reaction (qRT-PCR) to quantify the degree of cell differentiation. Western blot was performed to detect the protein expression of CH25H and the key molecules of mTOR signaling pathway. Subcutaneous ectopic osteogenesis experiment in nude mice and immunohistochemistry (IHC) were applied to verify the results of in vitro experiments. Results CH25H could increase osteogenesis and suppress adipogenesis. Besides, the mTOR signaling pathway was found upregulated when knocking down CH25H. When using rapamycin, a specific inhibitor of mTOR, the regulating effect of osteo-adipogenic regulation were partly reversed. These results indicated that CH25H was a key regulator of BMSCs osteo-adipogenic differentiation and the mTOR signaling pathway was the downstream mechanism. Animal experiments have also got consistent results. Conclusion CH25H could promotes osteogenesis and suppresses adipogenesis of bone marrow stromal cells via mTOR signaling pathway.
Femoral head necrosis is responsible for severe pain and its incidence is increasing. Abnormal adipogenic differentiation and fat cell hypertrophy of bone marrow mesenchymal stem cells increase intramedullary cavity pressure, leading to osteonecrosis. By analyzing gene expression before and after adipogenic differentiation, we found that Microfibril-Associated Protein 5 (MFAP5) is significantly down-regulated in adipogenesis whilst the mechanism of MFAP5 in regulating the differentiation of bone marrow mesenchymal stem cells is unknown. The purpose of this study was to clarify the role of MAFP5 in adipogenesis and therefore provide a theoretical basis for future therapeutic options of osteonecrosis. By knockdown or overexpression of MFAP5 in C3H10 and 3T3-L1 cells, we found that MFAP5 was significantly down-regulated as a key regulator of adipogenic differentiation, and identified the underlying downstream molecular mechanism. MFAP5 directly bound to and inhibited the expression of Staphylococcal Nuclease And Tudor Domain Containing 1, an essential coactivator of PPARγ, exerting an important regulatory role in adipogenesis.
In the current research, spinal cord injury was repaired through a moderate treadmill exercise and carica papaya leaf extract-loaded nanocomposite hydrogel. In vitro studies showed that the carica papaya extract-loaded nanocomposite hydrogel augmented the metabolic function of the cells and showed a gradual degradation. In vivo study showed that the developed treatment strategy promoted spinal cord injury repair as shown in histopathological studies and BBB assay.
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