miR-200b-3p/ERG/PTHrP axis mediates the inhibitory effect of ethanol on the differentiation of fetal cartilage into articular cartilage.

PURPOSE: This study aims to further explore cartilage development in prenatal ethanol exposure (PEE) offspring at different times to explore the specific time points and mechanism of ethanol-induced fetal cartilage dysplasia. METHODS: On gestational day (GD)14, GD17, and GD20, PEE fetal cartilage was evaluated by morphological analysis. RT-qPCR, immunohistochemistry, and immunofluorescence were used to detect the expression of cartilage marker genes and their regulatory factors. Bone marrow mesenchymal stem cells (BMSCs) were used to explore the effect of ethanol on the differentiation of chondrocytes. Additionally, we used inhibitors, overexpression plasmids and a luciferase reporter assay on GD17 chondrocytes to verify the mechanism. RESULTS: PEE significantly reduced cartilage matrix content and the expression of marker genes on GD17 and GD20 but had no effect on GD14. The inhibition of chondrogenic differentiation by PEE mainly occurred on GD14-17. Furthermore, the expression of miR-200b-3p was increased, while that of ERG and PTHrP was markedly reduced in PEE fetal cartilage. In vitro, ethanol (30-120 mM) inhibited the differentiation of BMSCs into chondrocytes in a concentration-dependent manner, accompanied by strong expression of miR-200b-3p and low expression of ERG and PTHrP. Moreover, PTHLH and ERG overexpressed, as well as a miR-200b-3p inhibitor reversed the inhibitory effect of ethanol on the differentiation of fetal chondrocytes. Furthermore, miR-200b-3p could target and negatively regulate ERG. CONCLUSIONS: PEE can significantly inhibit the development of articular cartilage, especially during articular cartilage formation. The mechanism is related to the decreased differentiation of fetal cartilage into articular cartilage mediated by the miR-200b-3p/ERG/PTHrP axis.

Isorhynchophylline ameliorates the progression of osteoarthritis by inhibiting the NF-κB pathway.

Osteoarthritis (OA), a progressive and degenerative joint disease, is characterized by cartilage degradation, synovitis, subchondral bone remodeling and osteophyte formation. Isorhynchophylline (IRN) is an oxindole alkaloid isolated from the traditional Chinese herb Uncaria rhynchophylla. In this study, we evaluated the protective effects of IRN on human OA chondrocytes. IRN treatment dose-dependently decreased the interleukin-1ß (IL-1ß)-induced expressions of nitric oxide (NO; p < 0.001), prostaglandin E2 (PGE2; p < 0.001), tumor necrosis factor alpha (TNF-α; p < 0.001), interleukin-6 (IL-6; p < 0.001), cyclooxygenase-2 (COX-2; p < 0.001) and inducible nitric oxide synthase (iNOS; p < 0.001) in chondrocytes. Meanwhile, the production of metalloproteinase 13 (MMP13; p < 0.001) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5; p < 0.001) was inhibited by IRN treatment. Molecular docking studies revealed that IRN directly interacted with the nuclear factor kappa B (NF-κB) complex, which was associated with a reduced level of NF-κB nuclear translocation and the inhibition of NF-κB signaling activity. Furthermore, administration of IRN generated marked in vivo protective effects during OA development. Collectively, our results demonstrate that IRN may exhibit therapeutic benefits against OA, potentially by ameliorating the inflammative and degenerative progression of OA via inhibiting the NF-κB pathway.

Autologous Dedifferentiated Osteogenic Bone Marrow Mesenchymal Stem Cells Promote Bone Formation in a Rabbit Model of Anterior Cruciate Ligament Reconstruction versus Bone Marrow Mesenchymal Stem Cells.

PURPOSE: This study aimed to verify whether transplantation of dedifferentiated osteogenic bone marrow mesenchymal stem cells (De-BMSCs) at the tendon-bone interface could result in more bone formation than BMSC transplantation in anterior cruciate ligament (ACL) reconstruction. METHODS: BMSCs from femur and tibia of New Zealand White rabbit were subjected to osteogenic induction and then cultured in osteogenic factor-free medium; the obtained cell population was termed De-BMSCs. Bilateral ACL reconstruction was performed in 48 adult rabbits. Three groups were established: control group with alginate gel injection, BMSCs group with the BMSCs injection, and De-BMSCs group with the De-BMSCs injection. At week 4 and 12 postoperatively, tendon-bone healing by histologic staining, micro-computed tomography examination, and biomechanical test were evaluated. RESULTS: The expression of α1 chain of type I collagen, osteocalcin, and osteopontin at the tendon-bone interface in the De-BMSCs group was greater than in the control or BMSCs group. The bone volume/total volume by micro-computed tomography scan was significantly greater in the De-BMSCs group than that in the control group (P = .013) or BMSCs group (P = .045) at 4 weeks, and greater than that in the control group (P = .014) or BMSCs group (P = .017) at 12 weeks. The tunnel area was significantly smaller in the De-BMSCs group than in the control group (P = .013) or BMSCs group (P = .044) at 12 weeks. The failure load and stiffness in De-BMSCs group were both significantly enhanced at 4 and 12 weeks than control group or De-BMSCs group. CONCLUSIONS: De-BMSCs transplantation can promote bone formation at the tendon-bone interface better than BMSCs transplantation in ACL reconstruction and increase the early biomechanical strength of the reconstructed ACL CLINICAL RELEVANCE: De-BMSCs transplantation is a potential choice for enhancing early bone formation in the tunnel in ACL reconstruction.

GR/Sp3/HDAC1/UGDH signaling participated in the maternal dexamethasone-induced dysplasia of the rat fetal growth plate.

Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2 mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.