Adipocyte-secreted microvesicle-derived miR-148a regulates adipogenic and osteogenic differentiation by targeting Wnt5a/Ror2 pathway.

AIMS: Adipocyte-secreted microvesicles (MVs)-derived microRNAs (miRNAs) are relevant to adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteonecrosis of the femoral head (ONFH). Our aims are to investigate the mechanism of adipocyte-derived MVs-miR-148a in ONFH. MATERIALS AND METHODS: Adipocyte-derived MVs were identified via transmission electron microscopy and specific markers expression. The adipogenic and osteogenic differentiation were investigated by Oil-Red O staining, alkaline phosphatase (ALP) activity, Alizarin Red S (ARS) staining and osteogenic or adipogenic factors levels. Genes and proteins expression were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The relationship between miR-148a and Wnt5a was tested via dual-luciferase reporter analysis. The adipogenic differentiation and osteogenic differentiation in methylprednisolone (MPS)-induced ONFH rat model were assessed via hematoxylin-eosin (HE) staining, and immunohistochemical staining of collagen I (COL I). KEY FINDINGS: Adipocyte-derived MVs promoted adipogenic differentiation via increasing Oil-Red O staining positive cells, adiponectin (Adipoq), acid-binding protein 2 (aP2) and peroxisome proliferator-activated receptor γ (PPAR-γ) levels, and repressed osteogenic differentiation of BMSCs via decreasing ARS staining positive cells, ALP, Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) levels. MiR-148a was present in adipocyte-derived MVs, and miR-148a knockdown inhibited adipogenic differentiation and promoted osteogenic differentiation. Furthermore, Wnt5a expression was regulated by miR-148a. MiR-148a overexpression facilitated adipogenic differentiation and suppressed osteogenic differentiation via regulating the Wnt5a/Ror2 pathway. Adipocyte-derived MVs promoted adipogenic differentiation and inhibited osteogenic differentiation in MPS-induced ONFH rat model. SIGNIFICANCE: Adipocyte-derived MVs-miR-148a promoted adipogenic differentiation and suppressed osteogenic differentiation via targeting the Wnt5a/Ror2 pathway.

Water sources of dominant species in three alpine ecosystems on the Tibetan Plateau, China.

Plant water sources were estimated by two or three compartment linear mixing models using hydrogen and oxygen isotope (deltaD and delta18O) values of different components such as plant xylem water, precipitation and river water as well as soil water on the Tibetan Plateau in the summer of 2005. Four dominant species (Quercus aquifolioides, Pinus tabulaeformis, Salix rehderiana and Nitraria tangutorum) in three typical ecosystems (forest, shrub and desert) were investigated in this study. Stable isotope ratios of the summer precipitations and the soil water presented variations in spatial and temporal scales. delta18O values of N. tangutorum xylem water were constant in the whole growth season and very similar to those of deep soil water. Water sources for all of the plants came from both precipitations and soil water. Plants switched rapidly among different water sources when environmental water conditions changed. Rainwater had different contributions to the plants, which was influenced by amounts of precipitation. The percentage of plant xylem water derived from rainwater rose with an increase in precipitation. Water sources for broad-leaved and coniferous species were different although they grew in the same environmental conditions. For example, the broad-leaved species Q. aquifolioides used mainly the water from deep soil, while 92.5% of xylem water of the coniferous species P. tabulaeformis was derived from rainwater during the growth season. The study will be helpful for us to fully understand responses of species on the Tibetan Plateau to changes in precipitation patterns, and to assess accurately changes of vegetation distribution in the future.

Anti-apoptotic effect of insulin in the control of cell death and neurologic deficit after acute spinal cord injury in rats.

Recent studies confirmed that the new cell survival signal pathway of Insulin-PI3K-Akt exerted cyto-protective actions involving anti-apoptosis. This study was undertaken to investigate the potential neuroprotective effects of insulin in the pathogenesis of spinal cord injury (SCI) and evaluate its therapeutic effects in adult rats. SCI was produced by extradural compression using modified Allen's stall with damage energy of 40 g-cm force. One group of rats was subjected to SCI in combination with the administration of recombinant human insulin dissolved in 50% glucose solution at the dose of 1 IU/kg day, for 7 days. At the same time, another group of rats was subjected to SCI in combination with the administration of an equal volume of sterile saline solution. Functional recovery was evaluated using open-field walking, inclined plane tests, and motor evoked potentials (MEPs) during the first 14 days post-trauma. Levels of protein for B-cell lymphoma/leukemia-2 gene (Bcl-2), Caspase-3, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were quantified in the injured spinal cord by Western blot analysis. Neuronal apoptosis was detected by TUNEL, and spinal cord blood flow (SCBF) was measured by laser-Doppler flowmetry (LDF). Ultimately, the data established the effectiveness of insulin treatment in improving neurologic recovery, increasing the expression of anti-apoptotic bcl-2 proteins, inhibiting caspase-3 expression decreasing neuronal apoptosis, reducing the expression of proinflammatory cytokines iNOS and COX-2, and ameliorating microcirculation of injured spinal cord after moderate contusive SCI in rats. In sum, this study reported the beneficial effects of insulin in the treatment of SCI, with the suggestion that insulin should be considered as a potential therapeutic agent.

Interleukin-6 protects annulus fibrosus cell from apoptosis induced by interleukin-1 beta in vitro.

OBJECTIVE: To investigate the effect of interleukin-6 (IL-6) on the apoptosis of annulus fibrosus (AF) cell induced by interleukin-1beta (IL-1beta). METHODS: Cultured AF cells were divided into 6 groups and treated with no drug, 10 ng/mL IL-6, 10 ng/mL IL-1beta, 10 ng/mL IL-1beta and Z-VAD-FMK (a caspase-9 inhibitor), 10 ng/mL IL-1beta and 10 ng/mL IL-6, 10 ng/mL IL-1beta and 100 ng/mL IL-6, respectively. After three days of culture, the apoptosis rate, the positive rates of caspase-3, -8, and -9 of AF cells were detected with flow cytometry. RESULTS: The apoptosis rates of cells in group 1 to 6 were 2.67% +/- 1.08%, 2.71% +/- 0.53%, 20.37% +/- 1.57%, 11.34% +/- 0.67%, 18.17% +/- 0.74%, and 9.42% +/- 1.08%, respectively. There was no significant difference between group 1 and 2, while the apoptosis rates of group 4, 5, and 6 were significantly lower than group 3 (P = 0.001, P = 0.172, and P = 0.001, respectively). Positive rates of caspase-3 in group 5 (12.35% +/- 0.64%) and 6 (9.26% +/- 0.36%) were significantly lower than group 3 (17.14% +/- 0.72%; P = 0.001 and P < 0.001, respectively). And positive rates of caspase-9 in group 5 (15.13% +/- 1.45%) and 6 (10.17% +/- 2.50%) were significantly lower than group 3 (19.4% +/- 0.98% ; P = 0.014 and P = 0.004, respectively). But there was not obvious change of caspase-8 activity after IL-6 was added. CONCLUSION: IL-6 is capable of protecting AF cells from IL-1beta induced apoptosis in vitro. Mechanism of the protection is related with the inhibition of caspase-3 and -9 activities.

Effects of basic fibroblast growth factor on biological characteristics of osteoblasts.

OBJECTIVE: To elucidate the effects of exogenous basic fibroblast growth factor (bFGF) on biological characteristics of rat osteoblasts cultured in vitro. METHODS: The osteoblasts isolated from a Sprague-Dawley rat and cultured in vitro were treated with different concentrations of bFGF (5-50 ng/ml) respectively. At 24 hours after treatment, the proliferating cell nuclear antigen was measured with immunocytochemistry, alkaline phosphatase (ALP) activity was determined and the expression of transforming growth factor beta 1 (TGF-beta(1)) was detected to observe the effects of bFGF on growth and differentiation of osteoblasts. RESULTS: bFGF (5-50 ng/ml) could obviously promote the growth of osteoblasts. The intracellular expression of TGF-beta(1) mRNA increased significantly, but the intracellular ALP content decreased. CONCLUSIONS: bFGF can obviously stimulate the proliferation of osteoblasts and promote the synthesis of TGF-beta(1), but cannot promote the differentiation of osteoblasts.

[Study of rat osteoblasts transfected by transforming growth factor-beta 1 gene].

OBJECTIVE: To investigate the effect of transforming growth factor-beta 1 (TGF-beta 1) gene transfer on the biological characteristics of osteoblasts. METHODS: The expression of TGF-beta 1 in the transfected osteoblasts was detected by in situ hybridization and assay of TGF-beta 1 activity in the supernatant (mink lung epithelium cell growth-inhibition test). The effects of gene transfer and supernatant of the transfected osteoblasts on the proliferation and alkaline phosphatase(ALP) activity of osteoblasts were detected by 3H-TdR and MTT. RESULTS: The results of in situ hybridization analysis suggested that the osteoblasts transfected by TGF-beta 1 gene could express TGF-beta 1 obviously. The complex medium, which was the mixture of serum-free DMEM and the activated supernatant according to 1:1, 1:2, 1:4, could inhibit growth of Mv-1-Lu evidently and the ratios of inhibition were 16.3%, 22.7%, 28.2% respectively. TGF-beta 1 gene transfer had no effect on the biological characteristics of osteoblasts, but the activated supernatant of transfected osteoblasts stimulated proliferation and inhibited ALP activity of osteoblasts. CONCLUSION: TGF-beta 1 gene transfer promotes the expression of TGF-beta 1 and the biological characteristics of transfected osteoblasts are stable, which is helpful for gene therapy of bone defects in vivo.