Image1_Bone marrow mesenchymal stem cell exosomes-derived microRNA-216a-5p on locomotor performance, neuronal injury, and microglia inflammation in spinal cord injury.TIF

Background: MicroRNA-216a-5p (miR-216a-5p) mediates inflammatory responses and neuronal injury to participate in the pathology of spinal cord injury (SCI). This study intended to explore the engagement of bone marrow mesenchymal stem cell exosomes (BMSC-Exo)-derived miR-216a-5p in locomotor performance, neuronal injury, and microglia-mediated inflammation in SCI rats.Methods: Rat BMSC or BMSC-Exo was injected into SCI rats. GW4869 treatment was adopted to suppress the exosome secretion from BMSC. Subsequently, miR-216a-5p-overexpressed BMSC-Exo (BMSC-miR-Exo) or negative-control-overexpressed BMSC-Exo (BMSC-NC-Exo) were injected into SCI rats.Results: The injection of BMSC or BMSC-Exo enhanced locomotor performance reflected by Basso, Beattie & Bresnahan score (p + cells (p +iNOS+ cells, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 (p Conclusion: BMSC-Exo-derived miR-216a-5p enhances functional recovery by attenuating neuronal injury and microglia-mediated inflammation in SCI, which may be attributable to its inhibition of the TLR4/NF-κB pathway.</p

JSI-124 inhibites the U87MG cells–induced tube formation of HUVECs.

<p>(A) U87MG cells were treated with or without JSI-124 for 8 hours and then incubated with fresh media without JSI-124 for 24 hours, followed by the collection of conditioned media (CM). (B) VEGF level was analyzed in the conditioned medium by ELISA.</p

JSI-124 inhibits cell viability and induces apoptosis in HUVECs.

<p>(A) Effect of JSI-124 on HUVECs under normal culture condition. HUVECs (5×10³/ well) were treated with different concentrations of JSI-124 for 24 and 48 h. (B) JSI-124 induced HUVECs apoptosis dose-dependently detected by flow cytometric assay. (C) JSI-124 induced GBM cells apoptosis dose-dependently detected by cell death detection ELISA^Plus assay. All data were expressed as mean ± SD. * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.01 versus control. (D) and (E) JSI-124 downregulated the level of p-STAT3, decreased the expression of Bcl-2 and Bcl-xL, and induced cleavage of caspase-3 and PARP in HUVECs in a dose-dependent manner by western blot analysis. GAPDH was shown as loading control.</p

JSI-124 Suppresses Invasion and Angiogenesis of Glioblastoma Cells <i>In Vitro</i>

<div><p>Glioblastoma multiforme (GBM) is one of the utmost malignant tumors. Excessive angiogenesis and invasiveness are the major reasons for their uncontrolled growth and resistance toward conventional strategies resulting in poor prognosis. In this study, we found that low-dose JSI-124 reduced invasiveness and tumorigenicity of GBM cells. JSI-124 effectively inhibited VEGF expression in GBM cells. In a coculture study, JSI-124 completely prevented U87MG cell–mediated capillary formation of HUVECs and the migration of HUVECs when cultured alone or cocultured with U87MG cells. Furthermore, JSI-124 inhibited VEGF-induced cell proliferation, motility, invasion and the formation of capillary-like structures in HUVECs in a dose-dependent manner. JSI-124 suppressed VEGF-induced p-VEGFR2 activity through STAT3 signaling cascade in HUVECs. Immunohistochemistry analysis showed that the expression of CD34, Ki67, p-STAT3 and p-VEGFR2 protein in xenografts was remarkably decreased. Taken together, our findings provide the first evidence that JSI-124 effectively inhibits tumor angiogenesis and invasion, which might be a viable drug in anti-angiogenesis and anti-invasion therapies.</p></div

JSI-124 inhibites the U87MG cells–induced migration of HUVECs.

<p>U87MG cells were cultured in the well (lower chamber). Cells were then treated with or without JSI-124 for 8 hours. After treatment, the medium from the lower chamber was replaced with fresh DMEM without JSI-124, and the insert containing a monolayer of HUVECs in DMEM was then placed into the well. After 8 hours, migrated cells were photographed and counted. (A) Schematic diagram of coculture assay. (B) Control. HUVECs were seeded onto the upper chamber, whereas the bottom chamber contains only DMEM without U87MG cells. (C) HUVECs cocultured with U87MG cells untreated with JSI-124. (D) HUVECs cocultured with U87MG cells pretreated with 100 nM. All data were expressed as mean ± SD. * <i>P</i> < 0.05, versus control.</p

JSI-124 inhibits tumor growth and angiogenesis in U87MG cells xenograft model.

<p>U87MG cells (5×10⁶ cells per mouse) were inoculated subcutaneously into the right flank of 5-wk-old female Babl/c nude mice. Tumors reached a mean volume of 90 to 120 mm³, mice were randomly assigned to JSI-124 (1 mg/kg/day, in 20% DMSO in PBS) or drug vehicle control (20% DMSO in PBS) and dosed i.p. with 100μl vehicle of drug once daily for 18 days. (A) JSI-124 significantly inhibited tumor growth measured by tumor volume. (B) JSI-124 had little toxicity in the amount exposed measured by mice body weight. (C) JSI-124 strongly suppressed solid tumors. (D) Immunohistochemical staining result of CD34, Ki67, p-VEGFR2 and p-STAT3 on tumor sections. Data were presented as means ± SD. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 versus control group.</p

JSI-124 inhibits VEGF expression in GBM cells.

<p>Cells were exposed to different doses of JSI-124 for 24 hours and VEGF protein released by GBM cells into the conditioned medium was measured by ELISA kit. All data were expressed as mean ± SD. * <i>P</i> < 0.05, versus control.</p

JSI-124 inhibits VEGF-induced migration, invasion and tubular structure formation of endothelial cells.

<p>(A) Effect of JSI-124 on VEGF-induced HUVECs proliferation determined by CCK-8 assay. HUVECs (5×10³/ per well) were starved with 0.1% FBS medium and then treated with or without VEGF (20 ng/mL) and different concentrations of JSI-124 for 24 h. (B) JSI-124 remarkably inhibited VEGF-induced endothelial cells migration measured by transwell migration assay. HUVECs were seeded in the upper chamber of transwell (coated no matrigel) and treated with different concentrations of JSI-124. The bottom chamber was filled with ECM supplemented with VEGF. After about 8 h, the migrated HUVECs passed through the membrane were quantified. (C) JSI-124 strongly suppressed VEGF-induced endothelial cells invasion measured by transwell invasion assay. HUVECs were seeded in the upper chamber of transwell (coated with 50% matrigel) and treated with different concentrations of JSI-124. The bottom chamber was filled with ECM supplemented with VEGF. After about 8 h, the migrated HUVECs passed through the membrane were quantified. (D) JSI-124 inhibits VEGF-induced tube formation of HUVECs determined by capillary-like tube formation assay. Cells (4×10⁴/ well) were placed in the 96-well plates coated with matrigel. After 4 h of incubation, tubular structure formation was captured under microscope. All data were expressed as mean ± SD. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 versus VEGF alone.</p

JSI-124 inhibits activation of VEGFR2 and JAK2/STAT3 signaling induced by VEGF in HUVECs.

<p>(A) JSI-124 suppressed the activation of VEGFR2 induced by VEGF in HUVECs in a dose-dependent manner by western blot analysis. (B) Inhibition p-VEGFR2 by JSI-124 resulted in an inhibition of JAK2/STAT3 pathway activation triggered by VEGF in HUVECs dose-dependently by western blot analysis. GAPDH was shown as loading control.</p

JSI-124 inhibits cell viability and induces apoptosis in GBM cells.

<p>(A) JSI-124 at high dose suppressed viability of GBM cell lines dose-dependently determined by CCK-8 assay. (B) JSI-124–mediated GBM cells apoptosis was detected by flow cytometric assay. (C) JSI-124–induced GBM cells apoptosis was measured by cell death detection ELISA^Plus assay. All data were expressed as mean ± SD. * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001 versus control.</p