Neuroprotective effects of isosteviol sodium through increasing CYLD by the downregulation of miRNA-181b.

NF-κB signaling pathway plays a critical role in cerebral ischemic stroke. MicroRNA-181b (miR-181b) induces the expression of NF-κB signaling pathways indirectly, and isosteviol sodium (STVNa) protects against ischemic injury via the inhibition of NF-κB-mediated inflammatory and apoptotic responses. However, the function of miR-181b and the actual relationship between STVNa and miR-181b in the ischemia-induced activation of NF-κB signaling pathways remains unclear. In this study, we found that miR-181b expression was significantly decreased in N2A neuroblastoma cells after CoCl2-induced hypoxic injury in vitro. We further found, via western blot analysis and quantitative polymerase chain reaction assay, that altering miR-181b expression could induce changes in one of its target proteins, cylindromatosis (CYLD). Specifically, upregulation and downregulation of miR-181b (through transfection of either pre- or small interfering miR-181b, respectively) could negatively regulate CYLD protein levels as well as N2A cell survival rate and apoptosis following CoCl2-induced injury. Furthermore, STVNa treatment following ischemic injury significantly downregulated the expression of miR-181b to alter apoptotic proteins downstream of the NF-κB signaling pathway through increasing CYLD protein levels in vivo and in vitro. STVNa also had a protective effect on CoCl2-injured N2A cells, increasing cell survival rate, inhibiting apoptosis, reducing the damage of mitochondrial membrane potential (MMP), and the generation of reactive oxygen species (ROS). Together, these results suggest that STVNa may downregulate miRNA-181b to protect mouse brain with ischemia stroke and against hypoxic injury in N2A cells by repressing NF-κB signaling pathways through the activation of CYLD, providing a novel therapy for ischemic stroke.

Simultaneous determination of bambuterol and its two major metabolites in human plasma by hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry.

In this study, a rapid and sensitive hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (HILIC-UPLC-MS/MS) method was developed for simultaneous determination of bambuterol and its two major metabolites monocarbamate bambuterol and terbutaline in human plasma. All samples were simply precipitated using acetonitrile and separated on a UPLC-HILIC column under gradient elution with a mobile phase consisting of acetonitrile and water with the addition of 10mm ammonium acetate and 0.1% formic acid at 0.4 mL/min. The analytes were detected by a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte, and the intra- and inter-day precisions were <12.8%. The analytical runtime within 4.0 min per sample made this method suitable for high throughput determination. The validated method was successfully applied to a clinical pharmacokinetic study of bambuterol in eight healthy volunteers. Furthermore, the effects of the chromatographic conditions on the retention of the analytes on HILIC were investigated, and the benefits of HILIC were evaluated by comparing with a C18 column. The results indicated that liquid-liquid partition and the electrostatic interactions played an important role in the retention of the analytes on HILIC in this study. And HILIC offered particular advantages over RPLC approach in the aspects of the peak symmetry, the column efficiency, and the column pressure.