The Neuroprotective Effect of Sophocarpine against Glutamate-Induced HT22 Cell Cytotoxicity.

Neuronal cell death and dysfunction of the central nervous system can be caused by oxidative stress, which is associated with the development of neurodegenerative diseases. Sophocarpine, an alkaloid compound derived from Sophora moorcroftiana (Benth.) Baker seeds, has a wide range of medicinal value. This study sought to determine how sophocarpine exerts neuroprotective effects by inhibited oxidative stress and apoptosis in mouse hippocampus neuronal (HT22) cells. 20mM glutamate-induced HT22 cells were used to develop an in vitro model of oxidative stress damage. The Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability. According to the instructions on the kits to detect reactive oxygen species (ROS) levels and oxidative stress indicators. HT22 cells were examined using immunofluorescence and Western Blotting to detect Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) expression. The expression of proteins and messenger RNA (mRNA) for heme oxygenase-1 (HO-1) was examined by Western Blotting and Quantitative real time polymerase chain reaction (qRT-PCR). Mitochondrial membrane potential (MMP) and Cell apoptosis were used by 5, 5', 6, 6'-Tetrachloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide (JC- 1) kit and Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay kit, respectively. Finally, the expression of pro-apoptotic proteins was detected by Western Blotting. The result demonstrated that sophocarpine (1.25 µM-10 µM) can significantly inhibit glutamate-induced cytotoxicity and ROS generation, improve the activity of antioxidant enzymes. Sophocarpine increased the expression of HO-1 protein and mRNA and the nuclear translocation of Nrf2 to play a cytoprotective role; however, cells were transfected with small interfering RNA targeting HO-1 (si-HO-1) reversed the above effects of sophocarpine. In addition, sophocarpine significantly inhibited glutamate induced mitochondrial depolarization and further inhibited cell apoptosis by reducing the expression level of caspase-related proteins.

Migration time correction for dual pressure capillary electrophoresis in semi-targeted metabolomics study.

Migration time fluctuation strongly affects peak alignment and identification of unknown compounds, making migration time correction an essential step in capillary electrophoresis (CE)-based metabolomics. To obtain more reliable information, metabolites with different apparent mobilities are analyzed by tandem mass spectrometry. Applying a small pressure is a common practice for reducing the analysis time of anions in a positive mode CE, known as the pressure-assisted CE. However, applying pressure may reduce the separation efficiency and can be undesirable for cation analysis. A simple way to address this issue is to increase the pressure after a certain time, during the separation. We term this practice as dual pressure CE. However, changing the pressure during the CE separation complicates migration time correction. Previous migration time correction methods were established based on a consistent electroosmotic flow and a constant pressure-driven bulk-flow velocity. We proposed a new correction method to support the peak alignment when dual pressure CE is used. A Python-based script was developed to implement dual pressure CE migration time correction for semi-targeted metabolomics study performed by a multiple reaction monitoring-based method. This script can help select suitable endogenous metabolites as correction markers, perform migration time correction, and conduct peak alignment. A case study showed that migration time precision of 156 metabolites in 32 samples can be improved from 4.8 to 11.4%RSD (relative standard deviation) to less than 1.8%RSD.

Responses of Spring Discharge to Different Rainfall Events for Single-Conduit Karst Aquifers in Western Hunan Province, China.

It is a challenge to describe the hydrogeological characteristics of karst aquifers due to the complex structure with extremely high heterogeneity. As the response of karst aquifers to rainfall events, spring discharge variations after precipitation can be used to identify the internal structure of karst systems. In this study, responses of spring discharge to different kinds of precipitations are investigated by continuously monitoring precipitation and karst spring flow at a single-conduit karst aquifer in western Hunan province, China. Recession curves were used to analyze hydrodynamic behaviors and separate recession stages. The results show that the shape of the recession curve was changed under different rainfall conditions. Recession processes can be divided in to three recession stages under heavy rain conditions due to water drainage mainly from conduits, fracture, and matrix at each stage, but only one recession stage representing drainage mainly from matrix in the case of light rain. With the change in amount and intensity of precipitation, the calculated recession coefficient at each stage changes in an order of magnitude. The influence of precipitation on the recharge coefficient and the discharge composition at each recession are discussed, and then the conceptual model diagram of water filling and releasing in the single-conduit karst aquifers is concluded. The findings provide more insight understand on hydraulic behaviors of karst spring under different types of rainfall events and provide support for water resource management in karst regions.

Inhibiting microRNA-424 in bone marrow mesenchymal stem cells-derived exosomes suppresses tumor growth in colorectal cancer by upregulating TGFBR3.

OBJECTIVE: MicroRNAs (miRNAs) have been demonstrated to be differently expressed in colorectal cancer (CRC) and were identified as biomarkers and therapeutic targets for CRC. We aimed to identify the effect of microRNA-424 (miR-424) on process of CRC. METHODS: Exosomes were obtained from bone marrow mesenchymal stem cells (BMSCs). MiR-424, transforming growth factor-ß receptor 3 (TGFBR3) vimentin, S100A4, p-Smad1 expression in tissues and cells was measured. After treated with miR-424 inhibitor or TGFBR3 overexpression plasmid, the migration, invasion, cell cycle distribution and apoptosis of Lovo cells and exosomes-transfected Lovo cells were determined. The subcutaneous tumor models were established and the tumor growth was observed. The target relation between miR-424 and TGFBR3 was confirmed. RESULTS: MiR-424 was upregulated while TGFBR3 was downregulated in CRC tissues. TGFBR3 was targeted by miR-424. Inhibited miR-424 or elevated TGFBR3 upregulated p-Smad1, indicating that TGFBR3 mediated the Smad1 pathway, thus regulating CRC progression. MiR-424 inhibition or TGFBR3 restoration also suppressed migration and invasion of CRC cells, arrested the CRC cells at G0/G1 phase, and promoted CRC cell apoptosis. Moreover, exosomal miR-424 from BMSCs promoted CRC development. CONCLUSION: Inhibited exosomal miR-424 from BMSCs inhibited malignant behaviors of CRC cells by targeting TGFBR3, thus suppressing the progression of CRC.

Relation between Ejection Mechanism and Ion Abundance in the Electric Double Layer of Droplets.

Charged droplets have been associated with distinct chemical reactivity. It is assumed that the composition of the surface layer plays a critical role in enhancing the reaction rates in the droplets relative to their bulk solution counterparts. We use atomistic modeling to relate the localization of ions in the surface layer to their ejection propensity. We find that ion ejection takes place via a two-stage process. First, a conical protrusion emerges as a result of a global droplet deformation that is insensitive to the locations of the single ions. The ions are subsequently ejected as they enter the conical regions. The study provides mechanistic insight into the ion-evaporation mechanism, which can be used to revise the commonly used ion-evaporation models. We argue that atomistic molecular dynamics simulations of minute nanodrops do not sufficiently distinguish the ion-evaporation mechanism from a Rayleigh fission. We explain mass spectrometry data on the charge state of small globular proteins and the existence of supercharged droplet states that have been detected in experiments.

Determination of urinary prostaglandin E2 as a potential biomarker of ureteral stent associated inflammation.

Ureteral stents are the most widely used surgical implant in urology. However, they may cause adverse effects to patients, including pain, discomfort, and inflammation. In this work, the inflammatory effect of stent placement and the associated elevation of cyclooxygenase-2 (COX-2) expression were observed. Furthermore, a capillary electrophoresis mass spectrometry (CE-MS) based approach was subsequently developed to quantify urinary prostaglandin E2 (PGE2), a COX-2 metabolite known to contribute to inflammatory renal diseases, to further interrogate the role of this pathway. Urine samples were cleaned and preconcentrated by solid-phase extraction (SPE), and an on-line sample stacking method was used for the enrichment of analytes. The accuracy, precision, and specificity of this method were validated. Standard addition methods were performed to assess the reliability of using deuterated internal standards (IS) in compensating the remaining matrix effect after SPE as well as the detector fluctuation. Through the analysis of 32 pig urine samples, a statistically significant increase of PGE2 was observed in the stented group compared to the unstented (P = 0.01) and the recovered (P = 0.004) groups. This work determined that stent placement may contribute to COX-2-dependent inflammation and developed a reliable CE-MS based methodology to quantify PGE2 in stented individuals that may further understand the biology of stent-associated inflammation and inform urologic patient management.

NIPAAm-MMA nanoparticle-encapsulated visnagin ameliorates myocardial ischemia/reperfusion injury through the promotion of autophagy and the inhibition of apoptosis.

The main method for the treatment of acute myocardial infarction (AMI) is percutaneous coronary intervention; however percutaneous coronary intervention will induce ischemia/reperfusion (IR) injury, resulting in the loss of cardiac function and cardiomyocyte death. An effective drug to target this condition is necessary. N-isopropylacrylamide and methacrylic acid were used to synthesize drug delivery nanoparticles (NP) containing the natural compound visnagin for IR injury treatment. It was demonstrated that NP containing fluorescein isothiocyanate localized to the site of myocardial IR, and that NP-visnagin treatment induced cardioprotection, reducing the size of the MI and ameliorating cardiac dysfunction through the induction of autophagy and the inhibition of apoptosis. In the future, visnagin may be suitable as a drug for IR injury treatment, and NP may be an effective drug delivery system.

Effects of recombinant adeno-associated virus-mediated CD151 gene transfer on the expression of rat vascular endothelial growth factor in ischemic myocardium.

The aim of this study was to observe the effects of cluster of differentiation (CD) 151 on the expression of vascular endothelial growth factor (VEGF) in ischemic myocardium by the injection of a recombinant adeno-associated virus (rAAV) vector carrying the human CD151 gene. A rat acute myocardial infarction model was established, and rAAV-CD151 was injected into the ischemic myocardium. Four weeks later, the ischemic myocardium was removed in order to detect the expression of exogenous CD151 mRNA by reverse transcriptase polymerase chain reaction. In addition, the expression of CD151 and VEGF was detected by western blot analysis to evaluate the effect of CD151 overexpression on VEGF expression. Four weeks after injection of the vector, exogenous CD151 mRNA was expressed in the myocardial tissues of the CD151 group, whereas it was not detected in sham surgery, model control or rAAV-green fluorescent protein (GFP) gene-treated groups. The expression levels of CD151 protein were significantly higher in the CD151 group compared with those in the other three groups (P<0.05). The VEGF expression level in the CD151 group was higher compared with those in the control and GFP groups (P>0.05). These results indicate that rAAV-CD151 effectively transfects rat myocardial tissues, and may promote angiogenesis of the ischemic myocardium, improve left ventricular function and increase VEGF expression to improve ventricular function.

Protein kinase B/Akt-dependent phosphorylation of glycogen synthase kinase-3beta in irradiated vascular endothelium.

The vascular endothelium plays a critical role in the response of cancer to ionizing radiation. Activation of the phosphoinositide-3-kinase/Akt pathway is one initial signaling event in irradiated endothelial cells. Specifically, a low dose of ionizing radiation (3 Gy) induces phosphorylation of Akt at Ser473 in the vascular endothelium within minutes of irradiation. However, signaling events that are downstream of Akt have not been well defined. Here, we show that phosphorylation of the Akt downstream target glycogen synthase kinase-3beta (GSK-3beta) at Ser9 also occurred within minutes of exposure to ionizing radiation. In addition, ionizing radiation caused the dissociation of GSK-3beta from the cell membrane, consistent with the inactivation of GSK-3beta enzyme activity. Overexpression of the dominant negative mutant Akt attenuated GSK-3beta phosphorylation at Ser9 and enhanced radiation-induced apoptosis. X-irradiated endothelial cells formed capillaries in both in vitro and in vivo models, whereas overexpression of the dominant negative mutant Akt inhibited capillary tubule formation. Studies using GSK-3beta antagonists showed that GSK-3beta activity was required for apoptosis in endothelial cells treated simultaneously with Akt antagonists and radiation. In mouse vascular models, radiation-induced microvascular destruction in response to Akt antagonists also required GSK-3beta function. These data indicate that on exposure of vascular endothelium to ionizing radiation, activation of Akt signaling contributes to GSK-3beta inhibition, which in turn promotes endothelial cell survival and capillary formation. Thus, pharmacologic regulation of Akt/GSK-3beta signaling may present a new approach to the radiation response in the tumor microvasculature.

SU11248 maintenance therapy prevents tumor regrowth after fractionated irradiation of murine tumor models.

Receptor tyrosine kinase activation contributes to cell viability during cytotoxic therapy. The novel broad spectrum receptor tyrosine kinase inhibitor, SU11248, inhibits vascular endothelial growth factor receptor 2, platelet-derived growth factor receptor, c-kit, and fetal liver tyrosine kinase 3. In this study, we maintained SU11248 plasma levels beyond the completion of radiotherapy to determine whether tumor regrowth can be delayed. The antiangiogenic effects of SU11248 were demonstrated using human umbilical vein endothelial cells in vitro. Apoptosis increased and clonogenic survival decreased when SU11248 was used in combination with radiation from 0 to 6 Gy on endothelial cells. In vivo tumor growth delay was increased in C57B6J mice with Lewis lung carcinoma or glioblastoma multiform (GL261) hind limb tumors. Mice were treated with daily i.p. injections (40 mg/kg) of SU11248 during 7 days of radiation treatment (21 Gy). Combined treatment with SU11248 and radiation significantly reduced tumor volume as compared with either treatment alone. Concomitant reduction in vasculature was confirmed using the dorsal vascular window model. The vascular length established using images taken from a consistent quadrant in the window show the combination therapy was more effective in destroying tumor vasculature than either treatment alone. SU11248 maintenance administration beyond the completion of radiotherapy results in prolongation of tumor control. In summary, SU11248 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. Moreover, inhibition of angiogenesis well beyond radiation therapy may be a promising treatment paradigm for refractory human neoplasms.