Hexarelin attenuates atherosclerosis via inhibiting LOX-1-NF-κB signaling pathway-mediated macrophage ox-LDL uptake in ApoE-/- mice.

Growth hormone secretagogues (GHS) have been proved to exert protective effects on the cardiovascular system, while their potential beneficial effects on macrophages in atherosclerosis (AS) are rarely been clarified. This study aimed to demonstrate whether hexarelin, a synthetic peptidyl GHS, can suppress AS progression via regulating the function of macrophages. AS was induced by chronic (3 months) feeding with high lipid diet in ApoE-/- mice. Mice were treated either with hexarelin (100 µg/kg s.c., q.d. for 3 months) (AS + Hex group) or saline (AS group). Age-matched C57BL/6 J mice were used as normal controls. AS and related signaling molecules in aortic tissues and RAW264.7 macrophages were identified with variant methods including histological staining, ELISA, western blotting, confocal microscopy and flow cytometry. AS significantly developed in ApoE-/- mice fed with high lipids diet. Hexarelin decreased serum TC, TG and LDL-c, increased serum HDL-c and attenuated the formation of atherosclerotic plaques and neointima compared with the AS group. Hexarelin decreased the aortic expressions of CD68 and LOX-1 which were elevated in the AS group. Hexarelin increased GHSR expression, suppressed ox-LDL uptake and LOX-1 expression and inhibited nuclear factor-kappa B (NF-κB) activation both in the aorta of ApoE-/- mice and in RAW264.7 macrophages. We conclude that hexarelin effectively attenuates AS progression in ApoE-/- mice by modulating circulatory lipids profile and inhibiting macrophage ox-LDL uptake via suppressing the LOX-1-NF-κB signaling pathway. The study supports the perspective of hexarelin as an anti-AS drug.

Investigation of the key factors on 3-hydroxypropionic acid production with different recombinant strains.

3-Hydroxypropionic acid (3-HP) is an important compound and precursor for a series of chemicals and polymeric materials. In this study, the 3-HP producing bacteria were constructed and studied for efficient synthesis of 3-HP. The results indicated that the instability of glycerol dehydratase (GDHt) affected the 3-HP production significantly, which was successfully solved by the expression of glycerol dehydratase reactivase (GdrB), with fivefold increase in 3-HP yield. Meanwhile, NAD+-regenerating enzymes GPD1 (glycerol-3-phosphate dehydrogenase) was expressed; however, the results showed 3-HP was significantly decreased from 56.73-4 mM, and malic acid was obviously increased. Analysis of the C flux distribution showed that the main reason for the results was the lack of NAD+. The addition of NAD+ further increased the 3-HP production to 23.87 mM, demonstrating that the "regeneration of NAD+" was the major factor for enhancing 3-HP production.

[Construction and identification of BKCaαsubunit expression plas-mid with double labeling of Flag and GFP].

OBJECTIVE: This study aimed to construct a large conductance calcium activated potassium channel α (BKCa) subunit plasmid with two tags by the overlapping PCR technique to set up a steady base for future ion channel study. METHODS: Based on the existing coding BKCa channel α subunit expression plasmid pcDNA3.1-hSlo, we constructed a double-tag expression plasmid, namely, pcDNA3.1-Flag-hSlo-GFP (Flag-hSlo-GFP). RESULTS: Flag tag was inserted into the S1-S2 extracellular loop of BKCa channel α subunit, and GFP tag was connected to the C-terminus of BKCa channel α subunit. Sequence of the constructed plasmid was confirmed successful. CONCLUSIONS: The expression plasmid Flag-hSlo-GFP was constructed successfully with overlapping PCR. Overlapping PCR is a valuable method for amplifying long size genes.

Activation of AMPA receptor promotes TNF-α release via the ROS-cSrc-NFκB signaling cascade in RAW264.7 macrophages.

The relationship between glutamate signaling and inflammation has not been well defined. This study aimed to investigate the role of AMPA receptor (AMPAR) in the expression and release of tumor necrosis factor-alpha (TNF-α) from macrophages and the underlying mechanisms. A series of approaches, including confocal microscopy, immunofluorescency, flow cytometry, ELISA and Western blotting, were used to estimate the expression of AMPAR and downstream signaling molecules, TNF-α release and reactive oxygen species (ROS) generation in the macrophage-like RAW264.7 cells. The results demonstrated that AMPAR was expressed in RAW264.7 cells. AMPA significantly enhanced TNF-α release from RAW264.7 cells, and this effect was abolished by CNQX (AMPAR antagonist). AMPA also induced elevation of ROS production, phosphorylation of c-Src and activation of nuclear factor (NF)-κB in RAW264.7 cells. Blocking c-Src by PP2, scavenging ROS by glutathione (GSH) or inhibiting NF-κB activation by pyrrolidine dithiocarbamate (PDTC) decreased TNF-α production from RAW264.7 cells. We concluded that AMPA promotes TNF-α release in RAW264.7 macrophages likely through the following signaling cascade: AMPAR activation â†’ ROS generation â†’ c-Src phosphorylation â†’ NF-κB activation â†’ TNF-α elevation. The study suggests that AMPAR may participate in macrophage activation and inflammation.

Tanshinone II-A sodium sulfonate (DS-201) enhances human BKCa channel activity by selectively targeting the pore-forming α subunit.

AIM: Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BKCa channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BKCa channels. METHODS: Human BKCa channels containing α subunit alone or α plus ß1 subunits were expressed in HEK293 cells. BKCa currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BKCa subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy. RESULTS: DS-201 (40-160 µmol/L) concentration-dependently increased the total open probability of BKCa channels in HEK293 cells, associated with enhancements of Ca(2+) and voltage dependence as well as a delay in deactivation. Coexpression of ß1 subunit did not affect the action of DS-201: the values of EC50 for BKCa channels containing α subunit alone and α plus ß1 subunit were 66.6±1.5 and 62.0±1.1 µmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 µmol/L) markedly increased the expression of α subunit without affecting ß1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 µmol/L) induced significant cytotoxicity. CONCLUSION: DS-201 selectively targets the pore-forming α subunit of human BKCa channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the ß1 subunit does not contribute to the action of DS-201.

Multi-walled carbon nanotubes impair Kv4.2/4.3 channel activities, delay membrane repolarization and induce bradyarrhythmias in the rat.

PURPOSE: The potential hazardous effects of multi-walled carbon nanotubes (MWCNTs) on cardiac electrophysiology are seldom evaluated. This study aimed to investigate the impacts of MWCNTs on the Kv4/Ito channel, action potential and heart rhythm and the underlying mechanisms. METHODS: HEK293 cells were engineered to express Kv4.2 or Kv4.3 with or without KChIP2 expression. A series of approaches were introduced to analyze the effects of MWCNTs on Kv4/Ito channel kinetics, current densities, expression and trafficking. Transmission electron microscopy was performed to observe the internalization of MWCNTs in HEK293 cells and rat cardiomyocytes. Current clamp was employed to record the action potentials of isolated rat cardiomyocytes. Surface ECG and epicardial monophasic action potentials were recorded to monitor heart rhythm in rats in vivo. Vagal nerve discharge monitoring and H&E staining were also performed. RESULTS: Induction of MWCNTs into the cytosole through pipette solution soon accelerated the decay of IKv4 in HEK293 cells expressing Kv4.2/4.3 and KChIP2, and promoted the recovery from inactivation when Kv4.2 or Kv4.3 was expressed alone. Longer exposure (6 h) to MWCNTs decreased the IKv4.2 density, Kv4.2/Kv4.3 (but not KChIP2) expression and trafficking towards the plasma membrane in HEK293 cells. In acutely isolated rat ventricular myocytes, pipette MWCNTs also quickly accelerated the decay of IKv4 and prolonged the action potential duration (APD). Intravenous infusion of MWCNTs (2 mg/rat) induced atrioventricular (AV) block and even cardiac asystole. No tachyarrhythmia was observed after MWCNTs administration. MWCNTs did not cause coronary clot but induced myocardial inflammation and increased vagus discharge. CONCLUSIONS: MWCNTs suppress Kv4/Ito channel activities likely at the intracellular side of plasma membrane, delay membrane repolarization and induce bradyarrhythmia. The delayed repolarization, increased vagus output and focal myocardial inflammation may partially underlie the occurrence of bradyarrhythmias induced by MWCNTs. The study warns that MWCNTs are hazardous to cardiac electrophysiology.

[Chemical constituents of flavonoids from Apocynum venetum].

OBJECTIVE: To study the chemical constituents of flavonoids from Apocynum venetum L. METHODS: The compounds from water extracts were isolated by HPD-100 MCI-gel, polyamide, silica and Sephadex LH-20. Their structrues were characterized by chemical methods and spectroscopic methods. RESULTS: The five compounds were identified to be trifolin (I), hyperroside (III), hyperin (V), isoquercetin-6'-o-acetate (VII) and isoquercetin (VIII). CONCLUSION: Compound I and compound VII are isolated from Apocynum venetum L. for the first time.

Research into methylation of p16 gene in the patients suffering from coal-burnt arsenism in Xingren County Guizhou Province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To probe into the situation and significance of p16 gene CPG island methylation in patients with arseniasis caused by coal-burning pollution.</p><p><b>METHODS</b>DNA was extracted using the Phenol-Chloroform method from leukocytes of 51 patients suffered from coal-burnt arsenism and 52 healthy volunteers. The quantity of the DNA was determined by UV spectrophotometry. Target DNA was denatured by NaOH, then the single strand DNA was modified by sodium bisulfite, converting all unmethylated (but not the methylated) cytosines to uracil. Subsequently a nested amplification with primers specific for methylated versus unmethylated DNA was performed, and PCR products were detected by gel electrophoresis.</p><p><b>RESULTS</b>Hypermethylation of the p16 CPG island was presented in 94.1% of the patients suffering from coal-burnt arsenism and in 73.1% of the healthy volunteers. There was statistical difference (P < 0.05) between them.</p><p><b>CONCLUSIONS</b>Methylation of p16 gene CPG island should have important pertinence in the metabolism of coal-burnt arsenism.</p>