Improvement of Pharmacokinetics Behavior of Apocynin by Nitrone Derivatization: Comparative Pharmacokinetics of Nitrone-Apocynin and its Parent Apocynin in Rats

Apocynin, a potent inhibitor of NADPH-oxidase, was widely studied for activities in diseases such as inflammation-mediated disorders, asthma and cardiovascular diseases. In our recent study, a novel nitrone derivative of apocynin, AN-1, demonstrated potent inhibition to oxidative injury and to high expression of gp91^phox subunit of NADPH-oxidase induced by tert-butyl hydroperoxide (t-BHP) in RAW 264.7 macrophage cells, and displayed promising preclinical protective effect against lipopolysaccharide (LPS)-induced acute lung injury in rats. In this work, the pharmacokinetic behaviors of AN-1 in Sprague-Dawley rats with single intravenous and intragastric doses were investigated for further development. Furthermore, apocynin’s pharmacokinetics remain lacking, even though its pharmacological action has been extensively evaluated. The pharmacokinetics of parent apocynin were also comparatively characterized. A simple HPLC method was developed and validated to determine both AN-1 and apocynin in rat plasma. The chromatographic separation was achieved on an Agilent HC-C18 column (250 mm×4.6 mm, 5 µm) at an isocratic flow rate of 1.0 mL/min, with the mobile phase of methanol and water (53∶47, v/v) and the UV detection set at 279 nm. Good linearity was established over the concentration range of 0.1–500 µg/mL for AN-1 and 0.2–100 µg/mL for apocynin. The absolute recovery, precision and accuracy were satisfactory. Compared with the parent compound apocynin, AN-1 yielded a much longer T_1/2 (AN-1 179.8 min, apocynin 6.1 min) and higher AUC_0–t (AN-1 61.89 mmol/L·min, apocynin 2.49 mmol/L·min) after equimolar intravenous dosing (0.302 mmol/kg). The absolute bioavailability of oral AN-1 was 78%, but that of apocynin was only 2.8%. The significant improvement of pharmacokinetic behavior might be accounted for the effective pharmacodynamic results we documented for the novel nitrone derivative AN-1.     

Performance of Shear Wave Elastography for Differentiation of Benign and Malignant Solid Breast Masses

Objectives

To perform a meta-analysis assessing the ability of shear wave elastography (SWE) to identify malignant breast masses.

Methods

PubMed, the Cochrane Library, and the ISI Web of Knowledge were searched for studies evaluating the accuracy of SWE for identifying malignant breast masses. The diagnostic accuracy of SWE was evaluated according to sensitivity, specificity, and hierarchical summary receiver operating characteristic (HSROC) curves. An analysis was also performed according to the SWE mode used: supersonic shear imaging (SSI) and the acoustic radiation force impulse (ARFI) technique. The clinical utility of SWE for identifying malignant breast masses was evaluated using analysis of Fagan plot.

Results

A total of 9 studies, including 1888 women and 2000 breast masses, were analyzed. Summary sensitivities and specificities were 0.91 (95% confidence interval [CI], 0.88–0.94) and 0.82 (95% CI, 0.75–0.87) by SSI and 0.89 (95% CI, 0.81–0.94) and 0.91 (95% CI, 0.84–0.95) by ARFI, respectively. The HSROCs for SSI and ARFI were 0.92 (95% CI, 0.90–0.94) and 0.96 (95% CI, 0.93–0.97), respectively. SSI and ARFI were both very informative, with probabilities of 83% and 91%, respectively, for correctly differentiating between benign and malignant breast masses following a “positive” measurement (over the threshold value) and probabilities of disease as low as 10% and 11%, respectively, following a “negative” measurement (below the threshold value) when the pre-test probability was 50%.

Conclusions

SWE could be used as a good identification tool for the classification of breast masses.     

Neurotoxicity of Perfluorooctane Sulfonate to Hippocampal Cells in Adult Mice

Perfluorooctane sulfonate (PFOS) is a ubiquitous pollutant and found in the environment and in biota. The neurotoxicity of PFOS has received much concern among its various toxic effects when given during developing period of brain. However, little is known about the neurotoxic effects and potential mechanisms of PFOS in the mature brain. Our study demonstrated the neurotoxicity and the potential mechanisms of PFOS in the hippocampus of adult mice for the first time. The impairments of spatial learning and memory were observed by water maze studies after exposure to PFOS for three months. Significant apoptosis was found in hippocampal cells after PFOS exposure, accompanied with a increase of glutamate in the hippocampus and decreases of dopamine (DA) and 3,4-dihydrophenylacetic acid (DOPAC) in Caudate Putamen in the 10.75 mg/kg PFOS group. By two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) analysis, seven related proteins in the hippocampus that responded to PFOS exposure were identified, among which, Mib1 protein (an E3 ubiquitin-protein ligase), Herc5 (hect domain and RLD 5 isoform 2) and Tyro3 (TYRO3 protein tyrosine kinase 3) were found down-regulated, while Sdha (Succinate dehydrogenase flavoprotein subunit), Gzma (Isoform HF1 of Granzyme A precursor), Plau (Urokinase-type plasminogen activator precursor) and Lig4 (DNA ligase 4) were found up-regulated in the 10.75 mg/kg PFOS-treated group compare with control group. Furthermore, we also found that (i) increased expression of caspase-3 protein and decreased expression of Bcl-2, Bcl-XL and survivin proteins, (ii) the increased glutamate release in the hippocampus. All these might contribute to the dysfunction of hippocampus which finally account for the impairments of spatial learning and memory in adult mice.     

Porous Allograft Bone Scaffolds: Doping with Strontium

Strontium (Sr) can promote the process of bone formation. To improve bioactivity, porous allograft bone scaffolds (ABS) were doped with Sr and the mechanical strength and bioactivity of the scaffolds were evaluated. Sr-doped ABS were prepared using the ion exchange method. The density and distribution of Sr in bone scaffolds were investigated by inductively coupled plasma optical emission spectrometry (ICP-OES), X-ray photoelectron spectroscopy (XPS), and energy-dispersive X-ray spectroscopy (EDS). Controlled release of strontium ions was measured and mechanical strength was evaluated by a compressive strength test. The bioactivity of Sr-doped ABS was investigated by a simulated body fluid (SBF) assay, cytotoxicity testing, and an in vivo implantation experiment. The Sr molar concentration [Sr/(Sr+Ca)] in ABS surpassed 5% and Sr was distributed nearly evenly. XPS analyses suggest that Sr combined with oxygen and carbonate radicals. Released Sr ions were detected in the immersion solution at higher concentration than calcium ions until day 30. The compressive strength of the Sr-doped ABS did not change significantly. The bioactivity of Sr-doped material, as measured by the in vitro SBF immersion method, was superior to that of the Sr-free freeze-dried bone and the Sr-doped material did not show cytotoxicity compared with Sr-free culture medium. The rate of bone mineral deposition for Sr-doped ABS was faster than that of the control at 4 weeks (3.28±0.23 µm/day vs. 2.60±0.20 µm/day; p<0.05). Sr can be evenly doped into porous ABS at relevant concentrations to create highly active bone substitutes.     

Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca²⁺-activated K⁺ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit.     

Involvement of MAPK/NF-κB Signaling in the Activation of the Cholinergic Anti-Inflammatory Pathway in Experimental Colitis by Chronic Vagus Nerve Stimulation

Background

Autonomic nervous system dysfunction is implicated in the etiopathogenesis of inflammatory bowel diseases (IBD). Therapies that increase cardiovagal activity, such as Mind-Body interventions, are currently confirmed to be effective in clinical trials in IBD. However, a poor understanding of pathophysiological mechanisms limits the popularization of therapies in clinical practice. The aim of the present study was to explore the mechanisms of these therapies against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats using a chronic vagus nerve stimulation model in vivo, as well as the lipopolysaccharide (LPS)-induced inflammatory response in human epithelial colorectal adenocarcinoma cells (Caco-2) by acetylcholine in vitro.

Methods and Results

Colitis was induced in rats with rectal instillation of TNBS, and the effect of chronic VNS (0.25 mA, 20 Hz, 500 ms) on colonic inflammation was evaluated. Inflammatory responses were assessed by disease activity index (DAI), histological scores, myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS), TNF-α and IL-6 production. The expression of Mitogen-activated protein kinases (MAPK) family members, IκB-α, and nuclear NF-κB p65 were studied by immunoblotting. Heart rate variability (HRV) analysis was also applied to assess the sympathetic-vagal balance. DAI, histological scores, MPO activity, iNOS, TNF-α and IL-6 levels were significantly decreased by chronic VNS. Moreover, both VNS and acetylcholine reduced the phosphorylation of MAPKs and prevented the nuclear translocation of NF-κB p65. Methyllycaconitine (MLA) only reversed the inhibitory effect on p-ERK and intranuclear NF-κB p65 expression by ACh in vitro, no significant change was observed in the expression of p-p38 MAPK or p-JNK by MLA.

Conclusion

Vagal activity modification contributes to the beneficial effects of the cholinergic anti-inflammatory pathway in IBD-related inflamed colonic mucosa based on the activation of MAPKs and nuclear translocation of NF-κB. Our work may provide key pathophysiological mechanistic evidence for novel therapeutic strategies that increase the cardiovagal activity in IBD patients.     

Association of Body Mass Index with Cause Specific Deaths in Chinese Elderly Hypertensive Patients: Minhang Community Study

Background

Most studies have suggested that elevated body mass index (BMI) was associated with the risk of death from all cause and from specific causes. However, there was little evidence illustrating the effect of BMI on the mortality in elderly hypertensive patients in Chinese population.

Methods

The information of 10,957 hypertensive patients at baseline not less than 60 years were from Xinzhuang, a town in Minhang district of Shanghai, was extracted from the Electronic Health Record (EHR) system. All study participants were divided into eight categories of baseline BMI (with cut-points at 18, 20, 22, 24, 26, 28 and 30 kg/m²). Relative hazard ratio of death from all cause, cardiovascular and non-cardiovascular cause by baseline BMI groups were calculated, standardized for sex, age, smoking, drinking, physical activity, systolic blood pressure, history of cardiovascular disorders, serum lipid disturbance, diabetes mellitus and antihypertensive drug treatment.

Results

During follow up (median: 3.7 years), 561 deaths occurred. Underweight (BMI<18 kg/m²) was associated with significantly increased mortality from all cause mortality (OR: 2.00; 95% CI: 1.43–2.79) and non cardiovascular mortality (OR: 2.76; 95% CI: 1.87–4.07), but not with cardiovascular mortality. For the cause specific analysis, the underweight was associated significantly with neoplasms (OR: 2.15; 95% CI: 1.16–4.00) and respiratory disorders (OR: 3.41; 95% CI: 1.64–7.06). The results for total mortality and specific cause mortality were not influenced by sex, age and smoking status.

Conclusion

Our study revealed an association between underweight and increased mortality from non-cardiovascular disorders in elderly hypertensive patients in Chinese community. Overweight and obesity were not associated with all cause or cause specific death.     

Antimicrobial Constituents of the Leaves of Mikania micrantha H. B. K

Background

To isolate plant-derived compounds with antimicrobial activity from the leaves of Mikania micrantha, to determine the compounds configuration, and to evaluate their antimicrobial activity against eight plant pathogenic fungi (Exserohilum turcicum, Colletotrichum lagenarium, Pseudoperonispora cubensis, Botrytis cirerea, Rhizoctonia solani, Phytophthora parasitica, Fusarium solani, and Pythium aphanidermatum,) and four plant pathogenic bacteria (gram negative bacteria: Ralstonia dolaanacearum, Xanthomonas oryzae pv. Oryzae, Xanthomonas Campestris pv. Vesicatoria, and Xanthomonas campestris pv. Citri), and four bacteria (gram positive bacteria: Staphyloccocus aureus, Bacillus subtilis, Micrococcus luteus, and Bacillus cereus).

Methods and Results

Antimicrobial constituents of the leaves of M. micrantha were isolated using bioactivity- guided fractionation. The antifungal activity of the isolated compounds was evaluated by the inhibit hypha growth method and inhibit spore germination method. Characterization of antibacterial activity was carried out using the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). MIC and MBC were determined by the broth microdilution method. Six compounds – deoxymikanolide, scandenolide, dihydroscandenolide, mikanolide, dihydromikanolide, and m - methoxy benzoic acid – have been isolated from leaves of Mikania micrantha H. B. K. Deoxymikanolide, scandenolide, and dihydroscandenolide were new compounds. The result of bioassay showed that all of isolated compounds were effective against tested strains and deoxymikanolide showed the strongest activity.

Conclusions and Significance

The leaves of M. micrantha may be a promising source in the search for new antimicrobial drugs due to its efficacy and the broadest range. Meanwhile, adverse impact of M. micrantha will be eliminated.     

Dynamics and Regulation of RecA Polymerization and De-Polymerization on Double-Stranded DNA

The RecA filament formed on double-stranded (ds) DNA is proposed to be a functional state analogous to that generated during the process of DNA strand exchange. RecA polymerization and de-polymerization on dsDNA is governed by multiple physiological factors. However, a comprehensive understanding of how these factors regulate the processes of polymerization and de-polymerization of RecA filament on dsDNA is still evolving. Here, we investigate the effects of temperature, pH, tensile force, and DNA ends (in particular ssDNA overhang) on the polymerization and de-polymerization dynamics of the E. coli RecA filament at a single-molecule level. Our results identified the optimal conditions that permitted spontaneous RecA nucleation and polymerization, as well as conditions that could maintain the stability of a preformed RecA filament. Further examination at a nano-meter spatial resolution, by stretching short DNA constructs, revealed a striking dynamic RecA polymerization and de-polymerization induced saw-tooth pattern in DNA extension fluctuation. In addition, we show that RecA does not polymerize on S-DNA, a recently identified novel base-paired elongated DNA structure that was previously proposed to be a possible binding substrate for RecA. Overall, our studies have helped to resolve several previous single-molecule studies that reported contradictory and inconsistent results on RecA nucleation, polymerization and stability. Furthermore, our findings also provide insights into the regulatory mechanisms of RecA filament formation and stability in vivo.     

Differential Responses of Hepatic Endoplasmic Reticulum Stress and Inflammation in Diet-Induced Obese Rats with High-Fat Diet Rich in Lard Oil or Soybean Oil

Scopes

To investigate the effects of high-fat diet enriched with lard oil or soybean oil on liver endoplasmic reticulum (ER) stress and inflammation markers in diet-induced obese (DIO) rats and estimate the influence of following low-fat diet feeding.

Methods and Results

Male SD rats were fed with standard low-fat diet (LF, n = 10) and two isoenergentic high-fat diets enriched with lard (HL, n = 45) or soybean oil (HS, n = 45) respectively for 10 weeks. Then DIO rats from HL and HS were fed either high-fat diet continuously (HL/HL, HS/HS) or switched to low-fat diet (HL/LF, HS/LF) for another 8 weeks. Rats in control group were maintained with low-fat diet. Body fat, serum insulin level, HOMA-IR and ectopic lipid deposition in liver were increased in HL/HL and HS/HS compared to control, but increased to a greater extent in HL/HL compared to HS/HS. Markers of ER stress including PERK and CHOP protein expression and phosphorylation of eIF2α were significantly elevated in HL/HL group while phosphorylation of IRE1α and GRP78 protein expression were suppressed in both HL/HL and HS/HS. Besides, inflammatory signals (OPN, TLR2, TLR4 and TNF-α) expressions significantly increased in HL/HL compared to others. Switching to low-fat diet reduced liver fat deposition, HOMA-IR, mRNA expression of TLR4, TNF-α, PERK in both HL/LF and HS/LF, but only decreased protein expression of OPN, PERK and CHOP in HL/LF group. In addition, HL/LF and HS/LF exhibited decreased phosphorylation of eIF2α and increased phosphorylation of IRE1α and GRP78 protein expression when compared with HL/HL and HS/HS respectively.

Conclusions

Lard oil was more deleterious in insulin resistance and hepatic steatosis via promoting ER stress and inflammation responses in DIO rats, which may be attributed to the enrichment of saturated fatty acid. Low-fat diet was confirmed to be useful in recovering from impaired insulin sensitivity and liver fat deposition in this study.