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Chen D.-B.,Chengdu Medical College | Cao K.,Chengdu Medical College | Yang J.,Hubei University of Medicine | Pan Q.,Chengdu Medical College | And 6 more authors.
Journal of Sichuan University (Medical Science Edition) | Year: 2013

Objective To clone the Noxa gene and to observe the apoptosis of A549 cells transfected with the recombinant plasmid of pcDNA-Noxa. Methods The Noxa gene was obtained by PCR, and was cloned into pcDNA3. 1(-). A549 cells were transfected with the recombinant plasmid of pcDNA-Noxa. Western blot analysis was performed to determine the overexpression of Noxa. A549 cells were stained with Hoechst 33258 to observe the apoptosis. Results The recombinant plasmid of pcDNA-Noxa was successfully constructed evidenced by endonuclease digestion and sequence analysis. The overexpression of Noxa was identified using Western blot analysis. The recombinant plasmid of pcDNA-Noxa induced apoptosis of A549 cells. Conclusion Noxa has exhibited potential pro-apoptotic activity against A549 cells. This study is a foundation for further research into pro-apoptotic activity of Noxa gene.

Yang M.,Guangzhou Medical College | Lin H.-B.,Guangdong Food and Drug Administration Center for Evaluation and Certification | Gong S.,Guangzhou Medical College | Chen P.-Y.,Guangzhou Medical College | And 3 more authors.
Cytokine | Year: 2014

Aim: Astragalus membranaceus is a Chinese medicinal herb and has been shown to improve hapten-induced experimental colitis. One of its major components is polysaccharides. We investigated the effect of Astragalus polysaccharides (APS) on expression of TNF-α, IL-1β and NFATc4 in a rat model of experimental colitis. Methods: The experimental colitis model was induced by TNBS. Forty five rats were divided into five groups (n=. 9): Normal control group, receiving ethanol vehicle with no TNBS during induction and IP saline injection during treatment; TNBS colitis model group (TNBS. +. IP saline), receiving only IP saline vehicle treatment; APS low dose group (TNBS. +. L-APS), receiving APS 100. mg/kg; APS high dose group (TNBS. +. H-APS), receiving APS 200. mg/kg; and positive control group (TNBS. +. Dexm), receiving dexamethasone 0.3. mg/kg. The clinical features, macroscopic and microscopic scores were assessed. The expressions of TNF-α, IL-1β and NFATc4 were measured by real-time PCR and ELISA assays. Results: Compared to normal control rats, TNBS. +. IP saline had significant weight loss, increased macroscopic and microscopic scores, higher disease activity index (DAI) up-regulation of TNF-α, IL-1β and NFATc4 mRNA expression and up-regulation of TNF-α and IL-1β protein expression. Compared to TNBS. +. IP saline, treatment with APS or dexamethasone significantly reduced DAI, partially but significantly prevented TNBS colitis-induced weight loss and improved both macroscopic and microscopic scores; high dose APS or dexamethasone significantly down-regulated TNF-α and IL-1β expressions (both mRNA and protein) and up-regulated NFATc4 mRNA and protein expression. The effect of high dose APS and dexamethasone is comparable. Conclusions: APS significantly improved experimental TNBS-induced colitis in rats through regulation of TNF-α, IL-1β and NFATc4 expression. © 2014 The Authors.

Bi B.-T.,Southern Medical University | Lin H.-B.,Guangdong Food and Drug Administration Center for Evaluation and Certification | Cheng Y.-F.,Southern Medical University | Zhou H.,Southern Medical University | And 4 more authors.
Neurochemistry International | Year: 2012

C-reactive protein (CRP) and β-amyloid protein (Aβ) are involved in the development of Alzheimer's disease (AD). However, the relationship between CRP and Aβ production is unclear. In vitro and in vivo experiments were performed to investigate the association of CRP with Aβ production. Using the rat adrenal pheochromocytoma cell line (PC12 cells) to mimic neurons, cytotoxicity was evaluated by cell viability and supernatant lactate dehydrogenase (LDH) activity. The levels of amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE-1), and presenilins (PS-1 and PS-2) were investigated using real-time polymerase chain reaction and Western blotting analysis. Aβ1-42 was measured by enzyme-linked immunosorbent assay. The relevance of CRP and Aβ as well as potential mechanisms were studied using APP/PS1 transgenic (Tg) mice. Treatment with 0.5-4.0 μM CRP for 48 h decreased cell viability and increased LDH leakage in PC12 cells. Incubation with CRP at a sub-toxic concentration of 0.2 μM increased the mRNA levels of APP, BACE-1, PS-1, and PS-2, as well as Aβ1-42 production. CRP inhibitor reversed the CRP-induced upregulations of the mRNA levels of APP, BACE-1, PS-1, and PS-2, and the protein levels of APP, BACE-1, PS-1, and Aβ1-42, but did not reversed Aβ1-42 cytotoxicity. The cerebral levels of CRP and Aβ1-42 in APP/PS1 Tg mice were positively correlated, accompanied with the elevated mRNA expressions of serum amyloid P component (SAP), complement component 1q (C1q), and tumor necrosis factor-α (TNF-α). These results suggest that CRP cytotoxicity is associated with Aβ formation and Aβ-related markers expressions; CRP and Aβ were relevant in early-stage AD; CRP may be an important trigger in AD pathogenesis. © 2011 Elsevier Ltd. All rights reserved.

Zhou W.,Guangdong Food and Drug Administration Center for Evaluation and Certification | Xie Z.,Guangdong Food and Drug Administration Center for Evaluation and Certification | Shao L.,Guangdong Inspection and Quarantine Technology Center
Chinese Journal of Chromatography (Se Pu) | Year: 2012

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for simultaneous determination of 13 antibiotics in oral hygiene products, including five tetracyclines, three macrolides, two quinolones, one β-lactam and two lincosamides. The sample was extracted with 0. 1% (volume percentage; same hereinafter) formic acid-acetonitrile (95: 5, v/v), then centrifuged; filtered and diluted. The target compounds were separated on a C18 column (l50 mm × 2. 1 mm; 5 μm) with a gradient elution of 0. 1% formic acid and acetonitrile as the mobile phases; and detected by tandem mass spectrometry in positive electrospray ionization and multiple reaction monitoring (MRM) mode. The quantification of 13 antibiotic was performed by the external standard method. The calibration curves showed good linearity in the range of 5.0-50. 0 μg/L with detection limits of 10. 0 mg/kg. The recoveries of Antibiotics in mouthwash and toothpaste samples at the three spiked levels of 10, 20 and I00 mg/kg were in the range of 80. 1% - 115% with the relative standard deviations in the range of 0. 94% -8. 69%. This method is accurate; reliable; simple; and suit-able for the analysis of antibiotics in oral hygiene products.

Cai Y.,Sun Yat Sen University | Cai Y.,Guangdong Food and Drug Administration Center for Evaluation and Certification | Xu C.,Sun Yat Sen University | Chen P.,Guangdong Food and Drug Administration Center for Evaluation and Certification | And 4 more authors.
Journal of Pharmacological and Toxicological Methods | Year: 2014

Introduction: Monolayers of Caco-2 cells have been widely accepted as one of the well-established in vitro models to predict intestinal drug permeability and absorption in humans. However, the procedure for culturing the traditional 21-day Caco-2 model is labor intensive and time consuming, which limits its wide application in drug development. The objective of the present study was to develop a rapid Caco-2 model with a 7-day cell culture process. Methods: A few modifications of the BIOCOAT® HTS Caco-2 Assay System were introduced including changing the cell seeding density, the composition of cell culture media, and the interval to change media. The monolayer structure was visualized through confocal microscopy. The transepithelial electrical resistance (TEER), apparent permeability coefficients (Papp), and functional activity of P-glycoprotein (P-gp) were determined and compared with the 21-day model. Moreover, pharmacokinetic studies were performed in animal models to evaluate the absolute bioavailability of oral doses (Foral) of different compounds. Results: Our newly developed 7-day Caco-2 model displayed comparable cellular morphology and integrity with the traditional 21-day model. No significant difference in paracellular and transcellular permeability was observed between the two systems. The efflux ratios of transporting digoxin, the prototypical substrate of P-gp in 21- and 7-day systems were 16.04 and 24.92, respectively. When the P-gp inhibitor verapamil was present, the efflux ratios of 21- and 7-day systems were 1.37 and 0.86, respectively, suggesting the comparability of the P-gp functional activity in both systems. Furthermore, pharmacokinetic studies of several compounds performed in animal models revealed that the absolute bioavailability of oral doses in vivo was well correlated with the Caco-2 permeability in vitro. Discussion: The novel system provides a rapid and economical option for assessing the drug permeability, and is applicable to the studies of intestinal drug absorption. © 2014 Elsevier Inc.

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