Xiujie Pan

Simultaneous detection of five antibiotics in milk by high-throughput suspension array technology

A new suspension array technology is proposed for the simultaneous quantitative determination of five antibiotics-tylosin, tetracycline, gentamicin, streptomycin, and chloramphenicol in milk. A novel treatment of milk samples for suspension array with diethyl ether was performed which greatly reduced the interference of the disturbing components in milk on the reaction results with no significant effect on detection sensitivity. Compared with using biotin labeled monoclonal antibody, using of secondary antibody-biotin make the detection sensitivity further improved. The minimum detectable concentration in samples of tylosin, tetracycline, gentamicin, streptomycin, and chloramphenicol were 0.3, 1.5, 4, 20, and 25 ng/ml, respectively, and the working ranges of samples were 6-400, 7-300, 8-200, 90-3000 and 70-8000 ng/ml, respectively. The mean recovery was 89.38-113.73% with a standard deviation within 16.62%. The suspension assay technology is powerful for the fast quantitative analysis of multi-antibiotics residue in milk.

Crotonaldehyde induces oxidative stress and caspase-dependent apoptosis in human bronchial epithelial cells

Crotonaldehyde is a widespread environmental pollutant and lipid peroxidation product. Crotonaldehyde is a risk factor for many diseases (e.g., chronic pulmonary inflammation). However, its toxicity and its mechanism of action have not been thoroughly investigated. The purpose of this study is to investigate crotonaldehyde-induced oxidative stress and mechanism of cell death in BEAS-2B cells. Crotonaldehyde caused decreases of intracellular reduced glutathione levels and increases of reactive oxygen species in a dose-dependent manner. Crotonaldehyde induced cell death by apoptosis, and gradually transitioned to necrosis at high dose of crotonaldehyde, as demonstrated by Annexin V-FITC/PI staining and cell morphology analysis. Crotonaldehyde-induced ATP decline observed in the study might partially account for the switch from apoptosis to necrosis. Mitochondria membrane potential, cytochrome c release, caspase-9, and caspase-3/7 activity were investigated, and the results suggest that crotonaldehyde-induced apoptosis was activated in a caspase-dependent way. Collectively, these results demonstrate crotonaldehyde induces cell oxidative stress and caspase-dependent apoptosis.

Inhibition of cyclooxygenase-2 by tetramethylpyrazine and its effects on A549 cell invasion and metastasis

Cyclooxygenase (COX)-2 plays an important role in tumorigenesis and has been implicated to be a critical factor for invasion and metastasis of lung cancer. Tetramethylpyrazine (TMP), an effective component of the traditional Chinese medicine Chuanxiong, has been traditionally used in treating neurovascular and cardiovascular diseases. Recently TMP has been reported to have beneficial effect in cancer patients. However, the function and the mechanism of TMP in lung cancer have not been elucidated to date. In this study, we investigated the in vitro and in vivo effect of TMP in tumorigenesis and whether COX-2 is a molecular target of TMP. We showed that TMP exhibited a dose- and time-dependent inhibition on A549 cell proliferation by suppressing cell cycle progression. In vitro treatment of A549 cells with TMP resulted in a significant inhibition of invasion, associated with reduced activities of COX-2 and MMP-2/TIMP-2. Furthermore, in vivo experiments showed that TMP significantly suppressed metastatic growth of A549 cells and COX-2 expression in metastatic nude mouse model. This preclinical study provides the first evidence for the novel anti-tumor effects of TMP as a COX-2 pathway inhibitor in human adenocarcinoma cell line A549. These studies suggest that TMP may serve as an effective agent for the treatment and chemoprevention of non-small cell lung cancer.

Transforming growth factor β3 attenuates the development of radiation-induced pulmonary fibrosis in mice by decreasing fibrocyte recruitment and regulating IFN-γ/IL-4 balance

Radiation-induced pulmonary fibrosis is a frequently occurred complication from radiotherapy of thoracic tumors. The transforming growth factor-β (TGF-β) superfamily plays a key regulatory role in pulmonary fibrosis. As TGF-β3 showed the potential anti-fibrotic properties especially in scar-less wound healing as opposed to the fibrotic function of TGF-β1, we sought to explore the role of TGF-β3 in radiation-induced pulmonary fibrosis. A single thoracic irradiation of 20 Gy was applied in mice to establish the model of radiation-induced pulmonary fibrosis and the mice were treated by intraperitoneal injections of recombinant TGF-β3 weekly after irradiation. We found that TGF-β3 decelerated the progress of radiation-induced pulmonary fibrosis and hindered the recruitment of fibrocytes to lung. In addition, Th1 response was suppressed as shown by diminished IFN-γ in bronchoalveolar lavage fluid (BALF) after irradiation, and enhancement of Th2 response was marked by increased IL-4 in BALF. TGF-β3 administration significantly attenuated these effects and increased the percentage of Tregs in lung during the progression of pulmonary fibrosis. Taken together, these data suggest that TGF-β3 might be involved in the regulatory mechanism for attenuation of radiation-induced pulmonary fibrosis.

Radiosensitization and growth inhibition of cancer cells mediated by an scFv antibody gene against DNA-PKcs in vitro and in vivo

BackgroundOverexpression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is commonly occurred in cancers and causes radioresistance and poor prognosis. In present study, the single-chain variable antibody fragments (scFv) targeting DNA-PKcs was developed for the application of radiosensitization in vitro and in vivo. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody.MethodsDNA-PKcs epitopes were predicted and cloned. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA damage repair was analyzed by comet assay and immunofluorescence detection of γH2AX foci. The radiosensitization in vivo was determined on Balb/c athymic mice transplanted tumours of HeLa cells.ResultsFour epitopes of DNA-PKcs have been predicted and expressed as the antigens, and a specific human anti-DNA-PKcs scFv antibody gene, anti-DPK3-scFv, was obtained by screening the phage antibody library using the DNA-PKcs peptide DPK3. The specificity of anti-DPK3-scFv was verified, in vitro. Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of γH2AX foci. Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation. Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy.ConclusionThe antiproliferation and radiosensitizing effects of anti-DPK3-scFv via targeting DNA-PKcs make it very appealing for the development as a novel biological radiosensitizer for cancer therapeutic potential.

Crotonaldehyde-exposed macrophages induce IL-8 release from airway epithelial cells through NF-κB and AP-1 pathways

Crotonaldehyde, a highly toxic α, β-unsaturated aldehyde, is a major component of cigarette smoke and a ubiquitous environmental pollutant. Crotonaldehyde exposure is known to have adverse effects on respiratory health, but the underlying mechanisms remain obscure. To examine the interaction between macrophages and airway epithelial cells after exposure to crotonaldehyde, BEAS-2B and A549 cells were treated with conditioned media from a human monocytic leukemia cell line (THP-1) cells stimulated with crotonaldehyde. We demonstrate that conditioned media from THP-1 cells stimulated with crotonaldehyde increased interleukin (IL)-8 production, enhanced nuclear factor (NF)-κB and AP-1 DNA-binding activity in BEAS-2B and A549 cells. Analysis of these conditioned media revealed marked increases in tumor necrosis factor (TNF)-α, IL-1β and IL-8 levels. Preincubation of conditioned media with either TNF-α- or IL-1β-neutralizing antibodies reduced IL-8 production. Furthermore, BEAS-2B and A549 cells directly treated with crotonaldehyde induced increase in IL-8 production. These data suggest that crotonaldehyde is capable of directly stimulating the production of IL-8 in both macrophages and airway epithelial cells. Crotonaldehyde-stimulated macrophages also amplify the inflammatory response by enhancing IL-8 release from airway epithelial cells mediated by NF-κB and AP-1 pathways through a mechanism involving TNF-α and IL-1β. These findings indicate that crotonaldehyde can cause lung inflammatory response via multiple mechanisms, and may contribute to chronic airway inflammation in smokers.

Gene expression profile and cytotoxicity of human bronchial epithelial cells exposed to crotonaldehyde

Crotonaldehyde is an environment pollutant and lipid peroxidation product. Crotonaldehyde produces adverse effects to humans and serves as a risk factor for human pulmonary diseases. Like acrolein and 4-hydroxynonenal, crotonaldehyde seems likely to alter many cell signaling cascades, including inflammatory responses. The purpose of this study was to investigate the genome-wide transcriptional responses of normal human bronchial epithelial cells exposed to crotonaldehyde. Using microarrays technology, the global changes in transcriptional level were analyzed. Prior to RNA extraction, cells were exposed to crotonaldehyde at 40 or 80 microM for 3 or 6h. Real-time quantitative polymerase chain reaction (qPCR) was performed to validate microarray data and cell cycle arrest was determined. The commonly differentially regulated genes in many biological processes were dysregulated including inflammatory responses, exogenous metabolism, cell cycle, heat shock responses, and antioxidant responses. Results in the present study screen out the important roles of HMOX1 in regulating other signaling cascades and ALDH1A3 in detoxifying exogenous toxicants. Collectively, our study demonstrated that crotonaldehyde altered gene expression profile in the genome-wide transcriptional level in normal human bronchial epithelial cells. And many of them represented potential mechanisms of crotonaldehyde causing cytotoxicity and tissue injury in the human lung.

LL-37 secreted by epithelium promotes fibroblast collagen production: a potential mechanism of small airway remodeling in chronic obstructive pulmonary disease

Emerging evidence suggests that the process of small airway remodeling is mediated by profibrotic growth factors produced by epithelium, which are capable of activating the underlying mesenchymal cells with excessive collagen production. It has been demonstrated that human cathelicidin antimicrobial protein LL-37 is highly expressed in small airway epithelium from COPD patients. However, it is unknown whether the increased levels of LL-37 in epithelium are involved in the pathogenesis of small airway remodeling in COPD. In this study, we examined the expression of LL-37 in small airways from smokers with COPD and controls (non-smokers and smokers without COPD) by immunohistochemistry, and then the association between LL-37 expression in epithelium and the structural changes of small airway remodeling was analyzed. In vitro, the effect of CSE-induced epithelial secretion of LL-37 on collagen production in human lung fibroblasts (HFL-1 cell line) was studied in a co-culture system. Finally, the signaling pathways involved in the effect of LL-37 on fibroblast collagen production were evaluated. The results showed that LL-37 immunoreactivity in airway epithelium was significantly elevated in smokers with COPD compared with controls. In addition, the magnitude of LL-37 expression in epithelium was positively correlated with airway wall thickness and collagen deposition. In vitro, CSE-induced epithelial secretion of LL-37 promoted fibroblast collagen production. Finally, we showed that formyl peptide receptor-like 1 (FPRL1)-dependent extracellular signal-regulated kinase (ERK) signaling pathway was essential for LL-37-induced collagen production in HFL-1 cells. These results suggest that after cigarette smoke exposure, the increased levels of LL-37 in airway epithelium could stimulate collagen production in the underlying lung fibroblasts and may contribute to small airway remodeling in COPD.

Crotonaldehyde induces apoptosis and immunosuppression in alveolar macrophages.

Crotonaldehyde, a highly toxic α, β-unsaturated aldehyde, is a major component of cigarette smoke (CS) and a ubiquitous environmental pollutant. Exposure to crotonaldehyde-rich pollutants such as CS is associated with suppression of respiratory host defense against infections. The aim of this study was to evaluate the apoptotic and immunological effects of crotonaldehyde exposure in a rat alveolar macrophage (AM) cell line, NR8383. Our studies showed that crotonaldehyde induced AM cell death mainly via the apoptotic process. Crotonaldehyde also decreased the phagocytic activity of AMs. Crotonaldehyde caused inhibition of NO, TNF-α, IL-1β and IL-12 production in AMs treated with lipopolysaccharide (LPS), which is probably related to inhibition of NF-κB activation. These results indicate that crotonaldehyde can cause adverse effects in AMs via multiple mechanisms, and may contribute to compromised lung immunological response in smokers.

The human cathelicidin LL-37 enhances airway mucus production in chronic obstructive pulmonary disease.

Airway mucus overproduction is a distinguishing feature of chronic obstructive pulmonary disease (COPD). LL-37 is the only member of human cathelicidins family of antimicrobial peptides and plays a central role in many immune and inflammatory reactions. Increasing evidence suggests the involvement of LL-37 in the pathogenesis of COPD. Here, we investigated the effects of LL-37 on airway mucus overproduction in COPD. We observed overexpression of both LL-37 and MUC5AC mucin (a major mucin component of mucus) in airways of COPD patients and found a correlation between them. We showed in vitro that LL-37 induces MUC5AC mucin production by airway epithelial NCI-H292 cells in the absence and presence of cigarette smoke extract, with TNF-α converting enzyme (TACE)-EGFR-ERK1/2 pathway and IL-8 required for the induction. Therefore, we concluded that LL-37 enhances the mucus production in COPD airways, thus contributing to the progression of COPD.