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ABSTRACT: Oxidative stress has been implicated as an important contributing factor in the pathogenesis of several pulmonary inflammatory diseases. Previous studies have indicated a relationship between oxidative stress and the attenuation of epithelial tight junctions (TJs). In Human Bronchial Epithelial-16 cells (16HBE), we demonstrated the degradation of zonula occludens-1 (ZO-1), and claudin-2 exhibited a great dependence on the activation of the transient receptor potential melastatin (TRPM) 2 channel, phospholipase Cγ1 (PLCγ1) and the protein kinase Cα (PKCα) signaling cascade.
International Journal of Molecular Sciences 01/2013; 14(5):9475-86. · 2.60 Impact Factor
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ABSTRACT: Multi-drug resistance leads to the failure of chemotherapy for cancers. Our previous study showed that overexpression of CA916798 led to multi-drug resistance. However, the underlying mechanisms remain unknown. In the current study, we observed that the levels of phosphorylated AKT, phosphorylated mTOR and CA916798 all increased in the drug resistant human adenocarcinoma samples and paralleled with the change of drug resistance. The results of immunofluorescence and Co-IP indicated that the positive correlation of CA916798 expression with AKT1 activation might be associated with drug resistance of lung adenocarcinoma. Furthermore, AKT1 stimulated CA916798 expression through mTOR pathway in both A549 and A549/CDDP cell lines, which was also observed in the xenografted tumor in nude mice. The results showed that CA916798 located in the downstream of PI3K/AKT/mTOR pathway. Inhibition of PI3K by LY294002 could efficiently reduce CA916798 expression and tumor size in vivo as well. Additionally, LY294002 combined with rapamycin inhibited CA916798 expression and tumor size stronger than LY294002 alone. Our findings may also provide a new explanation for synergistic anti-tumor effects of PI3K and mTORC1 inhibitors.
PLoS ONE 01/2013; 8(5):e62327. · 4.09 Impact Factor
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ABSTRACT: A series of novel berberine triazoles were synthesized and characterized by IR, NMR, MS and HRMS spectra. All target compounds and their precursors were screened for antimicrobial activities in vitro against four Gram-positive bacteria, four Gram-negative bacteria and two fungal strains. Bioactive assay indicated that most of the prepared compounds exhibited good antibacterial and antifungal activities with low MIC values ranging from 2 to 64μg/mL, which were comparable to or even better than the reference drugs Berberine, Chloromycin, Norfloxacin and Fluconazole. The competitive interactions between compound 5a and metal ions to Human Serum Albumin (HSA) revealed that the participation of Mg(2+) and Fe(3+) ions in compound 5a-HSA association could result in the concentration increase of free compound 5a, shorten the storage time and half-life of compound 5a in the blood, thus improving its antimicrobial efficacy.
Bioorganic & medicinal chemistry letters 12/2012; · 2.65 Impact Factor
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ABSTRACT: OBJECTIVE: To explore the variation of small ubiquitin-related modification (SUMO) level of glucocorticoid receptors (GRs) exposed to acrolein stimulation as well as the influence of the variation on mucus hypersecretion. METHODS: The recombinant plasmid was constructed by inserting small ubiquitin-like modifier 1 (SUMO1) cDNA into eukaryotic expression plasmid pcDNA3.1-EGFP with a flag sequence marker. The recombinant plasmid pcDNA3.1-EGFP-flag-SUMO1 was identified by enzyme digestion analysis. The 16HBE cells were cultured and randomized divided into 9 following groups: A. control; B. acrolein stimulated; C. dexamethasone incubated; D. acrolein stimulated and dexamethasone incubated; E. SUMO1 transfected; F. pcDNA3.1-EGFP vector transfected; G. SUMO1 transfected and acrolein stimulated; H. SUMO1 transfected, acrolein stimulated and dexamethasone incubated; I. pcDNA3.1-EGFP vector transfected, acrolein stimulated and dexamethasone incubated. Each group consisted of 5 parallel wells and experiments were repeated for 4 times for statistical analysis. The transfection efficiency was evaluated by the expression of enhanced green fluorescent protein (EGFP) via fluorescence microscope. The SUMO modification level of GRs was measured with co-immunoprecipitation and Western blot. The transcription level of mucin (MUC) 5AC was evaluated with reverse transcription-polymerase chain reaction (RT-PCR). The MUC5AC secreted in supernatant was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The SUMO modification level was much lower in group B (0.079 ± 0.023) than that in group A (0.446 ± 0.068) (t = -23.13, P = 0.000) and in group D (0.574 ± 0.018) than that in group C (0.843 ± 0.028) (t = -36.34, P = 0.000). The transcription level of MUC5AC was significantly lower in group H (0.330 ± 0.063) than group D (0.617 ± 0.190) (q = -7.80, P = 0.000). Through ELISA, there was no significance between groups G and B in the term of secretion level of MUC5AC. While the secretion level of MUC5AC was much lower in group H ((0.416 ± 0.092) µg/L) than group D ((0.663 ± 0.104) µg/L) and group G ((0.740 ± 0.343) µg/L) (q = -7.31, -9.59; P = 0.001, 0.000). CONCLUSION: Acrolein decreases the SUMO modification of GRs and reduces the inhibitory effect of dexamethasone on the transcription of MUC5AC.
Zhonghua yi xue za zhi 12/2012; 92(46):3291-3295.
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ABSTRACT: To explore the mechanisms of mucus hypersecretion in airway of rats induced by the synergies between cold air and cigarette smoke inhalation and understand the intervention effects of saussurea and budesonide in this process.
A total of 70 SD rats were randomly divided into 7 groups. Group A: control; Group B: cold stimulation group receiving cold air inhalation for 3 h daily for 40 d; Group C: cigarette smoke inhalation group receiving cigarette smoke inhalation for 0.5 h daily for 40 d; Group D: cigarette smoke inhalation + cold stimulation group; Group E: cigarette smoke inhalation + cold stimulation + saussurea (0.8 mg/kg saussurea intraperitoneally injected once daily for 40 d); Group F: cigarette smoke inhalation + cold stimulation + inhaled budesonide (0.5 mg/kg inhaled once daily for 40 d); Group G: cigarette smoke inhalation + cold stimulation + saussurea + inhaled budesonide. The relative quantities of TRPM8 mRNA within bronchial epithelium of each group were detected by real-time polymerase chain reaction (PCR) and TRPM8 protein was detected by immunohistochemical assay and Western blot. The levels of mucin (MUC) 5AC, interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay.
TRPM8 mRNA of groups A-G were 1.00 ± 0.00, 0.98 ± 0.07, 2.27 ± 0.29, 2.31 ± 0.30, 1.55 ± 0.38, 1.66 ± 0.40 and 1.31 ± 0.23; TRPM8 protein 0.16 ± 0.05, 0.16 ± 0.04, 0.22 ± 0.06, 0.25 ± 0.05, 0.17 ± 0.04, 0.18 ± 0.03, 0.15 ± 0.05, 0.25 ± 0.04, 0.24 ± 0.03, 0.58 ± 0.06, 0.56 ± 0.09, 0.41 ± 0.09, 0.39 ± 0.07 and 0.20 ± 0.06 respectively. TRPM8 mRNA and protein of groups C and D were significantly higher than those of group A. And groups E, F and G were significantly lower than those of group D (all P < 0.05). In BALF of groups A-G, MUC5AC were (57 ± 6), (69 ± 5), (66 ± 4), (185 ± 43), (142 ± 30), (147 ± 36) and (60 ± 11) µg/mg, IL-8 (58 ± 14), (146 ± 38), (134 ± 29), (379 ± 101), (262 ± 67), (294 ± 70) and (81 ± 27) ng/L, TNF-α(153 ± 28), (208 ± 90), (274 ± 64), (600 ± 113), (458 ± 96), (498 ± 84) and (169 ± 65) ng/L respectively. The values of groups B, C and D were significantly higher than those of group A (all P < 0.05) while groups E, F and G were significantly lower than those of group D (all P < 0.05). Cigarette smoke inhalation and cold stimulation synergistically enhanced the expression of MUC5AC, IL-8 and TNF-α. Saussurea and inhaled budesonide synergistically inhibited the expression of MUC5AC, IL-8 and TNF-α.
Cold air inhalation evokes the release of proinflammatory cytokines and MUC5AC via activated TRPM8 channel up-regulated by cigarette smoke inhalation. Saussurea and inhaled budesonide synergistically inhibits the above mentioned process.
Zhonghua yi xue za zhi 08/2012; 92(32):2283-7.
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ABSTRACT: To explore the role of neuregulin 1β (NRG-1β) in airway hypersecretion induced by interleukin (IL)-1β.
After stimulating the airway epithelial cell line HBE16 with IL-1β, the expressions of NRG-1β mRNA and mucin (MUC) 5AC mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), the proteins of NRG-1β and MUC5AC measured by enzyme-linked immunosorbent assay (ELISA) and phosphorylated erythroblastic leukemia viral oncogene homolog (ErbB)1-4 detected by Western blot. The cells were pre-treated with antibodies of ErbB1-4, inhibitors of p38 mitogen-activated protein kinase (MAPK), ERK1/2, mitogen- and stress-activated protein kinase (MSK)1 and antibody of cAMP-response element-binding protein (CREB). After the addition of stimulant NRG-1β, MUC5AC was measured by ELISA.
IL-1β could increase markedly the levels of NRG-1β mRNA and MUC5AC mRNA and also the proteins of NRG-1β and MUC5AC in a dose-dependent fashion. NRG-1β at concentrations of 1, 10, 100, 200 nmol/L increased the expression of MUC5AC (0.328 ± 0.055, 0.364 ± 0.086, 0.650 ± 0.134, 0.586 ± 0.068) versus the control group (0.227 ± 0.019). And the results had statistical significances (P < 0.05). The expressions of phosphorylated ErbB2 and ErbB3 stimulated by NRG-1β were positive while those of phosphorylated ErbB1 and ErbB 4 negative. After a pretreatment of antibodies of ErbB2, ErbB3, inhibitors of p38MAPK, ERK1/2, MSK1 and antibody of CREB and a stimulation of NRG-1β, the expression of MUC5AC decreased (0.221 ± 0.033, 0.238 ± 0.044, 0.386 ± 0.021, 0.352 ± 0.022, 0.294 ± 0.017, 0.252 ± 0.019) versus the NRG-1β group (0.650 ± 0.134). And the results had statistical significances (P < 0.05).
IL-1β may cause airway hypersecreton probably through the combination of NRG-1β with ErbB2 and ErbB3 heterodimers and the activation of MAPK/MSK1/CREB signal conduction.
Zhonghua yi xue za zhi 07/2012; 92(28):1988-91.
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ABSTRACT: Quercetin, a plant flavonoid, is a potent antioxidant and anti-inflammatory agent. Mucus hypersecretion is a common pathological change in chronic inflammatory diseases of the airway. We investigated the effect of quercetin on mucin 5AC (MUC5AC) expression induced by human neutrophil elastase (HNE) in airway epithelial cells and its molecular mechanisms. Human airway epithelial (HBE16) cells were pretreated with quercetin and were treated with HNE. We found that HNE induced a significant increase in the levels of MUC5AC and EGFR in cells treated only with HNE. Quercetin suppressed gene transcription and protein expression of MUC5AC in a dose-dependent manner, with significant inhibition from 40 μM. mRNA and protein expressions of EGFR decreased markedly when pretreated with quercetin. Among three MAPK proteins, only phosphorylated ERK1/2 protein expression increased significantly after treatment with HNE alone and decreased significantly after pretreatment with quercetin. HNE also activated phosphorylated PKC protein expression which was attenuated when pretreated with quercetin. These results suggest that quercetin can inhibit HNE-induced MUC5AC expression in human airway epithelial cells through PKC/EGFR/ERK signal transduction pathway. In the future, quercetin might be a valuable treatment for mucin hypersecretion in chronic inflammatory airway diseases in clinic.
International immunopharmacology 07/2012; 14(2):195-201. · 2.21 Impact Factor
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ABSTRACT: Mucus hypersecretion is a major pathophysiologic feature in chronic inflammatory airway diseases. Oxidative stress plays a pivotal role in this process. Recent studies have found that heparin has antioxidant effects which can reduce free radical damage. Here, we hypothesized that heparin has some influence on the expression of mucin 5AC (MUC5AC) induced by phorbol myristate acetate (PMA) in a bronchial epithelial cell line (HBE16), also we have investigated the potential mechanism involved in the process. We found that ROS, the mRNA of Duox1, EGFR and MUC5AC, as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC in the PMA group were significantly increased when compared with the control group (all P < 0.01). After pretreatment with heparin however, there was a significant decrease in ROS levels, the mRNA of Duox1, EGFR, and MUC5AC, and the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with the PMA group (all P < 0.01). MUC5AC protein in the supernatant was inhibited in a dose-dependent manner by heparin. Pretreatment with DMTU resulted in a significant decrease in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC when compared with the PMA group (all P < 0.01). When cells were pretreated with both heparin and DMTU, there was a further reduction in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with either the PMA group, heparin group, or DMTU group (all P < 0.01). Our results show that PMA can induce MUC5AC expression by activation of the Duox1-ROS-TACE-TGF-α-EGFR signaling pathway. Heparin can decrease the level of Duox1, ROS production and block the PMA-induced activation of EGFR, thus inhibiting the overexpression of mucin MUC5AC in a dose-dependent manner. In addition to reducing ROS production, heparin may also inhibit the expression of MUC5AC through other signal mechanisms.
Molecular and Cellular Biochemistry 01/2012; 360(1-2):383-91. · 2.06 Impact Factor
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ABSTRACT: To explore the effects of secretary leukocyte protease inhibitor (SLPI)-transfected bone marrow mesenchymal stem cells (BMSCs) transplantation on airway inflammation and mucus secretion in chronic obstructive pulmonary disease (COPD) rats.
Sixty rats were equally and randomly divided into negative control, COPD model, BMSCs and SLPI-transfected BMSCs groups. The COPD rat model was established in all rats with the exception of the negative control rats by smoking and intratracheal instillation of lipopolysaccharide (LPS). BMSCs with or without transfection of plasmid containing SLPI gene were delivered through caudal vein of rats at 30 days post-induction. The expression of SLPI was examined with Western blot. The levels of interleukin (IL)-8 and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay (ELISA). Goblet cell hyperplasia of lung pathological section was observed on.
Compared with the negative control group, the expression of SLPI increased significantly after the administration of SLPI-transfected BMSCs (0.79 ± 0.06 vs 0.24 ± 0.02, P < 0.05). The levels of IL-8 and TNF-α in BMSCs and SLPI-transfected BMSCs group were lower than those in the COPD model group. Compared with the negative control group, the administration of SLPI-transfected BMSCs resulted in a further decrease in IL-8 and TNF-α in bronchoalveolar lavage fluid [(17.6 ± 1.7) vs (36.6 ± 4.0) ng/L, P < 0.05]. SLPI-transfected BMSCs transplantation also significantly attenuated goblet cell hyperplasia in rats (P < 0.05).
There is a potential role for cell-based SLPI gene therapy in the treatment of COPD.
Zhonghua yi xue za zhi 12/2011; 91(48):3438-41.
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ABSTRACT: To explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) in cold-induced production of inflammatory factors in airway epithelial cells and related signal transduction mechanism.
The 16HBE human airway epithelial cells were stimulated with cold temperature (18°C). In intervention experiments, cells were pretreated with TRPM8 channel antagonist BCTC, protein kinase C (PKC) specific inhibitor calphostin C and transfected with TRPM8 shRNA or control shRNA respectively, and thereafter cold stimulation was applied. Cells were divided into 6 groups: a control group (incubated at 37°C), a cold stimulation group, a cold stimulation + BCTC group, a cold stimulation + TRPM8 shRNA group, a cold stimulation + control shRNA group, a cold stimulation + calphostin C group. Western blot was performed to show the extent of knockdown in TRPM8 protein expression in the TRPM8 shRNA transfected cells. Dynamics of relative concentration of intracellular Ca(2+) in the former 5 groups were measured by calcium imaging techniques. Images were taken at one frame per 10 seconds. The levels of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α mRNA and protein were detected by real-time PCR and ELISA respectively.
The highest relative concentration of intracellular calcium in cold stimulation group (2.36 ± 0.24) was higher than that of control group (1.01 ± 0.02) (t = 12.52, P < 0.01). BCTC and TRPM8 shRNA reduced intracellular calcium (1.05 ± 0.09, 1.08 ± 0.09), compared with single cold stimulation group (t = 6.69 and 9.12, all P < 0.01). IL-6, IL-8, TNF-α mRNA and protein in cold stimulation group[0.66 ± 0.16, 0.77 ± 0.15, 0.73 ± 0.09 and (92 ± 13) ng/L, (125 ± 22) ng/L, (88 ± 12) ng/L ] were significantly higher than those in control group [0.37 ± 0.08, 0.32 ± 0.07, 0.48 ± 0.10 and (52 ± 8) ng/L, (50 ± 9) ng/L, (61 ± 8) ng/L] (t = 3.20 - 6.26, all P < 0.05). IL-6 mRNA, IL-8 mRNA, TNF-α mRNA and protein in cold stimulation + BCTC group [0.42 ± 0.09, 0.52 ± 0.13, 0.52 ± 0.12 and (72 ± 8) ng/L, (92 ± 14) ng/L, (68 ± 11) ng/L], cold stimulation + TRPM8 shRNA group [0.41 ± 0.10, 0.49 ± 0.08, 0.50 ± 0.08 and (60 ± 12) ng/L, (89 ± 14) ng/L, (68 ± 11) ng/L] and cold stimulation + calphostin C group [0.40 ± 0.07, 0.44 ± 0.09, 0.47 ± 0.08 and (69 ± 9) ng/L, (86 ± 15) ng/L, (61 ± 10) ng/L] were significantly lower than those in cold stimulation group (t = 2.47 - 4.21, all P < 0.05). IL-6 mRNA, IL-8 mRNA, TNF-α mRNA and protein in cold stimulation + control shRNA group [0.61 ± 0.10, 0.69 ± 0.11, 0.64 ± 0.13 and (89 ± 13) ng/L, (118 ± 20) ng/L, (79 ± 13) ng/L] showed no significant change, compared with cold stimulation group (t = 0.35 - 1.12, all P > 0.05).
Cold temperature may induce Ca(2+) influx and up-regulate IL-6, IL-8, and TNF-α expression in 16HBE cells by activating the TRPM8 ion channels, and this is via a signaling pathway involving PKC.
Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases 10/2011; 34(10):757-61.
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ABSTRACT: Hypermethylation of the Wnt inhibitory factor-1 (WIF-1) promoter has been implicated in the overactivation of the Wnt pathway in human lung cancer. Curcuminoids exert anti-cancer effects and have been reported to act as hypomethylating agents. Previously, we have investigated and compared the demethylation effects of three curcuminoids and observed that bisdemethoxycurcumin exhibited the strongest demethylation potency. In this study, we used lung cancer cell lines with WIF-1 promoter hypermethylated as a model to study the demethylating effect of bisdemethoxycurcumin on WIF-1 restoration, Wnt signaling activity and cell death. Bisdemethoxycurcumin directly suppressed the activity of DNA methyltransferase-1 (DNMT1) but did not influence DNMT1 expression. In addition, it induced WIF-1 promoter demethylation and protein re-expression. WIF-1 restoration in lung cancer cells down-regulated nuclear β-catenin and the canonical Wnt cascade. Furthermore, we also showed that down-regulation of Wnt signaling by WIF-1 was required for bisdemethoxycurcumin-induced apoptosis in certain lung cancer cell types. This report is the first to show that bisdemethoxycurcumin induces apoptosis by reactivating WIF-1 from a silenced state. Our results provide new insights into the anti-cancer actions of bisdemethoxycurcumin.
Current cancer drug targets 09/2011; 11(9):1098-110. · 5.13 Impact Factor
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ABSTRACT: Mucus hypersecretion is a major manifestation in patients with chronic inflammatory airway diseases, and MUC5AC protein is a major component of airway mucus. Earlier studies have demonstrated that neutrophil elastase (NE), a serine protease, mainly produced by neutrophils, stimulates the production of MUC5AC from airway epithelial cells. The microRNA miR-146a has been linked to inflammatory diseases. However, the role of miR-146a in the NE-induced MUC5AC expression remains unclear. Here, we show that NE exerts a dose- and time-dependent induction of both MUC5AC and miR-146a in human bronchial epithelial cells (16HBE). Ectopic expression of miR-146a in 16HBE cells inhibited the stimulation of MUC5AC by NE, while, conversely, depletion of endogenous miR-146a enhanced the MUC5AC production. Knockdown of intrinsic miR-146a activated both c-Jun N-terminal kinase (JNK) and nuclear factor-kappaB (NF-κB) signaling pathways. Moreover, targeting JNK or NF-κB by specific chemical inhibitors blocked the upregulation of MUC5AC by miR-146a silencing. Taken together, our data highlight a negative feedback role for miR-146a in the control of MUC5AC production from airway epithelial cells stimulated by NE, which may be associated with the inactivation of JNK and NF-κB signaling.
Molecular and Cellular Biochemistry 07/2011; 358(1-2):249-55. · 2.06 Impact Factor
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ABSTRACT: Cold air stimulus is a major environmental factor that exacerbates chronic inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. At the molecular level, cold is detected by transient receptor potential melastatin 8 (TRPM8). To date, TRPM8 expression has not been characterized in the airway epithelium of patients with COPD. The role of TRPM8 channels in a series of airway responses induced by cold stimuli and the molecular and biochemical pathways of TRPM8 in regulating cold-induced responses are largely unknown.
We sought to explore the role of TRPM8 in cold air-provoked mucus hypersecretion and the potential signaling pathway involved in this process.
The expression of TRPM8 in the bronchial epithelium was examined by means of immunohistochemistry, RT-PCR, and Western blotting. TRPM8 receptor function and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) were characterized by means of Ca(2+) imaging and spatiotemporal dynamics of phospholipase C (PLC) δ1-pleckstrin homology-green fluorescent protein, respectively. The expression of MUC5AC mRNA and MUC5AC mucin protein was measured by using real-time PCR and ELISA, respectively. Four serine residues in the myristoylated alanine-rich C kinase substrate (MARCKS)-phosphorylation site domain were mutated to identify the function of MARCKS in TRPM8-mediated airway mucus hypersecretion.
TRPM8 protein and mRNA expression were significantly increased in patients with COPD compared with expression seen in healthy subjects. Cold produced robust increases in intracellular Ca(2+) levels and promoted translocation of PLCδ1-pleckstrin homology-green fluorescent protein. Cold increased expression of MUC5AC mRNA and intracellular and secreted MUC5AC protein in a nonsustained way. Phosphorylation site domain-mutant MARCKS cDNA hindered MUC5AC secretion induced by cold.
These results indicate that the TRPM8 receptor is involved in cold-induced mucus hypersecretion through the Ca(2+)-PLC-PIP2-MARCKS signaling pathway.
The Journal of allergy and clinical immunology 07/2011; 128(3):626-34.e1-5. · 9.17 Impact Factor
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ABSTRACT: A series of novel fluconazoliums were synthesized and their bioactive evaluation as potential antibacterial and antifungal agents were described. Some target compounds displayed good and broad-spectrum antimicrobial activities with low MIC values ranging from 0.25 to 64 μg/mL against all the tested strains, including three Gram-positive bacteria (Staphylococcus aureus, MRSA and Bacillus subtilis), three Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Bacillus proteus) as well as two fungi (Candida albicans and Aspergillus fumigatus). Among all tested title compounds, the octyl, dichlorobenzyl, naphthyl and naphthalimino derivatives gave comparable or even better antibacterial and antifungal efficiency in comparison with the reference drugs Fluconazole, Chloromycin and Norfloxacin.
European journal of medicinal chemistry 07/2011; 46(9):4391-402. · 3.27 Impact Factor
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ABSTRACT: A series of new coumarin-based benzotriazole derivatives were successfully synthesized via a multi-step sequence of cyclization, etherification and N-alkylation, and were confirmed by 1H NMR, IR, MS spectra as well as elemental analyses. All these synthesized coumarin compounds were evaluated for in vitro antimicrobial activities against four Gram-positive bacteria, four Gram-negative bacteria and three fungi by two fold serial dilution technique. The bioactive assay showed that all these prepared coumarin benzotriazoles could inhibit the growth of the tested bacterial and fungal strains. Title compounds 11a-11e and 13a-13c were more active than chloromycin on Proteus vulgaris ATCC 6896. Coumarin benzotriazoles 11a and 11b displayed comparable antibacterial efficacy against Staphylococcus aureus ATCC 25923 and Micrococcus luteus ATCC 4698 in comparison with reference drug chloromycin. Compared to fluconazole, compounds 11a-11d displayed stronger inhibition on Aspergillus fumigatus ATCC 96918. Moreover, coumarin-based benzotriazoles in combination with antibacterial chloromycin or antifungal fluconazole, showed notable antimicrobial efficacy with less dosage and broader antimicrobial spectrum. More importantly, fluconazole-insensitive A. fumigatus and methicillin-resistant Staphylococcus aureus N 315 (MRSA) were sensitive to these combined drugs.
Yao xue xue bao = Acta pharmaceutica Sinica 07/2011; 46(7):798-810.
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ABSTRACT: Scutellarin is a flavonoid extracted from a traditional Chinese herb, Erigeron breviscapus. The present study investigated the effect of scutellarin on MUC5AC mucin production and the possible mechanism. Human bronchial epithelial 16 (HBE16) cells were pretreated with scutellarin for 60 min, and then exposed to human neutrophil elastase (HNE) or interleukin (IL)-13 for 12 hr. RT-PCR and ELISA were performed to measure the amount of MUC5AC mucin production. The results showed that scutellarin inhibited MUC5AC expression both in mRNA and protein level induced by HNE in a concentration-dependent manner. However, scutellarin failed to inhibit MUC5AC mucin production induced by IL-13. To investigate the intracellular mechanisms associated with the effect of scutellarin on MUC5AC mucin production, western blotting was carried out to examine the phosphorylation of protein kinase C (PKC), signal transducer and activator of transcription 6 (STAT6) and extracellular signal-regulated kinase 1/2 (ERK1/2). The phosphorylation of PKC and ERK1/2 was attenuated after treatment with scutellarin, whereas STAT6 was not significantly affected. Therefore, it is suggested that scutellarin down-regulates MUC5AC mucin production on HBE16 cells via ERK-dependent and PKC-dependent pathways.
Journal of Korean medical science 06/2011; 26(6):778-84. · 0.84 Impact Factor
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ABSTRACT: Mucus hypersecretion is a common pathological change in chronic inflammatory diseases of the airway. These conditions are usually accompanied by chronic mechanical stress due to airway constriction. Our objective was to study the molecular mechanisms and physical effects of chronic mechanical stress on mucin 5AC (MUC5AC) expression in airway epithelial cells. We exposed normal human bronchial epithelial (NHBE) cells cultured at an air-liquid interface to different degrees of chronic compressive mechanical stress (10, 20, 30 cmH(2)O) for 7 days(1 h per day). MUC5AC protein content was detected by enzyme-linked immunosorbent assay (ELISA). MUC5AC mRNA expression was detected by reverse transcription PCR (RT-PCR) and real-time PCR. The effects of chronic mechanical stress on phosphorylated ERK1/2 (p-ERK1/2), phosphorylated JNK (p-JNK), phosphorylated P38 (p-P38), and phosphorylation of FAK at Tyr397 (p-FAK-Y397), were assessed by Western blot. We also assessed the impact of, an EGFR kinase inhibitor (AG1478), an ERK kinase inhibitor (PD-98059), and short interfering RNA (siRNA) targeted to FAK. We found that transcriptional and protein expression levels of MUC5AC were elevated significantly in the 30 cmH(2)O compressive stress group. p-ERK1/2 increased significantly in response to compressive stress and PD-98059 could attenuated stress-induced MUC5AC expression. p-FAK-Y397 increased significantly in response to compressive stress and FAK siRNA attenuated stress-induced ERK activation strongly. AG1478 attenuated stress-induced ERK activation and MUC5AC expression significantly, but incompletely. Combination of FAK siRNA and AG1478 led to complete attenuation of ERK activation and MUC5AC expression. These results suggest that chronic mechanical stress can enhance MUC5AC expression in human bronchial epithelial cells through the ERK signal transduction pathway. Both FAK and EGFR mediate the mitogenic response induced by mechanical stress in human bronchial epithelial cells through an ERK signaling cascade.
Molecular Biology Reports 05/2011; 39(2):1019-28. · 2.93 Impact Factor
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ABSTRACT: To investigate the mechanism of azithromycin (AZT) inhibiting the airway mucous hypersecretion through matrix metalloproteinase 9 (MMP9).
After culturing, the airway epithelial cells of line HBE16 were stimulated with neutrophil elastase (NE) and pretreated with AZT and epidermal growth factor receptor (EGFR) antagonist BIBX1522. Then the cells were divided into 4 groups: control group, NE-stimulated group, AZT-pretreated & NE-stimulated group and BIBX1522-pretreated & NE-stimulated group. The mucin (MUC)5AC mRNA and MMP9 mRNA levels were analyzed by RT-PCR (reverse transcription-polymerase chain reaction). And the MUC5AC protein content in supernatant was detected by ELISA (enzyme-linked immunosorbent assay). Gelatin zymogrphy was employed to assay the MMP9 activity. Western blot was used to detect the protein levels of MMP9, prozymogen MMP9 (pro-MMP9), tissue inhibitors of metalloproteinases 1 (TIMP-1), phosphorylated EGFR (p-EGFR) and phosphorylated external-signal regulated kinase (p-ERK).
As compared with the control group, the levels of MUC5AC and MMP9 gene transcription (0.83 ± 0.17, 0.79 ± 0.16) and protein expression (0.84 ± 0.15, 0.88 ± 0.16) in the NE stimulated group were obviously higher than those of the control group (all P < 0.01). And the activity of MMP9 increased (392.33 ± 18.33, P < 0.01) while the protein level of pro-MMP9 decreased (0.17 ± 0.10, P < 0.01). But the expression of TIMP-1 showed no significant change. The protein expressions of p-EGFR and p-ERK increased (0.86 ± 0.23, 0.85 ± 0.22, both P < 0.01); as compared with the NE-stimulated group, there was a reduction of MUC5AC and MMP9 gene transcription (0.36 ± 0.15, 0.41 ± 0.09, both P < 0.01) and protein expression levels (0.30 ± 0.08, 0.37 ± 0.14, both P < 0.01) in the AZT-pretreated and NE-stimulated group. The MMP9 activity decreased (295.33 ± 14.54, P < 0.01), the protein levels of pro-MMP9 and TIMP-1 increased (0.46 ± 0.14, 0.67 ± 0.17, both P < 0.05), p-ERK protein level decreased (0.40 ± 0.19, P < 0.01) while the expression of p-EGFR showed no significant decline. As compared with the NE-stimulated group, except for the expressions of MUC5AC mRNA (0.37 ± 0.14), MMP9 mRNA (0.37 ± 0.13), p-EGFR (0.36 ± 0.13) and p-ERK (0.37 ± 0.18) decreasing (all P < 0.01) in BIBX1522-pretreated and NE-stimulated group, the other results had no obvious change.
AZT may suppress the activity and production of MMP9 in HBE16 cells so as to inhibit the airway mucous hypersecretion.
Zhonghua yi xue za zhi 03/2011; 91(10):689-93.
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ABSTRACT: To investigate the transcriptional regulation of osmotic response element binding protein (OREBP) to heat shock proteins (HSP)70 and its effect on mucus secretion under hypertonic conditions.
Human bronchial epithelial HBE16 cells were cultured in vitro in hypertonic medium for different times. Western blot was used to analyze the levels of OREBP and HSP70-2 protein. The mucin(MUC)5AC protein content in supernatant were detected by enzyme linked immunosorbent assay (ELISA). Different deletions and mutants of HSP70-2 promoter in upstream region were cloned in the reporter pGL3-Basic plasmid. These reporter plasmids were co-transfected with OREBP siRNA and the promoter activities detected with dual luciferase assay to study the ORE site in the HSP70-2 promoter and its impact on MUC5AC.
Compared with control group (0.21 ± 0.05, 0.15 ± 0.06, 0.13 ± 0.04), the levels of OREBP, HSP70-2 and MUC5AC in supernatant significantly increased (0.54 ± 0.07, 0.20 ± 0.08, 0.17 ± 0.04) after HBE16 cells were exposed to 600 mOsm/L hypertonic mediums for 3 h (2.57, 1.33 and 1.31 fold in protein respectively) (all P < 0.05), and their expression contents increased in a time-dependent manner, for 6 h (2.81, 3.07 and 3.77 fold) (all P < 0.01), for 9 h (3.57, 5.13 and 4.00 fold) (all P < 0.01), for 12 h (4.24, 5.33 and 4.54 fold) (all P < 0.01). After a knock-down of OREBP by RNAi for 48 h, the levels of OREBP, HSP70-2 and MUC5AC significantly decreased (0.36 ± 0.08; 0.33 ± 0.08; 0.24 ± 0.05) versus the control group (0.95 ± 0.27, 0.75 ± 0.22, 0.58 ± 0.22) (protein inhibition ratio at 62%, 56% and 59% respectively) (P < 0.05, P < 0.01, P < 0.05). Luciferase assay indicated that an important ORE site in HSP70-2 promoter was in the region from -353 to -66. The inactivation of ORE site at -93 by site-directed mutagenesis led to a complete loss of HSP70-2 promoter activity (P < 0.01).
One ORE site at -93 in the HSP70-2 promoter and OREBP were found to play essential roles in inducing the HSP70-2 transcription and MUC5AC hypersecretion in human bronchial epithelial cells in response to hypertonicity.
Zhonghua yi xue za zhi 03/2011; 91(8):549-53.
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ABSTRACT: To explore the effects of sphingosine kinase 1 (SphK1) on mucin (MUC)5AC overexpression under the induction of tumor necrosis factor-α (TNF-α).
The HBE16 airway epithelial cells were cultured, pretreated with SphK1-specific inhibitor DMS or transfected with SphK1 siRNA. Then each group was stimulated by a certain concentration of TNF-α. The relative contents of SphK1, COX-2, p-P38, p-ERK and IκBα were detected by Western blot while the levels of MUC5AC, PGE2 and cAMP analyzed by enzyme linked immunoassay. The transcription activities of cAMP-response-element-binding protein and nuclear factor κB (NF-κB) were detected by luciferase reporter gene detection system. The mRNA expressions of SphK1 and MUC5AC were detected by RT-PCR (reverse transcription-polymerase chain reaction). And the intracellular MUC5AC protein was detected by immunofluorescence.
The levels of protein and mRNA expression of SphK1 and MUC5AC were obviously elevated [(0.69 ± 0.12, 0.86 ± 0.07), (0.60 ± 0.09, 0.79 ± 0.05)]. They were all significantly higher than those in the control group (0.26 ± 0.03, 0.33 ± 0.04, 0.18 ± 0.06, 0.22 ± 0.10). There was an elevation of COX-2, PGE2, cAMP, p-P38, p-ERK, CREB and NF-κB activity (all P < 0.01); the production of IκBα protein was lower than that in the control group (P = 0.003). DMS decreased the levels of MUC5AC mRNA expression (0.37 ± 0.06) and protein production (0.27 ± 0.04), lowered the production of COX-2, PGE2, cAMP, p-P38 and p-ERK, inhibited the activity of CREB and NF-κB and increased the IκBα production (all P < 0.01). Transfection of SphK1 siRNA showed the similar effects as DMS (all P < 0.01).
SphK1 plays an important role in regulating the MUC5AC expression under the induction of TNF-α. And the activation of critical signal factors in that process is dependent on SphK1.
Zhonghua yi xue za zhi 02/2011; 91(6):391-5.
