Triptolide, a type of diterpenoid, is the active compound of Tripterygium wilfordii; it plays roles in anti-inflammatory and immune response regulation. Our objective was to investigate the mechanism of the inhibitory effect of triptolide on interleukin-13 (IL-13) gene expression in activated T lymphocytes. Understanding the molecular mechanism by which triptolide exerts a therapeutic function may be useful in developing a pharmaceutical treatment for asthma.Peripheral blood mononuclear cells (PBMC) and Hut-78 cells were stimulated with anti-CD3/CD28 with or without co-incubation with triptolide. The alteration of IL-13 messenger RNA (mRNA), expression and protein level were analysed using real-time reverse transcription polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay, respectively. The intracellular distribution profile of transcription factor GATA3 and nuclear factor of activated T cells (NFAT1) were analysed by Western blotting. The binding rates of GATA3 and NFAT1 to the promoter sequence of IL-13 were analysed by chromatin immunoprecipitation (ChIP) PCR.In PBMC, the release of IL-13 was dependent on anti-CD3/CD28 stimulation. Its release could be inhibited by triptolide at the concentration of 500 nmol. In Hut-78 cells, IL-13 mRNA and protein expression were increased with anti-CD3/CD28 stimulation and significantly inhibited by incubation with 28 nmol triptolide. This concentration of triptolide also significantly inhibited the nuclear translocation of GATA3 and NFAT1 reducing the binding rate to the IL-13 gene promoter.Triptolide inhibits IL-13 gene transcription and protein expression by inhibiting GATA3 and NFAT1 nuclear translocation and their binding rates to the IL-13 gene promoter region.
Objective:To observe the effects of dexamethasone on expression of the transcription factor GATA-3 in asthmatic rats.Methods:Thirty-six SD rats(SPF)were randomly divided into three groups:normal control group(A),asthmatic model group(B)and dexamethasone group(C).The asthmatic model of SD rats was established by immunization with intraperitoneally injected and inhaled ovalbumin(OVA).The effects of the asthmatic model were evaluated by using animal respirator.The expression of GATA-3 in the lungs of SD rats was measured with immunohistochemistry method.Results:The results of the expiratory airway resistance(Re)of each group challenged by acetylcholine showed that the model was successfully established.Expression of GATA-3 in B and C group was significantly higher than that in A group(P 0.01).Compared with B group,expression of GATA-3 in C group was significantly lower(P 0.01).Conclusion:The over-expression of GATA-3 exists in bronchial asthmatic rats.Dexamethasone can reduce airway hyperresponsiveness and inhibit the expression of GATA-3.So dexamethasone can correct Th1/Th2 imbalance in asthmatic rats and has effects on asthma.
Objective To investigate the inhibitory effects of aerosol budesonide on the expression of indoleamine 2,3 dioxygenase (IDO),airway inflammation,and airway hyperresponsiveness in a murine model of bronchial asthma (asthma).Methods 18 BALB/c mice were randomly divided into 3 groups,including the control group,ovalbumin (OVA) group and budesonide group.Mice were sensitized and challenged by OVA.24 h after the last challenge,airway responsiveness to acetylcholine chloride (Ach) was measured.Hematoxylin & eosin staining was used to assess the inflammatory cell infiltrates.Levels of Th2 cytokines (IL-4 and IL-13) in bronchoalveolar lavage fluid (BALF),and total IgE and OVA-specific IgE (OVA-sIgE) in serum were relevantly detected by ELISA.The protein expression of IDO was determined by western blot analysis.Results The airway resistance in the OVA group was obviously increased in a dose-dependent manner following administration of Ach,whereas only a slight increase could be detected in the control group.Treatment with budesonide led to a sharp decrease in airway resistance compared with the OVA group (P ＜0.05).Total IgE and OVA-sIgE in serum,total inflammatory cells and differential eosinophils as well as Th2 cytokines in BALF were significantly increased in the OVA group.These inflammatory indices were remarkably decreased by treatment with budesonide (P ＜0.05).The pulmonary expression of IDO was apparently declined in the OVA group.Treatment with budesonide enhanced the pulmonary expression of IDO in comparison with the OVA group (P ＜ 0.05).Conclusions Budesonide could inhibit the airway inflammation and hyperresponsiveness by upregulating the expression of IDO in allergic asthma.
Key words:
Bronchial asthma; Indoleamine 2,3 dioxygenase; Budesonide; Dendritic cell; Regulatory T cell
Lung cancer has the highest mortality among malignant tumor worldwide,of which the prognosis is closely related to its clinical stage in the diagnosis.So early discovery,early diagnosis and early treatment are the main measure to decrease case fatality of lung cancer.The high-risk individuals with lung cancer have high expression of hnRNPA2/B1 and hTERT in exfoliated cells of their sputum.Combined detection should contribute to find the patients who are at early and treatable stage to reduce potential mortality.So combined detection of hnRNPA2/B1 and hTERT in sputum samples from lung cancer patients is a profitable approach to the early diagnosis of lung cancer.
Nuclear factor kappaB (NF-kappaB) overactivation, requiring phosphorylation and degradation of its inhibitor IkappaBalpha, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IkappaBalpha, carrying an IkappaBalpha gene from human placenta, we optimized a novel IkappaBalpha mutant (IkappaBalphaM) gene, constructed and characterized its replication-deficient recombinant adenovirus (AdIkappaBalphaM), and tested whether AdIkappaBalphaM-mediated overexpression of IkappaBalphaM could inhibit the NF-kappaB activation in endothelial cells.IkappaBalphaM gene (203 - 1003 bp) encoding 267 amino acids, acquired by site-directed deleting N-terminal phosphorylation sites of serine 32/36, was subcloned into the pShuttle and pGEM-T vectors for further polymerase chain reaction (PCR), restriction digestion, deoxyribonucleic acid (DNA) sequencing and homology analyses. Subsequent to inserting the expression unit of pShuttle-IkappaBalphaM, containing cytomegalovirus (CMV) promoter, IkappaBalphaM complementary DNA (cDNA) and polyadenylic acid (PolyA) signals, into the type 5 adenovirus (Ad5) vector, the resultant AdIkappaBalphaM was packaged in human embryonic kidney (HEK) 293 cells by cotransfection with lipofectamine. Western blot analysis and electrophoretic mobility shift assay were utilized to detect the AdIkappaBalphaM-mediated overexpression of IkappaBalphaM in HEK293 cells and its suppressive effect on phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB activation in human umbilical vein endothelial (ECV304) cells, respectively.The relevant nucleotides and deduced amino acids of 801 bp IkappaBalphaM gene were consistent with those of IkappaBalpha gene (GenBank accession number: M69043). The titer of the prepared AdIkappaBalphaM was 4.0 x 10 (12) plaque-forming units (pfu)/L. Moreover, the IkappaBalphaM gene was overexpressed in HEK293 cells, and potently inhibited the PMA-induced NF-kappaB activation in ECV304 cells dose-dependently.AdIkappaBalphaM is a novel vector for both efficient transfer and specific overexpression of IkappaBalphaM gene, as well as potent inhibition of NF-kappaB activity, providing a promising strategy for gene therapy of asthma.
Objective:To investigate the effects of luteolin on airway remodeling and the expression of IL-13Rα2 in the airway of chronicity asthmatic mice.Methods:Thirty-two BALB/c mices were randomly divided into four groups.The mouse asthma models(asthma group)were established by sensitization and challenge with ovalbumin(OVA),eight mices interfered with luteolin 30 min before challenge(luteolin group),eight mices administrated dexamethasone by intraperitoneal injection 30 min before challenge(dexamethasone group).The healthy control mice were treated with saline instead of OVA.Lung tissu were collected 24 h after the last challenge.The left lung tissue was enbedded in paraffin and sectioned.Henatoxylin and esin(HE)staining were performed,Histologocial changes were detected.The expressions of genes and their prouducts of IL-13Rα2 in airway were detected using RT-PCR and immunohistochemistry(ISH).The expressions of genes of TGF-β in lung tissue were detected using RT-PCR.Results:①HE-stained lung tissue showed there were airway remodeling in each group,the thickness of airway in luteolin group was significantly decreased compared with asthma group and increased compared with normal group.②Wat/Pi and Wam/Pi in luteolin group and dexamethasone group were significantly lower than those in asthma group.③RT-PCR showed that expression of IL-13R2mRNA and TGF-β1mRNA in luteolin group and dexamethasone group were significantly lower than those in asthma group.④ISH Showed that expression of IL-13Rα2 in luteolin group and dexamethasone group were significantly lower than those in asthma group.Conclusion:Luteolin might inhibit the thicking of airway wall and airway smooth muscle proliferation,and inhibit airway remodeling.Its possible mechanisms might be of inhibiting the expression of IL-13Rα2 in the lung tissue.
Much evidence suggests that respiratory syncytial virus (RSV) infection prolongs airway hyperresponsiveness (AHR) and exacerbates asthma by enhancing airway inflammation. However, the characteristic of airway inflammation and kinetics of airway dysfunction occurred in the central and peripheral airways were not fully delineated. The objective of this study was to investigate the effect of RSV on the allergic airway inflammation in different size airways and to elucidate its possible mechanism. Using a murine model of prior ovalbumin (OVA) sensitization and subsequent RSV challenge, lung resistance (RL), and dynamic compliance (Cdyn) was conducted by barometric whole-body plethysmography. Histological examinations were carried out. Differential cells count in bronchoalveolar lavage (BAL) fluid, serum anti-OVA IgE, and IgG1 were measured. Cytokine mRNA expression in lung tissue were determined. RSV triggered a significant increase in RL and reduction in Cdyn, as well as greatly prolonged the recovery of Cdyn more than that of RL in OVA-sensitized mice. Also, RSV resulted in more severe peripheral airway inflammation which exhibit as globe cell hyperplasia and CD8+ T cell infiltration. Furthermore, the number of lymphocytes, neutrophils and macrophages in BAL fluid, serum anti-OVA IgE and IgG1 were remarkably increased. Additionally, mice increased relative expression of cytokines IL-4, IL-13, and IFN-γ, but not IL-5, IL-17, and IL-17F. These findings demonstrated that RSV could selectively affect pathologic processes that contribute to altered airway function in the central and peripheral airways in OVA-sensitized mice. These processes may be involved in goblet cell hyperplasia and CD8+ T cell infiltration in peripheral airways.
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