[Inhibition of 1,25-(OH)2D3 on passively sensitized human airway smooth muscle cells].

OBJECTIVE: To investigate the effects of 1,25-(OH)(2)D(3) on the proliferation of passively sensitized human airway smooth muscle cells (HASMCs) and their expressions of MMP-9 and a disintegrin and metalloprotease 33(ADAM33). METHODS: HASMCs were passively sensitized with 10% serum from asthmatic patients. MTT colorimetry assay was used to examine the effect of 1,25-(OH)(2)D(3) on cell proliferation at different concentrations (10(-10) mol/L, 10(-9) mol/L, 10(-8) mol/L, 10(-7) mol/L).By this way, its optimal inhibitory concentration was determined. And then the effects of 1,25-(OH)(2)D(3) at the optimal concentration on cell proliferation was examined by the same MTT assay and cell cycle analysis by flow cytometry. The expressions of MMP-9 and ADAM33 in HASMCs were studied by real-time quantitative RT-PCR and Western blotting analysis. RESULTS: (1) Inhibition of cell proliferation by 1,25-(OH)(2)D(3) was barely detectable at 10(-10) mol/L. But with the increasing concentration ranging from 10(-9) mol/L to 10(-7) mol/L, 1,25-(OH)(2)D(3) markedly inhibited the cell proliferation concentration-dependently and reached the maximum effect at the concentration of 10(-7) mol/L. Accordingly, 10(-7) mol/L was chosen as the optimal concentration of 1,25-(OH)(2)D(3) for the following study. (2) At the concentration of 10(-7) mol/L, 1,25-(OH)(2)D(3) inhibited the cell proliferation of passively sensitized HASMCs in a time-dependent manner and hampered the G(1)/S transition. (3) 1,25-(OH)(2)D(3) pretreatment attenuated the MMP-9 and ADAM33 protein levels in passively sensitized HASMCs by (63.4 ± 3.6)% and (50.9 ± 2.9)%, respectively (P < 0.01). (4) 1,25-(OH)(2)D(3) significantly inhibited the MMP-9 and ADAM33 mRNA levels in passively sensitized HASMCs by (52.2 ± 2.5)% and (67.8 ± 3.2)%, respectively (P < 0.01). CONCLUSION: 1,25-(OH)(2)D(3) has a direct inhibitory effect on passively sensitized HASMCs in vitro, including the inhibition of cell proliferation and the expressions of MMP-9 and ADAM33, which maybe associated with the beneficial role of 1,25-(OH)(2)D(3) in the prevention and therapy of asthmatic airway remodeling.

Suppression of allergic airway inflammation in a mouse model by Der p2 recombined BCG.

Allergic asthma is a chronic inflammatory disease mediated by T helper (Th)2 cell immune responses. Currently, immunotherapies based on both immune deviation and immune suppression, including the development of recombinant mycobacteria as immunoregulatory vaccines, are attractive treatment strategies for asthma. In our previous studies, we created a genetically recombinant form of bacille Calmette-Guerin (rBCG) that expressed Der p2 of house dust mites and established that it induced a shift from a Th2 response to a Th1 response in naive mice. However, it is unclear whether rBCG could suppress allergic airway inflammation in a mouse model. In this article we report that rBCG dramatically inhibited airway inflammation, eosinophilia, mucus production and mast cell degranulation in allergic mice. Analysis of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels in bronchoalveolar lavage fluid (BALF) and lung tissue revealed that the suppression was associated with a shift from a Th2 response to a Th1 response. At the same time, rBCG induced a CD4(+) CD25(+) Foxp3(+) T-cell subtype that could suppress the proliferation of Th2 effector cells in vitro in an antigen-specific manner. Moreover, suppression of CD4(+) CD25(+) T cells could be adoptively transferred. Thus, our results demonstrate that rBCG induces both generic and specific immune responses. The generic immune response is associated with a shift from a Th2 to a Th1 cytokine response, whereas the specific immune response against Der p2 appears to be related to the expansion of transforming growth factor-beta (TGF-beta)-producing CD4(+) CD25(+) Foxp3(+) regulatory T cells. rBCG can suppress asthmatic airway inflammation through both immune deviation and immune suppression and may be a feasible, efficient immunotherapy for asthma.

Notch signaling regulates the FOXP3 promoter through RBP-J- and Hes1-dependent mechanisms.

Evidence has shown that Notch signaling modulates CD4(+)CD25(+) regulatory T-cells (Tregs). As transcription factor Foxp3 acts as a master molecule governing the development and function of Tregs, we investigated whether Notch signaling might directly regulate Foxp3 expression. Here, we provide evidence that Notch signaling can modulate the FOXP3 promoter through RBP-J- and Hes1-dependent mechanisms. A conserved RBP-J-binding site and N-box sites were identified within the FOXP3 promoter. We show that the Notch intracellular domain (NIC), the active form of Notch receptors, activates a reporter driven by the FOXP3 promoter. Dissection of the FOXP3 promoter revealed bipartite effects of the RBP-J-binding site and the N-boxes: the RBP-J-binding site positively, while the N-boxes negatively regulated the FOXP3 promoter activity. Moreover, in freshly isolated Tregs, NIC-RBP-J complex is bound to the FOXP3 promoter in Tregs. Our results suggest that Notch signaling might be involved in the development and function of Tregs through regulating Foxp3 expression.

[To explore the mechanisms of neurogenic inflammation and airway hyperresponsiveness of rat by inhaled sulfur].

AIM: To explore the physiopathological mechanisms of airway injury and the effect on the airway responsiveness of rat by inhaled sulfur dioxide(SO2). METHODS: Sixteen SD male rats were divided randomly into 2 groups (n = 8): the control group and SO2 group. The control group was exposed o pure air. SO2 group was exposed to SO2 of the content 1.0 mg/(m(3) x h) 6h daily for consecutive 3 d. At 4th day, we determined the airway responsiveness, collected the bronchoalveolar lavage fluid (BALF), plasma and lung tissue. Then we counted the total cellular score in BALF, measured the plasma SP content and made the immunohistochemistry staining on the lung tissue (HE and SP methods). RESULTS: Compared with the control group, the total cellular score in BALF and plasma SP content in SO2 group's increased significantly ( P < 0.01). HE staining showed there were a great deal of inflammatory cells infiltration under the tunica mucosa bronchiorum; and SP immunohistochemistry staining indicated there were significant changes in numbers of SP-IR positive fibers of SO2group. CONCLUSION: Exposure to low concentration of SO2 would injure healthy rat's airway, and induce airway hyperresponsiveness, neurogenic inflammation is one of its critical pathophysiological mechanisms.

[Respiratory syncytial virus infection enhances airway hyperresponsiveness in guinea pigs and the underlined mechanism].

AIM: To study the relation between Respiratory Syncytial Virus infection and asthma development by measuring airway responsiveness (AR) and M2R function. METHODS: Guinea pigs (n = 34) were randomly divided into 4 groups: Hep-2/NS group (group A, n = 9), RSV/NS group (group B, n =9), Hep-2/OVA group (group C, n = 8) and RSV/OVA group(group D, n = 8). On day 21 after infection we tested AR and M2R. Then counted eosinophils in BALF and observed pathological change. RESULTS: Intraairway pressure(IP mmH20) of group B had no significant difference with group A(P > 0.01), and the extent of IP decrease also had no difference between groups A and B (P > 0. 05), but IP of C group were much higher than group A (P<0.05), with extent of IP decrease lower than group A (P < 0.05). And IP of group D were higher than group C (P < 0.01), with the extent of IP decrease much lower than group C (P < 0.05). CONCLUSION: RSV infection could enhance OVA-induced M2R dysfunction, then develop AHR.

[CIAPIN1 expression in human lung cancer tissues and inhibitory effects of the gene on human pulmonary carcinoma NCI-H446 cells].

AIM: To explore CIAPIN1 gene expression in lung carcinoma tissues and its regulatory in the growth of NCI-H446. METHODS: Fifty-one formalin-fixed paraffin-embedded specimens of primary lung cancer and their non-cancerous counterparts were detected by SABC immunohistochemistry method. Adenoviral vector construction was recombined and the gene was transduced into NCI-H446 cells. The expression of CIAPIN1 protein was identified by Western blot. Trypan blue staining was used to count the alive cells and to draw a cellular growth curve. The changes of cell cycle and apoptosis were analyzed by flow cytometry. RESULTS: The positive rate of CIAPIN1 expression in cancer tissues (39.2%)was much lower than that in non-cancerous counterparts (100%, P<0.05). In Ad-CIAPIN1 group, the growth of transfected NCI-H446 cells in vitro was significantly inhibited. In addition the Ad-CIAPIN1-induced cell apoptosis and a predominant arrest in the G1/S phase (P<0.01) were observed. CONCLUSION: The down-regulation of CIAPIN1 expression in tumor is associated with the development of lung carcinoma. Transduction of NCI-H446 CIAPIN1-negative cell, with Ad-CIAPIN1 can inhibit cell growth, suggesting that CIAPIN1 can be a new tumor-related suppressor gene.

Inhibition of tumor angiogenesis by HMGB1 A box peptide.

High mobility group box 1 protein (HMGB1) is a highly conserved, ubiquitous non-histone nuclear protein, which participates in maintaining nucleosome structure, regulation of gene transcription, and modulating the activity of steroid hormone receptors. Substantial evidence demonstrated that HMGB1 could be secreted into the extracellular milieu, acts as a proinflammatory cytokine and mediates the downstream inflammatory responses in endotoxemia, arthritis and sepsis. Recently, several reports suggested that HMGB1 plays a key role in tumor angiogenesis through multiple mechanisms, including up-regulation of proangiogenic factors, promoting endothelial progenitor cells homing to ischemic tumor tissues and induction of endothelial cell migration and sprouting. And blockade of HMGB1 binding to the receptor for advanced glycation end products (RAGE) with anti-HMGB1 antibody, soluble RAGE or anti-RAGE neutralizing antibody has been proved to inhibit angiogenesis efficiently. Since HMGB1 A box peptide could antagonize the HMGB1 whole length protein by competitively binding to RAGE and has been considered as a HMGB1 specific antagonist, we postulate that the HMGB1 A box peptide could function as an anti-angiogenic agent to inhibit tumor angiogenesis. In our opinion, if the hypothesis proved to be practical, HMGB1 A box peptide could be widely used in clinical settings to treat malignant tumors.

[Effect of IL-4 on ABCG2 expression in human lung adenocarcinoma cell lines].

AIM: To investigate the effect of IL-4 on the expression of ABCG2 in lung adencarcinoma cell lines. METHODS: The ABCG2 mRNA and protein expression was determined by semi-quantitative RT-PCR and Western blot respectively. RESULTS: The ABCG2 mRNA and protein was detectable in lung adencarcinoma cell lines A549 and SPC-A-1. But IL-4 stimulation did not significantly affect the ABCG2 mRNA and protein expression in lung adencarcinoma cell lines. CONCLUSION: IL-4 does not regulate ABCG2 expression in human lung adencarcinoma cell lines.

Comparative proteomic study of acute pulmonary embolism in a rat model.

Pulmonary embolism (PE) is a common, potentially fatal disease, whose blood clots originate from the deep venous system of the lower extremities. PE is of clinical importance because of the considerable mortality and morbidity. In this study, at first we established a rat PE model by injecting 3-4 emboli into the left jugular vein. Before collecting the lung tissues, we perfused them with saline through the right jugular vein and at the same time cut off the right carotid to remove the blood. Then we separated and identified differentially expressed proteins in lung tissues at different time points using the techniques of 2-DE and MS. After image analysis of 2-DE gels, 46 protein spots of interest were excised from the gels and identified by MALDI-TOF-MS. Thirty-two protein spots of them found their corresponding protein candidates in the database. These proteins are associated with distinct aspects of PE such as the contractive function of smooth muscles, metabolism of energy, collagen and toxicant, cellular differentiation, apoptosis and injury, blood pressure adjustment, maintaining of acid-base balance, and so on. Ten of the identified proteins were validated by semiquantitative RT-PCR, and three of them were further validated by Western blot analysis. The differential expression patterns of these proteins suggest the distinct roles they may play in different stages of the rat PE model, and information from this study may be helpful to uncover the pathophysiologic molecular mechanisms involved in PE.

[The effects of rBCG expressing Der p2 in the form of lipoprotein on murine immune response].

AIM: To investigate the effects of rBCG vaccination containing foreign antigen Der p2 in the form of lipoprotein on murine immune response. METHODS: 6 to 8 weeks old and newborn BALB/c mice were vaccined intraperitoneally with 10(6) CFU rBCG or BCG. At the same time, the control group was injected with saline. Six weeks later, all animals were injected with Der p2 (20 microg). After two weeks later, the concentrations of IL-4 and IFN-gamma in the serum and splenocyte culture supernatant (STLCS) were determined by ELISA, and Th subgroups were determined by double fluorescent staining and flow cytometry. RESULTS: After vaccination, the serum and STLCS from both rBCG-immunized and BCG-immunized group of adult and newborn BALB/c mice had significantly higher level of IFN-gamma and lower level of IL-4 than those from control groups. Besides, there was the larger percentage of CD4 (+) IFN-gamma (+) cells in spleen from rBCG-vaccined and BCG-vaccined mice than that from control group. However, the percentage of CD4 (+) IL-4 (+) cells in spleen cells from rBCG-vaccined and BCG-vaccined group was lower than that from control group. Moreover, the level of IFN-gamma in STLCS from rBCG-immunized was significantly higher, compared with that from BCG-immunized mice. At the same time, the percentage of CD4 (+) IFN-gamma (+) cells in spleen from rBCG-vaccined mice was larger than that from BCG-vaccined group. CONCLUSION: Both rBCG and BCG could stimulate Th1 predominant immune response, when injected intraperitoneally into adult or newborn BALB/c mice, The Der p2 expressed on the cell wall of BCG can work as the component of BCG and be recognized by the immune system of mice, therefore stimulates Der p2-specific Th1 predominant immune response. These data indicate that recombinant BCG-expressing antigens can be used as the antigen-specific vaccines against allergic diseases by regulating the balance of Th1/Th2.

[Exploration of serum endothelin-1 as a marker of early radiation lung injury].

AIM: To explore the possibility of endothelin-1(ET-1) as a serological marker of early diagnosis and progression of radiation induced lung injury. METHODS: One hundred and ninety female rats were randomly divided into control group (group C) and experimental groups, namely, radiation group (group R), fluvastatin treatment group (group Flu), retinoic acid treatment group (group Ra) and dexemethasone treatment group (group Dex). The chests of rats in experimental groups were exposed to radiation by linear accelerator after anesthesia. The radiation dose for each rat was 15Gy, 2Gy per minute, and radiation distance was 1 meter. The next day after radiation, fluvastatin (20 mg. kg(-1). d(-1) ) was administered orally in group Flu, retinoic acid (20 mg. kg(-1). d(-1)) in group Ra and dexemethasone (3.33 mg. kg(-1). d(-1)) in group Dex. The rats in group C and group R were medicated with the equal volume of normal saline. On the 5th, 15th, 30th, and 60th day after radiation, five rats were randomly chosen from each group respectively. The sera were harvested by decapitation or cardiopuncture and at the same time, lung tissues were cut off. The levels of serum ET-1 and LN were detected by radioimmunological assay(RIA). The pathologic changes of lung tissue were observed under light microscope. RESULTS: Compared to the control group, serum ET-1 level began to increase on the 5th day after exposure to radiation and reached the peak on the 60th day in group R. The levels of laminin and hyaluronic acid began to rise on the 30th day and the 60th day respectively. The elevation of serum ET-1 level in group R was obviously earlier than that in other groups and correlated to extent of lung injury. CONCLUSION: The serum ET-1 can be used as a marker of early diagnosis and dynamic changes of radiation lung injury.

[Construction of antisense recombinant adenoviral vector for c-myc and its antiproliferative effect on rat lymphocytes].

AIM: To observe the antiproliferative effect of antisense recombinant adenoviral vector for c-myc on rat thymus lymphocytes. METHODS: Antisense and sense bacterial plasmids for c-myc were constructed. Bacterial plasmids and El detected adenoviral plasmid were cotransfected into 293 cells. Recombinant adenoviral vectors were obtained after cotransfection. The antiproliferative effects were assayed by MTS. The expression of c-myc mRNA was detected by RT-PCR. RESULTS: The results showed that antisense recombinant adenoviral vector for c-myc could inhibit rat thymus lymphocytes proliferation. The expression of c-myc mRNA was decreased after antisense recombinant adenoviral vector for c-myc was transfected into cells. CONCLUSION: Recombinant antisense adenoviral vector for c-myc could inhibit rat thymus lymphocytes proliferation.

[NF-kappaB expression in lung tissue of acute lung injury rat model and the influence by antioxidant N-acetylcysteine].

AIM: To observe the NF-kappaB expression in the lung tissue of LPS-induced acute lung injury(ALI) rat model and the influence of N-acetylcysteine (NAC) on NF-kappaB expression. METHODS: The expression of NF-kappaB in lung tissue in ALI rat model and the influence of NAC on NF-kappaB expression were detected by immunohistochemical (ABC) staining and Western blot. RESULTS: There were a small amount of sporadic NF-kappaB cells in airway epithelium and interstitium in normal control group. In contrast, nuclear NF-kappaB expression-positive cells increased obviously in airway mucosa, lung interestium, alveolar cavity and vascular wall of ALI rats. NF-kappaB(+) cells were mainly airway mucosa epithelial cells, infiltrating inflammatory cells, alveolar epithelial cells, and vascular endothelial cells. The NF-kappaB expression-positive cells in NAC therapy group notably decreased compared with ALI group and control group(P<0.01). Western blot analysis showed that the expression of NF-kappaB was different at various time points, reaching the peak at 3 h and then decreased (P<0.01) after LPS induced lung injury. CONCLUSION: In LPS induced acute lung injury rat model, the NF-kappaB nuclear expression increased obviously in airway mucosa, lung interestium and alveolar cavity. Most cells in lung tissue participated in the activation of NF-kappaB. NAC could alleviate inflammation by inhibiting activation of NF-kappaB.

[The changes of expression level of matrix metalloprotease 9 and its inhibitor (TIMP-1) in murine pulmonary fibrosis model].

AIM: To evaluate the balance between matrix metalloprotease-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) in bleomycin-induced pulmonary fibrosis in mice. METHODS: Pulmonary fibrosis model was induced by bleomycin in mice. The histological images of lungs were studied by HE staining. Alveolar macrophages(AMs) were separated by anti-CD68 mAbs using immunomagnetic beads(d0.05). The concentration of MMP-9 secreted by AMs was gradually decreased on day 1, 3, 7, 14 and 28, and levels of TIMP-1 gradually increased. CONCLUSION: There was an imbalance between macrophage-derived MMP-9 and TIMP-1 in bleomycin-induced pulmonary fibrosis in mice.

[Cloning and expression of the extracellular domain of calcium-activated chloride channel in mouse airway goblet cells].

AIM: To clone and express the extracellular domain of murine calcium-activated chloride channel (mCLCA3) in airway goblet cell of mouse. METHODS: According to the gene sequence of mCLCA3 the PCR primers for N-terminal, middle and C-terminal extracellular domains were designed. Using recombinant plasmid pcDNA3.1(-)/mCLCA3 as template, the DNAs coding for the three extracellular domains were amplified. And then the DNAs encoding N-terminal and C-terminal extracellular domains were inserted into expression vector pRSET-A, while the middle extracellular domain DNA was inserted into pGEX-T1. E.coli. BL21(DE3) were transformed with the three recombinant plasmids, respectively, and were induced with IPTG for expression. RESULTS: DNA sequencing showed that the cloned DNAs encoding extracellular domains were identical with those in GenBank (GenBank accession No. NM-017474 ). The 3 domains were expressed in E.coli and most of the expressed products existed in the form of inclusion body. CONCLUSION: The expression of three extracellular domains of mCLCA3 lays the foundation for further preparing anti-mCLCA3 antibody and exploring the mechanism of modulation of mCLCA3.

[Inhibitive effect on airway mucus overproduction with DNA vaccine based on xenogeneic homologous calcium-activated chloride channel in asthmatic mice].

OBJECTIVE: To observe the inhibitive effect on airway mucus overproduction with DNA vaccine based on human calcium-activated chloride channel 1 (hCLCA1) in asthmatic mice, who own mCLCA3 being xenogeneic homology of hCLCA1 in airway goblet cell. METHODS: The DNA vaccine was made with hCLCA1 gene inserted into pSecTag2B, and then BALB/c mice were vaccinated by i.m. once every two weeks. When serum antibody showed binding activity to mCLCA3 with ELISA analysis, asthma will be induced with ovalbumin in the vaccinated mice. To detect mucus production, lung sections were PAS stained and their MUC5AC mRNA levels were investigated by reverse transcription polymerase chain reaction (RT-PCR). Mice in control groups were injected with pSecTag2B/mCLCA3, pSecTag2B and saline, respectively. RESULTS: Antiserum of vaccine group after three times vaccination showed good binding activity to three mCLCA3 extracellular domains (ED), and the activity to N-terminal C-terminal ED was stronger than middle-ED. After induced to asthma, the number of goblet cell and MUC5AC mRNA level in vaccine group were lower than these in control group. CONCLUSION: hCLCA1 DNA vaccine can induce mouse to produce serum antibody binding itself mCLCA3, and thus airway mucus overproduction of asthmatic mouse is effectively inhibited.

[The inhibiting effect of niflumic acid on airway hyperresponsiveness in asthmatic mice].

OBJECTIVE: To evaluate the inhibiting effect of niflumic acid (NFA), an inhibitor of calcium-activated chloride channel (ClCa) on airway epithelium, on the airway hyperresponsiveness in asthmatic mice. METHODS: BALB/c mice were randomly divided into an asthma group (A group), a NFA prevention asthmatic group (B group) and a sham-challenged group (C group). The airway pressure time index (APTI) and the content of ET-1 and NO in bronchoalveolar lavage fluid (BALF) in all groups were measured. With the isolated tracheal rings with integral epithelium or epithelium removed from the asthma group (A(1) group and A(2) group) and the sham-challenged group (C(1) group and C(2) group), the contractile responsiveness of various rings to methacholine (mACh) was examined, and its change was observed when the rings were exposed to NFA beforehand. RESULTS: Compared with A group (1.62 +/- 0.14), the APTI in B group (1.21 +/- 0.07) was reduced remarkably (P < 0.01), and the contents of ET-1 [(103 +/- 9) ng/L] and NO [(48.5 +/- 3.2) micromol/L] in BALF of A group were significantly higher than those in B group, [(53 +/- 5) ng/L, (23.7 +/- 2.5) micromol/L (P < 0.01), respectively]. The ratios of maximum contractility in A(1), A(2), C(1) and C(2) groups were (3.79 +/- 0.44), (2.15 +/- 0.21), (1.26 +/- 0.14) and (2.06 +/- 0.18), respectively. The contractility of A(1) group was highest among all groups (all P < 0.01), but could be effectively decreased by NFA. CONCLUSIONS: By inhibiting the special ClCa on the airway epithelium, NFA can inhibit the production of ET-1 and NO by epithelium and thus exert preventive effect on airway hyperresponsiveness in asthma.

[Construction and identification of the E.coli-BCG shuttle vector expressing lipoprotein Der p2 on cell wall of mycobacterium vaccae].

AIM: To construct the E.coli-BCG shuttle vector carrying and expressing dust mite antigen gene Der p2 on cell wall of mycobacterium vaccae. METHODS: The gene fragment encoding 19 kDa antigen and the upstream control element (19-ss) was amplified by PCR from the mycobacteria tuberculosis H37Rv.Subsequently, the 19-ss gene was cloned into the E.coli-BCG shuttle vector pOLYG, which can schlepped and expressed exogenous antigen gene on cell wall of mycobacteria and containing the Der p2 gene. The expression of Der p2 gene in mycobacterium vaccae determined by indirect immunofluorescence staining. RESULTS: Sequencing proved that the cloned sequence of 19-ss gene was correct. The constructed E.coli-BCG shuttle vector (pCW) containing 19-ss gene had function of shuttle between E.coli and mycobacteria, and mediated the expression of antibiotic resistance gene. Indirect immunofluorescence staining indicated that the Der p2 gene was expressed in the form of lipoprotein on surface of the mycobacterium vaccae. CONCLUSION: E.coli-BCG shuttle vector has been constructed successfully, which can express exogenous antigen gene as a chimeric protein on cell wall.