Large-conductance calcium-activated potassium channel (BK(Ca)) and voltage-gated potassium channel Kv1.5 play an important role in the pathogenesis of bronchial hyperresponsiveness (BHR). It is known that cigarette smoke can induce BHR, however, the role of BK(Ca) and Kv1.5 expression in it remains to be further elucidated. The purpose of the present study was to investigate the direct effects of cigarette smoke extract (CSE) on BK(Ca) and Kv1.5 expression, and the role of protein kinase C (PKC) isoforms activation in primary cultured rat bronchial smooth muscle cells (BSMCs). Primarily cultured rat BSMCs were treated with 5% CSE, the expression and translocation of PKC isoforms were measured by Western blot, and the mRNA and protein levels of BK(Ca) and Kv1.5 alpha-subunits were determined by semi-quantitative RT-PCR and Western blot, respectively. The results showed that 5% CSE induced the translocation of PKCepsilon, PKCeta, PKCtheta from soluble fraction to particulate fraction, and reduced mRNA and protein expressions of BK(Ca) and Kv1.5 alpha-subunits. The decreased expression of potassium channels was partly restored by PKC inhibitor, BIM or Goe6983. In summary, CSE may activate PKC isoforms epsilon, eta, theta, thereby down-regulate the expressions of BK(Ca) and Kv1.5 in BSMCs.

AIM: To investigate if hydrogen peroxide may alter COX-2 gene expression in pulmonary artery endothelial cells(PAECs) and how CaMKⅡ functions in this process.METHODS: Cultured pulmonary arterial endothelial cells were treated with different concentrations of hydrogen peroxide for different durations.The cells survival rates were measured by CCK-8 after the cells were treated by hydrogen peroxide.The level of COX-2 mRNA and protein were measured by RT-PCR and Western blotting,respectively.RESULTS: The results showed that hydrogen peroxide up-regulated COX-2 mRNA and protein levels in a concentration-and time-dependent manners.Incubation with 100 μmol/L H2O2 for 4 h increased COX-2 mRNA and protein level to 256.01%±22.36%(P0.05) and 216.65%±21.52%(P0.05),respectively.The up-regulatory effects were prevented by KN-93,a selective CaMKⅡ inhibitor,at concentration of 10 μmol/L.CONCLUSION: These results suggest that hydrogen peroxide up-regulates COX-2 gene transcription in PAECs by a signaling pathway involving CaMKⅡ.

AIM:To investigate the role of potassium channel expression alteration in chronic cigarette smoking-induced increase in pulmonary vascular responsiveness,the effect of chronic cigarette smoking on large-conductance calcium-activated potassium channel(BKCa) and voltage-dependent delayed rectifier potassium channel(Kv1.5) expression in rat pulmonary smooth muscle cells were investigated in vivo.METHODS: HE staining,immuno-histochemistry and in situ hybridization techniques were used.RESULTS:(1) Chronic cigarette smoking downregulates the protein and mRNA expression of BKCa in pulmonary arterial smooth muscles.(2) Chronic cigarette smoking downregulated the protein and mRNA expression of Kv1.5 in pulmonary arterial smooth muscles.(3) In big artery,BKCa decreased more makedly than Kv1.5,but in small artery,both of them decreased equally.CONCLUSION: Chronic cigarette smoking downregulates the levels of BKCa and Kv1.5 in rat pulmonary arterial smooth muscle cells in vivo,which maybe contribute to the mechanism of cigarette smoking-induced increase in pulmonary vascular responsiveness.

AIM: To investigate the effect of histamine receptor antagonist on airway remodeling and acid-base imbalance in asthma of guinea pig. METHODS: Guinea pigs were divided into 5 groups: the normal control group, the asthma model group, the continued asthma model group, histamine group and histamine receptor antagonist group. For each group, the content of histamine, Na+, Cl-, PaO2, PaCO2, pH, AB, SB in serum, and thickness of airway mucosa and smooth muscle cell layer were measured and compared with each other. RESULTS: (1) According to the content of histamine in serum and thickness of airway mucosa and smooth muscle, the order was: the histamine groupcontinued asthma model groupthe asthma model groupthe normal control group (P0.01), and the histamine receptor antagonist groupthe continued asthma model group (P0.01,P0.05). (2) For PaO2, the order was: the asthma model groupthe normal control group (P0.01). For PaO2, pH, AB, SB, the order was: the histamine groupthe continued asthma model groupthe asthma model group (P0.01), and the histamine receptor antagonist groupthe continued asthma model group (P0.01), but for PaCO2, the order was conversed. Airway remodeling, increase in histamine in serum, respiratory acidosis and metabolic acidosis in asthmatic guinea pig were observed. Exogenous histamine accentuated the change, however, histamine receptor antagonist attenuated it. CONCLUSION: Histamine may take part in the airway remodeling of asthma. Histamine receptor antagonist can prevent and ameliorate airway remodeling and acid-base imbalance in asthma of guinea pig.

A minority of people are either in good health or ill,while a majority are in a stae of subhealth.In this article,the concepts of health,disease and subhealth,the manifestations,the causes and the mechanisms of the development of diseases and subhealth are discussed.

OBJECTIVE:To investigate the effect of exercise stress on chronic cigarette smoking induced downregulation of large conductance calcium-activated potassium channel (BKca) and voltage-dependent delayed rectifier potassium channel (Kv1.5) expression in pulmonary arterial smooth muscle cells of rats.METHODS:Rats were divided into three groups: the normal control group, the smoking control group and the smoking + exercise group. The plasma cortisol level, the potassium channel expression and the pathological changes in lung tissue were determined with HE staining, the immunohistochemistry and the in-situ hybridization.RESULTS:(1) In the smoking + exercise group, the plasma cortisol level was determined immediately after exercise [(1528.7 +/- 469.7) ng/L] and was higher than that determined before exercise [(672.4 +/- 235.7) ng/L] (P < 0.01); (2) The HE staining showed that the chronic pulmonary inflammatory response in the smoking control group was severe while it was mild in the smoking + exercise group; (3) The mRNA and protein expression (OD value) of BKca in the smoking control group (mRNA: 0.2206 +/- 0.0415 for big artery and 0.3935 +/- 0.1378 for small artery; protein: 0.2634 +/- 0.1219 for big artery and 0.0995 +/- 0.0851 for small artery) were less than those in the normal control group. The mRNA expression of BKca in the smoking + exercise group (OD value) (0.5022 +/- 0.1134 for big artery and 0.6408 +/- 0.2135 for small artery) was higher than that in the smoking control group; (4) The mRNA and protein expression of Kv1.5 in the smoking control group (OD value) (mRNA: 0.9354 +/- 0.3290 for big artery and 0.5012 +/- 0.1170 for small artery; protein: 1.1112 +/- 0.3310 for big artery and 0.4736 +/- 0.1250 for small artery) were less than those in the normal control group. The protein expression of Kv1.5 in the smoking + exercise group (0.7445 +/- 0.2690) in small artery was higher than that in the smoking control group.CONCLUSION:Proper exercise stress can decrease inhibition effect of the chronic smoking on the expression of potassium channel BKca and Kv1.5, which perhaps partly results from exercise induced increase of cortisol secretion.

AIM: To investigate the expression of hypoxia inducible factor-1alpha (HIF-1α) and the role of HIF-1α in tumor necrosis factor alpha (TNF-α) production in rat alveolar macrophages activated by lipopolysaccharide (LPS). METHODS: HIF-1α function was inhibited by using the method of HIF-1α decoy. Western blotting and semiquantitative RT-PCR were applied to determine the expression of HIF-1α protein and mRNA, respectively. The production of TNF-α was determined with ELISA. RESULTS: The content of HIF-1α protein in LPS group (1.95±0.57) and HIF-1α decoy group (1.89±0.59) were 4.8 times and 4.6 times higher than that in control group (0.41±0.14), respectively. The expression of HIF-1α mRNA showed no difference among three groups (F=3.14,P0.05). The production of TNF-α in LPS group was higher than that in control group (61 ng/L vs 156 ng/L, q=5.12, P0.05) and HIF-1α decoy group (90 ng/L vs 156 ng/L, q=4.63, P0.05), respectively. However, the content of TNF-α in HIF-1α decoy group was still higher than that in control group (61 ng/L vs 94 ng/L, q=4.47, P0.05). CONCLUSION: The enhanced stability of HIF-1α protein results in the marked upregulation of its protein and HIF-1α is contributed to the production of TNF-α in LPS-stimulating rat alveolar macrophages. It is indicated that HIF-1α plays important role in the pathogenesis of chronic inflammation involved in diseases such as COPD.

Objective:To investigate the expression of interleukin-7 in human liver and stomach,identify the splice variants of IL-7.Methods:Reverse transcription-polymerase chain reaction(RT-PCR) was used for investigating the expression of IL-7.According to IL-7 gene cDNA,the primers were designed and synthesized,then the splice variants of IL-7 in human cancer tissues was identify,cloned into vector and sequenced.Results:The results showed that(1)the expression of IL-7 mRNA was nearly equal between human liver and stomach,(2)two new bands of IL-7 was obtained from liver cancer and gastric carcinoma,one lacks exon 4,another exon 5.Conclusion:IL-7 gene is expressed highly in both human liver and stomach.In addition,human liver cancer and gastric carcinoma can produce splicing variants of IL-7 by alternative splicing.

本科研组承担的8项国家级科研项目、4项省市级项目较系统地研究了缺氧性肺血管收缩反应的特性、机制和调控,主要成果有以下5个方面:

Aim: To find out if the two aspects of asthma (chronic airway inflammation and bronchial hyperresponsiveness) are related to hypersensitivity of calcium signaling in bronchial epithelial cells. Methods: Porcine bronchial epithelial cells (PBEC) were divided into sensitized (S) and nonsesitized (N) groups. In group S, the cells were preincubated with serum from ovalbumin sensitized guinea pigs. In group N, the cells were preincubated with serum from nonsensitized guinea pigs. Single cell calcium imaging and ELISA-based NF-κB activity were used to evaluate the histamine-stimulated intracellular free calcium level and NF-κB activity, respectively. Results: First, 0.1 μmol/L histamine could induce [Ca 2+ ] i oscillations in PBEC of group S, but not in group N. Second, 1 μmol/L histamine could induce [Ca 2+ ] i oscillations of PBEC in both group S and group N. The [Ca 2+ ] i oscillation frequency of PBEC was significantly higher in group S than in group N, though the [Ca 2+ ] i oscillation amplitude showed no difference between the two groups. Finally, when 10 μmol/L histamine was used to stimulate PBEC, a transient initial increase followed by a sustained elevation (FSE) of [Ca 2+ ] i was observed in PBEC in both groups. The amplitude of the FSE of [Ca 2+ ] i in PBEC was significantly higher in group S than in group N. The subsequent NF-κB activity was in accordance to the calcium oscillation frequency evoked by histamine, but not to the amplitude. Conclusion: It was suggested that the increased sensitivity of calcium signaling in bronchial epithelial cells might contribute to the exorbitant inflammation or increased susceptibility in asthmatic airway epithelial cells.

AIM: To investigate the expression of FIZZ1/RELMα in lung tissue of chronic cigarette smoking rat,and to determine the relationship between airway inflammation and airway hyperresponsiveness.METHODS: Made rat model of chronic cigarette smoking was used.The expression of FIZZ1/RELMα in lung tissue was determined by immuno-histochemistry and in situ hybridization.RESULTS: In control rats,FIZZ1/RELMα protein and mRNA expressions were observed at low levels.In cigarette smoking rats,FIZZ1/RELMα expression increased in all the cells especially in bronchial smooth muscle cells,vascular wall cells and alveolar epithelial cells.CONCLUSION: FIZZ1/RELMα is a secreted peptide specifically expressed in lung.Cigarette smoking induces its upregulation,which possibly contributes to cigarette smoking-induced airway hyperresponsiveness.

Objective To investigate the effect of dexamethasone inhalation on cigarette smoking induced airway hyperresponsiveness and the alteration of potassium channel BKca expression in airway smooth muscle cells. Methods The rat as cigarette smoking model were treated with dexamethasone inhalation,to detect the pathological alteration of rat lung tissue was examined by HE staining,mRNA and protein expression of BKca were determined with in-situ hybridization and immuno-histochemistry techniques. Results (1)Cigarette smoking increased airway responsiveness,the airway responsiveness of dexamethasone treating group lower that of smoking group (P0.05),but higher than that of normal control group; (2) HE staining showed,that there was slight pulmonary inflammatory response in cigarette smoking group,no pulmonary inflammatory response in dexamethasone treating group; (3) The protein and mRNA expression of BKca in cigarette smoking group was less than that in normal control group in BSMC of both Bronchioli and Bronchi,dexamethasone treating lead to increase of the protein and mRNA expressionof BKca in contrast to cigarette smoking group. Conclusions Cigarette smoking induced increase of airway responsiveness isn’t parallel with pulmonary inflammatory response,dexamethasone inhalation can decrease cigarette smoking induced airway hyperresponsiveness,partly resulting from inhibition of potassium channel BKca expression in airway smooth muscle cells induced by cigarette smoking.

目的:观察组胺和低氧对培养的猪肺动脉和主动脉内皮细胞eNOS mRNA和蛋白质表达的影响.方法: 采用半定量RT-PCR和免疫细胞化学的方法.结果: (1)组胺可使肺动脉内皮细胞eNOS mRNA表达增加,在10-5mol/L作用24 h达高峰,为对照组的178.2%±7.7%(P<0.01),eNOS蛋白质表达也上调,为对照组的173%±47%(P<0.01),主动脉内皮细胞与肺动脉内皮细胞相似,可在组胺10-6mol/L作用24h达高峰,为对照组的177.4%±14.2%(P<0.01),eNOS蛋白质表达也上调,为对照组的165%±54%(P<0.01);(2)急性低氧12 h肺动脉内皮细胞eNOS mRNA表达增加,24 h达高峰,为对照组的151.0%±9.1%(P<0.01);蛋白质表达水平也增高到常氧对照组的216%±44%(P<0.01),而主动脉内皮细胞eNOS mRNA与蛋白质均未受低氧影响.结论: 组胺可使肺动脉和主动脉内皮细胞eNOS表达增加,但两者反应无明显差异;低氧使肺动脉内皮细胞eNOS表达上调,而对主动脉内皮细胞无明显影响.

Calcium oscillation may regulate gene transcription in a frequency-decoding manner during agonist stimulation, which provides an indicator of transcription level in cells. To determine whether persistent exposure to hypoxia may sensitize or blunt cell response to histamine, the effects of 24 h subacute mild hypoxia on histamine-stimulated calcium oscillation frequency were examined in pulmonary artery endothelial cells (PAECs). The results are: (1) 24 h subacute mild hypoxia significantly increased the histamine-stimulated calcium oscillation frequency in PAECs. The averaged frequency of calcium oscillation in posthypoxic PAECs was significantly higher than that in normoxic ones. (2) NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI, 10 mu mol/L), abolished histamine-stimulated calcium oscillations both in normoxic and posthypoxic PAECs. (3) Xanthine oxidase inhibitor, oxypurinol (100 mu mol/L), did not affect the calcium oscillation frequency in normoxic PAECs. However, it significantly decreased the elevation of calcium oscillation frequency in posthypoxic PAECs. These results demonstrated that, during pulmonary disease related to persistent hypoxia, PAECs become more sensitive to histamine. During histamine stimulation, NADPH oxidase plays a critical role in generating calcium oscillations, while xanthine oxidase may contribute to, at least in part, the increase of calcium oscillation frequency in posthypoxic PAECs.

目的研究良性运动应激对慢性吸烟引起的大鼠气道高反应性的影响.方法复制大鼠慢性吸烟模型后,进行运动训练,测定大鼠气道反应性、血浆皮质醇浓度,行苏木精-伊红染色观察肺组织病理变化.结果①吸烟组的气道反应性明显高于正常对照组(P＜0.05),而吸烟运动组的气道反应性显著低于吸烟组(P＜0.01),且与正常对照组无明显差异;②吸烟运动组运动后血浆皮质醇浓度明显高于运动前(P＜0.01),但全部运动结束后次日晨测定的血浆皮质醇浓度与运动前无明显差异(P＞0.05);③苏木精-伊红染色显示,吸烟组肺组织出现明显的慢性炎症反应,吸烟运动组比吸烟组炎症反应轻.结论适当的良性运动应激能降低由吸烟引起的气道高反应性.

目的研究不同剂量的延迟电压门控性钾通道(Kv)阻断剂四氨基吡啶(4-aminopyridine,4-AP)对大鼠气道反应性的影响.方法用大鼠离体气管张力实验观察不同剂量4-AP对不同浓度组胺所致气管张力变化的影响.结果单纯不同剂量4-AP诱导大鼠离体气管产生浓度依赖性收缩反应;0.05 mmol/L 4-AP与不同浓度组胺收缩气管呈相加作用;0.15 mmol/L 4-AP与组胺之间呈协同作用,即随着组胺浓度的增加,支气管收缩反应增加的幅度超过两者的相加作用;而0.45 mmol/L 4-AP反而使支气管对组胺发生舒张反应.结论不同剂量4-AP 对气管反应性的影响极不相同.

目的观察缺氧对培养的猪肺动脉内皮细胞基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)的表达的影响.方法采用RT-PCR、Western blot、底物胶电泳(酶谱图)、免疫细胞化学等方法.结果肺动脉内皮细胞缺氧24h可使MMP-2的mRNA表达和蛋白分泌减少,酶活性减弱,与常氧组比较有显著性差异, MMP-9无明显变化.结论缺氧时MMP-2降低,溶解细胞外基质的能力降低,可能是缺氧性肺血管构型重组的机制之一.

Primary cell culture, techniques of gene transfection, gelatin zymography, and Western blot were used to investigate the effect of hypoxia on the secretion of MMP-2 and MMP-9 in pulmonary artery endothelial cells (PAEC) and smooth muscle cells (PASMC), and the role of HIF-1, Our results showed that (1) after exposure to hypoxia for 24 h, the protein content and activity of MMP-2 in the PAEC medium as well as these of MMP-2 and MMP-9 in PASMC medium (P<0. 01) decreased significantly in contrast to those in normoxic group (P′<0.05); (2) after transfection of wild type EPO3′-enhancer, a HIF-1 decoy, the content and activity of MMP-2 and MMP-9 in hypoxic mediums became higher than those in normoxic group (P<0.01), while transfection of mutant EPO3′-enhancer didn't affect the hypoxia-induced down-regulation. It is concluded that hypoxia could inhibit the secretion and activity of MMP-2 and MMP-9 in PAEC and PASMC, which could be mitigated by the transfection of EPO3′-enhancer and that HIF-1 pathway might contribute to hypoxia-induced down-regulation of MMP-2 and MMP-9.

目的:探讨电压依赖性钾通道在吸烟诱发气道高反应中的作用.方法:以特异性钾通道阻断剂4-aminopyridine(4-AP)为工具药,用支气管环等长张力实验测定吸烟对组胺收缩支气管作用的影响.结果:(1)吸烟组支气管环对组胺的反应明显高于对照组(P＜0.05);(2)4-AP使支气管收缩的作用吸烟组显著强于对照组(P＜0.01);(3)用4-AP后,支气管对组胺的收缩反应增强,吸烟组增强的幅度大于对照组(P＜0.01).结论:电压门控钾通道(Kv)参与吸烟所致气道高反应.

Hypoxia is a commenest pathological process. The cellular oxygen sensors and signal transduction involved in hypoxic responses are so far not fully elucidated. There are many theories. Perhaps the oxygen sensors are different among different kinds of cells, and between acute and chronic hypoxic responses. The mitochondria cytochrom-oxidase-H_2O_2 might be the principal pathway leading to the constrictive response of pulmonary smooth muscle cells to hypoxia. The heme protein - reactive oxygen pathway might mediate the response of glomus cells in carotid body to hypoxia. The NADPH oxidase might be the oxygen sensor in airway chemoreceptor. As for chronic hypoxic responses, the oxygen dependent regulation of hypoxia-inducible factors by prolyl and asparaginyl hydroxylation has been paid great attention in recent years.