MUC5AC EXPRESSION UP-REGULATION GOBLET CELL HYPERPLASIA IN THE AIRWAY OF PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Objective To determine the number of goblet cells, the change of MUC5AC expression in chronic obstructive pul monary disease (COPD) patients and the relationship of smoking with goblet cell, MUC5AC, and lung function. Methods Eighteen patients undergoing lung resections for a solitary peripheral carcinoma were classified by lung function as having COPD. Twenty patients with normal lung function served as the control group. Normal lobe bronchioles far away from the lesion site were taken for paraffin section. Goblet cells were identified by AB/PAS staining and the ex pressionof MUC5AC in the paraffin's section was tested by immunohistochemistry. Results Goblet cell hyperplasia was observed in the COPD group. The positive rate of goblet cell in COPD group (0.20% ± 0.10%) was significantly higher than that in the normal lung function group (0.13% ± 0.06%, P ＜ 0.05). The positive rate of MUC5AC expression in the COPD group (0.27% ± 0.09%) was higher than that in the normal lung function group (0.20% ± 0.10%, P ＜0.05).The positive rate of goblet cell in smokers (27.93% ± 9.00%) of the COPD group and normal lung function group was higher than that in non-smokers (17.70% ± 9.37%, P ＜ 0.05), while MUC5AC expression had no significant difference between smokers and non-smokers (17.88% ± 6.44% and 10.88% ± 7.10%, respectively). Conclusion For COPD patients with declined lung function, there were goblet cell hyperplasia and increased expres sion of MUC5AC. MUC5AC expression up-regulation may due to goblet cell hyperplasia. Smoking may be an important factor for goblet cell hyperplasia.     

血红素加氧酶-1对人气道黏液高分泌的影响

目的 探讨血红素加氧酶-1(HO-1)对炎性刺激时人气道上皮细胞黏液分泌的影响.方法 通过人中性粒细胞弹性蛋白酶(HNE)刺激人肺腺癌细胞A549,构建炎性刺激下气道黏液高分泌模型,给予HO-1诱导剂氯化高铁血红素(Hemin)及HO-1抑制剂锌原卟啉(ZnPP Ⅸ)干预,观察黏蛋白5AC(MUC5AC)、表皮生长因子受体(EGFR)、磷酸化EGFR(p-EGFR)、HO-1及双功能氧化酶1(Duox1)的表达.将A549细胞分为对照组、HNE刺激组、Hemin干预组和ZnPP Ⅸ干预组.用四甲基偶氮唑盐法(MTT)测定Hemin及ZnPP Ⅸ对细胞活性的影响;逆转录-聚合酶链反应(RT-PCR)方法检测各组HO-1、Duox1、EGFR 及MUC5AC mRNA的转录水平;Western blot法检测p-EGFR、EGFR、Duox1和HO-1蛋白的表达;酶联免疫吸附测定法(ELISA)检测细胞裂解液中MUC5AC蛋白表达水平的变化;并用细胞免疫化学激光共聚焦显微镜观察不同条件下MUC5AC蛋白表达的变化.结果 HNE刺激组MUC5AC mRNA积分吸光度值和蛋白水平分别为(0.845±0.044)和(192.68±4.71)μg/mg,EGFR mRNA和蛋白的积分吸光度值分别为(0.868±0.039)和(0.623±0.052),均较对照组[(0.462±0.053)、(104.95±6.82)μg/mg和(0.528±0.054)、(0.297±0.055)]明显升高(均P＜0.01);p-EGFR蛋白水平亦明显升高,且HO-1及Duox1的mRNA和蛋白水平也较对照组显著增高(均P＜0.01).给予Hemin预处理后,与HNE刺激组相比,HO-1 mRNA及蛋白明显增多,p-EGFR蛋白水平、Duox1、EGFR及MUC5AC的mRNA和蛋白水平均降低(均P＜0.01).给予ZnPP Ⅸ预处理后,与对照组相比,HO-1 mRNA及蛋白水平无明显增高(P＞0.05),而p-EGFR蛋白水平、Duox1、EGFR及MUC5AC的mRNA和蛋白水平均明显增高(均P＜0.01).结论 HO-1可通过下调Duox1的表达,阻断EGFR活化上游的配体依赖信号途径,减少EGFR活化,抑制MUC5AC的表达,此机制为气道黏液高分泌疾病的防治提供了新的方向.     

Murine calcium-activated chloride channel family member 3 induces asthmatic airway inflammation independently of allergen exposure

Background Expression of murine calcium-activated chloride channel family member 3 （mCLCA3） has been reported to be increased in the airway epithelium of asthmatic mice challenged with ovalbumin （OVA）. However, its role in asthmatic airway inflammation under no OVA exposure has not yet been clarified. Methods mCLCA3 plasmids were transfected into the airways of normal BALB/c mice. mCLCA3 expression and airway inflammation in mouse lung tissue were evaluated. Cell differentials and cytokines in bronchoalveolar lavage fluid （BALF） were analyzed. The expression of mCLCA3 protein and mucus protein mucin-5 subtype AC （MUC5AC） were analyzed by Western blotting. The mRNA levels of mCLCA3, MUC5AC and interleukin-13 （IL-13） were determined quantitatively. Results mCLCA3 expression was not detected in the control group while strong immunoreactivity was detected in the OVA and mCLCA3 plasmid groups, and was strictly localized to the airway epithelium. The numbers of inflammatory cells in lung tissue and BALF were increased in both mCLCA3 plasmid and OVA groups. The protein and mRNA levels of mCLCA3 and MUC5AC in the lung tissue were significantly increased in the mCLCA3 plasmid and OVA groups compared to the control group. The level of IL-13, but not IL-4, IL-5, IFN-y, CCL2, CCL5 or CCL11, was significantly increased compared with control group in BALF in the mCLCA3 plasmid and OVA groups. The level of IL-13 in the BALF in the mCLCA3 plasmid group was much higher than that in the OVA group （P 〈0.05）. The level of mCLCA3 mRNA in lung tissue was positively correlated with the levels of MUC5AC mRNA in lung tissue, IL-13 mRNA in lung tissue, the number of eosinophils in BALF, and the content of IL-13 protein in BALE The level of IL-13 mRNA in lung tissue was positively correlated with the number of eosinophils in BALF and the level of MUC5AC mRNA in lung tissue. Conclusion These findings suggest that increased expression of a single-gene, mCLCA3, could simulate an asthma attack, and its mechanism may involve mCLCA3 overexpression up-regulating IL-13 expression.     

Role of extracellular signal-regulated kinase 1/2 in cigarette smoke-induced mucus hypersecretion in a rat model

Background  Airway mucus hypersecretion is an important pathophysiological feature of chronic obstructive pulmonary disease, which is closely associated with cigarette smoking. However, the signal transduction pathway from the cell surface to the nucleus through which cigarette smoke causes upregulation of mucin gene expression is not well known. This study was designed to investigate the role of extracellular signal-regulated Kinase 1/2 (ERK 1/2) in airway mucus hypersecretion induced by cigarette smoke in rats.

Methods  A rat model of airway mucus hypersecretion was induced by exposure to cigarette smoke for 4 weeks．Rats exposed to inhalation of cigarette smoke or normal saline were given an intraperitoneal injection of U0126, a specific MEK1 kinase inhibitor, at doses of 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg for 14 days. Expression of MUC5AC mRNA and protein, ERK 1/2 and phosphorylated-ERK 1/2 (p-ERK 1/2) were detected by RT-PCR, immunohistochemistry and Western blotting.

Results  Cigarette smoke significantly increased airway goblet cells metaplasia, induced the overexpression of MUC5AC mRNA and protein in bronchial epithelia, and increased the ratio of p-ERK 1/2 and ERK 1/2. U0126 significantly attentuated the expression of MUC5AC mRNA and protein induced by cigarette smoke (P <0.05). Moreover, there was a significant positive correlation between the ratio of p-ERK1/2 to ERK1/2 and the expression of MUC5AC mRNA and protein (P <0.05).

Conclusions  Inhibition of ERK 1/2 by U0126 decreased the ratio of p-ERK 1/2 to ERK 1/2 and expression of MUC5AC mRNA and protein. ERK 1/2 may play an essential role in cigarette smoke-induced mucus hypersecretion in vivo.

     

Clinical study of inflammatory factors in sputum induced early after lung volume reduction surgery

Background The aim of this study was to prospectively study the changes in neutrophil elastase （NE）, fibroblast growth factor 9 （Fgf9）, matrix metalloproteinase-9 （MMP-9）, tissue inhibitor of metalloproteinase 1 （TIMP-1） in sputum induced during the early period after lung volume reduction surgery （LVRS）. Methods From April to October 2005, ten consecutive patients with chronic obstructive pulmonary disease （COPD） underwent LVRS. Ten non-small cell lung cancer patients （stage II-Illa） received Iobectomy as a control group. The induced sputum was collected from both groups at six different times （two weeks before operation and postoperatively at 1, 2, 4, 6 and 10 days）. The level of NE, Fgf9, MMP-9 and TIMP-1 were measured using enzyme-linked immunosorbent assay. Results The pulmonary function （FEV~%） and arterial blood gases （PaO2 and PaCO2） were significantly different belween the groups. There were no significant differences in age, ejection fraction （EF）, and operation duration, but hemoglobin in the LVRS group was statistically higher than in the controls. At certain times, there were significant differences in NE, MMP-9, TIMP-1 and MMP-9/TIMP-1 （P 〈0.05） but not in Fgf9 between the two groups. The levels of NE and TIMP-1 were maximal at 2 days postoperatively and that of MMP-9 and MMP-9/TIMP-1 at 4 days postoperatively in the LVRS group. In the control group, maximal levels of NE and TIMP-1 occurred at 2 days postoperatively and that of MMP-9 and MMP-9/TIMP-1 at 1 day postoperatively. Ten days after surgery, all values of the control group were not significantly different from the baseline. In the LVRS group, the levels were significantly different from the pre-operative values （P 〈0.05） apart from TIMP-1. Conclusion The levels of NE, MMP-9, TIMP-1 and MMP-9/TIMP-1 of the LVRS group were different from those of the control group. The time course of these chanties may be related to LVRS and the underlying process of COPD.     

激活蛋白-1对香烟烟雾提取物诱导气道 黏蛋白5AC基因表达的调控作用

目的：了解激活蛋白-1（AP-1）参与调控香烟烟雾诱导的气道黏蛋白（MUC）5AC表达的作用机制,探讨此过程中AP-1活化的可能信号途径。 方法：体外培养人气道上皮细胞BEAS-2B,给予香烟烟雾提取物（CSE）刺激,干预实验用c-Jun 的显性负性突变体TAM67转染细胞阻断AP-1的DNA结合活性;用SP600125和PD98059预处理分别阻断c-Jun氨基端激酶（JNK）和细胞外信号调节激酶（ERK）的活性。以ELISA法检测MUC5AC蛋白含量,Western印迹检测磷酸化JNK（p-JNK）、磷酸化ERK（ p-ERK）和磷酸化P38（ p-P38）含量,RT-PCR检测MUC5AC mRNA 表达水平, EMSA测定AP-1的 DNA结合活性。 结果：CSE（1 g/L）刺激后MUC5AC mRNA表达水平和蛋白含量明显高于对照组（均P＜0.01）,AP-1 的DNA结合活性也明显强于对照组（P＜0.01）,伴随p-ERK和p-JNK蛋白含量显著增加,与对照组相比,差异有统计学意义（均P＜0.01）,而P38含量无明显变化(P＞0.05);与CSE组相比,TAM67转染细胞后MUC5AC mRNA表达水平和蛋白含量均显著降低 (P＜0.01);与CSE组相比,用SP600125或PD98059处理细胞后可明显抑制AP-1的 DNA结合活性 (均P＜0.05),并下调MUC5AC蛋白含量和mRNA的表达（均P＜0.05）。 结论：AP-1参与调控CSE诱导的气道MUC5AC基因表达的过程,此过程是由JNK和ERK信号转导通路激活AP-1,再由AP-1与MUC5AC启动子上AP-1 DNA反应元件结合后在转录水平上调MUC5AC的表达而实现的。     

中性粒细胞弹力蛋白酶引起黏蛋白5AC高表达的信号转导机制

目的研究中性粒细胞弹力蛋白酶（NE）通过表皮生长因子受体（EGFR）引起黏蛋白（MUC）5AC高表达的上游信号通路。方法培养人气道BEAS@B上皮细胞,给予NE刺激,以肿瘤坏死因子转换酶抑制剂-1（TAPI-1）及活性氧（ROS）清除剂DMTU为干预因素。RT-PCR检测干预前后细胞中MUCSACmRNA含量;酶联免疫吸附测定法（ELISA）检测培养上清中MUCSAC、可溶性转化生长因子（TGF）-α蛋白含量;Westem印迹分析磷酸化表皮生长因子受体（P-EGFR）蛋白水平。结果NE刺激后引起MUCSAC基因转录和蛋白表达水平明显高于未给予NE刺激的对照组（P〈0．01）,同时伴随可溶性TGF-α及p-EGFR蛋白水平的增高,与对照组相比,差异有统计学意义（均P〈0．01）;加入浓度为20umol/L的肿瘤坏死因子转换酶抑制剂TAPI-1后可明显下调上述指标的表达水平（均P〈0．01）,DMTU也同样降低了MUC5AC的基因转录、产物水平以及可溶性TGF-α蛋白相对含量,与单纯NE刺激组比较差异均有统计学意义（P〈0．01）,但对单纯外源性TGF-α刺激组引起的MUCSAC基因转录和蛋白合成增加无明显作用（均P〉0．05）。结论NE能刺激细胞产生ROS,再活化肿瘤坏死因子转换酶（TACE）引起黏蛋白产生的增多,组成EGFR通路上游的主要信号分子,故ROS／TACE／TGF-α前体／EGFR转导途经是NE引起气道黏液高分泌的重要机制。     

透明质烷和CD44参与调节气道黏液高分泌的分子机制

目的了解透明质烷（HA）和CD44在气道黏液高分泌中的作用,探索信号因子活化表皮生长因子（EGF）／表皮生长因子受体（EGFR）信号通路的分子机制。方法体外培养BEAS-2B气道上皮细胞,给予中性粒细胞弹性蛋白酶（NE）刺激,以活性氧（ROS）清除剂（DMTU）、透明质酸酶（Hase）、CD44抗体和组织激肽释放酶抑制剂（PI）作为干预因素。RT—PCR检测黏蛋白（MUC）5ACmRNA表达。ELISA法检测MUC5AC及EGF蛋白含量;Westernblotting分析磷酸化EGFR（p-EGFR）蛋白水平。结果NE刺激后MUC5AC、EGF、P—EGFR蛋白及MUC5ACmRNA表达均较对照组显著增高（P〈0．01）,给予DMTU干预后上述指标水平均明显降低（P〈0．01）;在加入DMTU后用Hase处理细胞,上述指标水平均较DMTU组明显升高（P〈0．05）,EGF蛋白含量增高更为明显（P〈0．01）;CD44抗体或PI处理后上述指标水平均明显低于NE组（P〈0．05）,EGF蛋白含量降低更为显著（P〈0．01）。结论NE可刺激细胞产生ROS,后者可裂解HA致其解聚,并使之与CD44结合而活化组织激肽释放酶（TK）,由活化的TK对前体EGF加工处理成EGF,由此启动EGFR信号级联反应,上调MUC5AC基因的表达。     

基质金属蛋白酶及其组织抑制物在子痫前期患者胎盘中的基因表达

目的研究子痫前期患者胎盘组织中基质金属蛋白酶(MMP-9、MMP-2)及其组织抑制物(TIMP-1、TIMP-2)基因mRNA表达,探讨其与子痫前期发病、进展的关系.方法采用半定量RT-PCR方法对30例正常晚期妊娠和50例子痫前期患者(包括24例轻度和26例重度)胎盘组织中MMP-2、MMP-9、TIMP-1和TIMP-2的mRNA水平进行检测.结果子痫前期组胎盘组织的MMP-9基因表达水平显著下降(P＜0.01),重度子痫前期组MMP-9基因表达水平尤低(0.07±0.05).尽管随病情的加重MMP-2mRNA水平呈现逐渐下降趋势,但没有统计学差异(P＞0.05).子痫前期组胎盘组织中的TIMP-1mRNA水平明显高于正常晚期妊娠者(P＜0.01),重度子痫前期组更高(1.49±0.21).子痫前期组胎盘组织TIMP-2mRNA表达显著降低(1.14±0.36),P＜0.01),并有随病情加重逐渐下降的趋势.结论随子痫前期患者病情的加重,胎盘组织的促浸润基因(MMP-2和MMP-9)表达水平降低,抑侵润基因(TIMP-1)的表达水平升高.子痫前期患者浸润相关基因的表达水平与子痫前期病情密切相关.     

Modulation of Matrix Metalloproteinase and TIMP-1 Expression by TGF-β1 in Cultured Human RPE Cells

In order to investigate the effects of TGF-β1 on the expression of MMP-2, -9 and TIMP1 in human retinal pigment epithelial (RPE) cells, the third-sixth passage cultured RPE cells were treated with TGF-β~ at different concentrations (0.01, 0. 1, 1.0, 10 ng/mL), the expression of MMP-2, -9 and TIMP-1 mRNA was detected by semi-quantitative RT-PCR assays. MMP-2, -9 and TIMP-1 mRNA were expressed in the cultured RPE cells. The values of MMP-2/β-actin in the cells treated with 0.1, 1.0, 10 ng/mL TGF-β1 were 1.04±0.04, 1.07±0.02 and 1.11±0.03, respectively, significantly higher than in the control group (0. 96±0.03, P＜0. 05-0. 01). The expression of MMP-2 mRNA could be up-regulated by TGF-β1, in a dose-dependent manner. The expression of MMP-9 mRNA in the cultured RPE cells was slightly up-regulated by various TGF-β1 concentrations treatment. The values of TIMP-1/β-actin in the cells treated with 0.01 and 0. 1 ng/mL TGF-β1 were 0. 85±0.01 and 0.97 ± 0.02 respectively, significantly lower than in the control group (1.07±0.04, P＜0.01), indicating that the expression of TIMP-1 mRNA was down-regulated by TGF-β1 at low concentrations. But along with the increase of TGF-β1 concentrations (1.0 and 10 ng/mL), the expression of TIMP-1 mRNA was slightly up-regulated, not significantly different from that in the control group (P＞0.05). It was concluded that TGF-β1 might play an important role in the up-regulation of the expression of MMP-2 in RPE cells and result in a directional shift in the balance between MMP and TIMP. This may be facilitated for RPE cells to migrate in the pathogenesis of vitreoretinopathy.     

自发性高血压大鼠Klotho与MMP-9, TIMP-1和 PAI-1基因的表达及福辛普利和缬沙坦对其的干预作用

目的：观察自发性高血压大鼠（spontaneously hypertensive rats,SHR）肾脏组织中Klotho、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、基质金属蛋白酶组织抑制剂-1（tissue inhibitor of metalloproteinase-1,TIMP-1）和纤溶酶原激活物抑制剂-1（plasminogen activator inhibitor,PAI-1）基因的表达状况,探讨其在高血压肾间质纤维化中的作用及福辛普利和缬沙坦的肾保护作用机制。 方法：22周龄雄性SHR 20只随机分为4组（每组5只）：高血压组（SHR组）、fosinopril组［Fos组,10 mg/( kg·d)灌胃］,Valsartan组［Val组,50 mg/( kg·d)灌胃］和fosinopril+valsartan组［Fos＋Val组,fosinopril 10 mg/( kg·d)+valsartan 50 mg/( kg·d)灌胃］。22周龄雄性Wistar Kyoto大鼠（WKY组）5只作为对照组。用药物干预连续喂养8周,分别用RT-PCR和Western 印迹检测肾脏组织中Klotho,MMP-9,TIMP-1和PAI-1 mRNA和蛋白的表达水平。 结果：SHR组肾脏组织Klotho mRNA和蛋白表达较WKY组明显下调(P＜0.01),而MMP-9,TIMP-1和PAI-1mRNA和蛋白的表达较WKY组均明显上调(P＜0.01);用fosinopril和/或valsartan干预后,可上调Klotho mRNA在Fos,Fos＋Val组(P＜0.01）和Val组(P＜0.05）的表达,上调Klotho 蛋白在Val,Fos＋Val组(P＜0.01）和Fos组(P＜0.05）的表达。同时下调MMP-9,TIMP-1,PAI-1 mRNA和蛋白在Fos,Val,Fos＋Val组的表达(P＜0.01）。相关分析表明：Klotho mRNA和蛋白的表达与MMP-9,TIMP-1,PAI-1 mRNA (r值分别为-0.864,-0.725和-0.785,P＜0.01)和蛋白（r值分别为-0. 614,-0.579和-0.552,P＜0.05）的表达均呈负相关。 结论：高血压肾损害与抗衰老基因Klotho和基质降解相关基因MMP-9,TIMP-1,PAI-1关系密切;Klotho基因与MMP-9,TIMP-1和PAI-1基因的表达呈负相关。上调抗衰老基因Klotho表达的药物fosinopril 和/或valsartan可改善Klotho-MMPs/TIMPs-PAI-1的表达,这可能是其抗肾纤维化的重要机制。     

晚期糖基化终末产物受体促进甲苯二异氰酸脂哮喘小鼠MUC5AC表达及气道黏液高分泌

目的 探讨晚期糖基化终末产物受体(RAGE)信号对甲苯二异氰酸脂(TDI)哮喘小鼠黏蛋白MUC5AC表达及气道黏液分泌的影响.方法 在体内水平上建立TDI小鼠哮喘模型,实验分4组:对照组、TDI溶剂(AOO)致敏激发组、TDI致敏激发组、RAGE抑制剂处理组.采用糖原染色评估各组间气道黏液分泌情况,Western blotting及免疫组化法检测MUC5AC表达水平.同时通过Western blotting检测各组间细胞外调节蛋白激酶(ERK)通路磷酸化水平.在体外水平配制TDI-人血清白蛋白(TDI-HSA)复合物,刺激人支气管上皮细胞(16HBE)及经慢病毒转染空载体和shRNA-RAGE载体的人支气管上皮细胞,检测各组MUC5AC及ERK通路分子表达情况,加入ERK抑制剂U0126后,进一步评估MUC5AC mRNA表达情况.结果 与对照组相比,TDI哮喘组小鼠糖原染色阳性率及MUC5AC表达升高(P<0.05),p-ERK表达增多(P<0.05),RAGE抑制剂处理组糖原染色阳性率及MUC5AC表达较TDI组减少(P<0.05).同时,p-ERK表达下降(P<0.05).体外水平,与对照组相比,TDI-HSA处理组MUC5AC mRNA及p-ERK表达增多(P<0.05),sh RNA-RAGE组MUC5AC mRNA及p-ERK表达下调(P<0.05),ERK抑制剂预处理组,MUC5AC mRNA表达下调(P<0.05).结论 TDI小鼠哮喘模型下RAGE可能通过激活ERK通路促进黏蛋白MUC5AC的表达及黏液高分泌的发生.