Dexamethasone protects airway epithelial cell line NCI-H292 against lipopolysaccharide induced endoplasmic reticulum stress and apoptosis

Background  Endoplasmic reticulum (ER) stress and ER stress-mediated apoptosis were reported to be involved in the pathogenesis of several diseases. In a recent study, it was reported that the ER stress pathway was activated in the lungs of lipopolysaccharide (LPS)-treated mice. It was also found that the C/EBP homologous protein (CHOP), an apoptosis-related molecule, played a key role in LPS-induced lung damage. The aim of this study was to verify whether LPS could activate the ER stress response in airway epithelial cells and which molecule was involved in the pathway. This study was also aimed at finding new reagents to protect the airway epithelial cells during LPS injury.

Methods  ER stress markers were observed in LPS-incubated NCI-H292 cells. SiRNA-MUC5AC was transfected into NCI-H292 cells. The effects of dexamethasone and erythromycin were observed in LPS-induced NCI-H292 cells.

Results  LPS incubation increased the expression of ER stress markers at the protein and mRNA levels. The knockout of MUC5AC in cells attenuated the increase in ER stress markers after incubation with LPS. Dexamethasone and erythromycin decreased caspase-3 activity in LPS-induced NCI-H292 cells.

Conclusions  LPS may activate ER stress through the overexpression of MUC5AC. Dexamethasone may protect human airway epithelial cells against ER stress-related apoptosis by attenuating the overload of MUC5AC.

     

Elafin对气道上皮细胞黏蛋白5AC的调节作用

目的探讨天然内生多肽Elafin对气道上皮细胞黏蛋白5AC(MUC5AC)的调节作用。方法通过培养气道上皮细胞,选择采用香烟烟雾提取物(CSE)以及脂多糖(LPS)作为刺激因素,使用细胞免疫荧光法以及ELISA法,分别检测各组气道上皮细胞MUC5AC的胞内蛋白以及胞外蛋白分泌的表达情况。结果单纯LPS刺激组细胞内的MUC5AC蛋白的分泌水平为(0.785±0.005)μg/ml,单纯CSE刺激组细胞内的MUC5AC蛋白的分泌水平为(0.803±0.005)μg/ml,均较对照组明显增多(均P0.01)。在LPS刺激前予Elafin预处理组细胞内的MUC5AC蛋白的分泌水平为(0.363±0.003)μg/ml,显著低于单纯LPS刺激组(P0.01)。在CSE刺激前予Elafin预处理组细胞内的MUC5AC蛋白的分泌水平为(0.406±0.006)μg/ml,显著低于单纯CSE刺激组(P0.01)。单纯予Elafin处理组细胞内的MUC5AC蛋白的分泌水平先后为(0.176±0.002)μg/ml及(0.193±0.004)μg/ml(P0.05),均较对照组显著降低。结论 Elafin可以通过阻断气道黏蛋白的生成及分泌,从而达到抑制气道黏液高分泌的目的。     

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.     

Inhibition of RAW264.7 Macrophage Inflammatory Cytokines Release by Small Haparin RNAi Targeting TLR4

In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-α) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37±8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-αreleased by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-αrelease by RAW264.7 cells evoked by LPS.     

不可分型流感嗜血杆菌脂肽P4诱导气道上皮细胞分泌黏蛋白的机制

目的 研究不可分型流感嗜血杆菌脂肽P4诱导气道上皮细胞分泌黏蛋白MUC5AC的作用及机制。方法培养人气道上皮细胞NCI-H292,用不同浓度的P4孵育细胞,检测粘蛋白5AC(MUC5AC)的分泌及mRNA表达、ROS的产生及肿瘤坏死因子α转化酶(TACE)的活性;分析Duox1 p47^phox和P67^phox亚基亚细胞转位和表皮生长因子受体(EGFR)的磷酸化。结果0、30、50和100 ng/mL P4作用NCI-H292细胞24 h后,可诱导其分泌MUC5AC并表达其mRNA,并促进p47^phox和P67^phox亚基转位至细胞膜、增高细胞内ROS的含量,同时可上调TACE的酶活性及诱导EGFR磷酸化。NADPH氧化酶抑制剂可抑制ROS产生;而ROS抑制剂处理则可降低TACE的酶活性;沉默TACE表达后可抑制EGFR磷酸化,而EGFR抑制剂处理可降低MUC5AC分泌。结论P4经Duox1/ROS/TACE/ EGFR诱导人NCI-H292细胞分泌MUC5AC。     

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。     

铜绿假单胞菌诱导气道上皮细胞产生MUC5AC的分子机制

目的探讨铜绿假单胞菌（Pseudomonasaeruginosa,Pa）诱导气道上皮细胞表达产生粘蛋白MUCSAC的影响,并探讨其可能的分子机制。方法体外培养NCI—H292细胞,用Pa感染后,采用ELISA检测MUC5AC的产生情况;并用商品化的MMP-9活性检测试剂盒检测其产生以及酶活性情况。同时,采用EGFR,P13K,NADPH,ROS和MMP特异性抑制剂AG1478,LY294002,DPI,NAC和GM6001预处理NCI—H292细胞,观察MUC5AC以及MMP-9的产生情况。结果Pa能以时间依赖性方式诱导NCI—H292细胞产生MMP-9．并增加其活性。EGFR活化后,经P13K途径激活Racl并诱导MMP-9的表达,最终诱导MUC5AC产生。结论Pa经EGFR／P13K／Racl／NADPH／ROS／MMP-9通路诱导NCI—H292细胞产生MUC5AC。     

TIM-1对哮喘小鼠气道MUC5AC及Th2细胞因子表达的影响

目的 研究T细胞免疫球蛋白-黏蛋白-1(TIM-1)表达对哮喘小鼠气道MUC5AC及Th2细胞因子的作用,探讨气道黏液高分泌的机制。方法 30只健康雌性C57BL/6小鼠,按随机数字表法分为正常组、哮喘组和TIM-1抗体组,每组10只。检测小鼠外周血单个核细胞（PBMCs）TIM-1+细胞比例、气道MUC5AC mRNA表达、肺泡灌洗液（BALF）中IL-13、IL-4、IL-5表达及黏液细胞和细胞内黏液量的改变。结果（1）哮喘组及TIM-1抗体组小鼠外周血PBMCs中TIM-1+ 细胞比例（11.20%,5.11%）均显著高于正常组（0.64%,P<0.05);而TIM-1抗体组明显低于哮喘组（P<0.05）。（2）哮喘及TIM-1抗体小鼠气道黏液细胞MUC5AC mRNA相对表达(17.3±1.4,5.6±0.3)及IL-13[(16.80±0.63) ng/ml,(5.70±0.64)ng/ml]、IL-4[（614.72±117.39）pg/ml,（325.78±86.54）pg/ml]、IL-5[（1681.13±613.55）pg/ml,（513.42±86.87）pg/ml]表达量均显著高于正常组[1, (1.09±0.25)ng/ml,（17.56±3.01）pg/ml,（30.78±9.67）pg/ml ],TIM-1抗体组小鼠上述指标显著低于哮喘组（P<0.05）。（3）气道MUC5AC及IL-13表达与TIM-1+细胞表达均呈正相关,r1=0.946,P1=0.004; r2=0.984,P2=0.000. 结论 哮喘小鼠外周血TIM-1+细胞升高,可致气道黏液过度分泌;抑制TIM-1表达可减少哮喘气道黏液高分泌。调节TIM-1表达有可能成为减少黏液高分泌及治疗哮喘的新途径。     

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。     

肿瘤坏死因子转换酶在炎性气道黏液高分泌中的作用

目的：探讨肿瘤坏死因子转换酶(TNF-α converting enzyme,TACE)对炎症条件下气道上皮细胞形成黏液高 分泌过程的影响。方法：通过不同剂量人中性粒细胞弹性蛋白酶(human neutrophil elastase,HNE)刺激人肺腺癌的气道 上皮细胞A549,构建炎症条件下气道黏液高分泌模型,以TACE抑制剂-1(TNF-α converting enzyme inhibitor-1,TAPI-1) 进行干预,观察TACE、黏蛋白(mucin,MUC)5AC的表达。将A549细胞分为5组：对照组,HNE(15,25,50 nmol/L) 组,TAPI-1组。RT-PCR检测各组TACE和MUC5AC mRNA的表达;Western印迹检测TACE蛋白的表达;酶联免疫吸附 测定法(ELISA)观察MUC5AC蛋白的表达。结果：各剂量HNE处理后,TACE和MUC5AC的mRNA和蛋白表达均较对照 组明显升高(P<0.01),且呈现剂量依赖性。而给予TAPI-1预处理后,与HNE刺激组比,各指标明显下调(P<0.01)。结 论：TACE参与了黏液高分泌的细胞转导途径,并可促成炎性气道黏液高分泌的形成。     

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。     

加味麻杏石甘汤治疗慢性阻塞性肺疾病急性加重的机制研究

目的探讨加味麻杏石甘汤对慢性阻塞性肺疾病急性加重(AECOPD)的作用机制。方法40只清洁级Wistar大鼠随机分为空白对照组、模型组、加味麻杏石甘汤组、克拉霉素组。空白对照组正常喂养,模型组、加味麻杏石甘汤组、克拉霉素组采取气道滴注脂多糖(LPS)联合烟熏的方法建立AECOPD模型,并连续30 d分别给予生理盐水、加味麻杏石甘汤(8.7 g/kg)、克拉霉素混悬液(50 mg/kg)灌胃。实验第31天,测大鼠肺功能,取大鼠肺组织,制作病理切片,行AB-PAS染色观察杯状细胞,并采用免疫组化法对肺组织中黏蛋白5AC(MUC5AC)、中性粒细胞弹性蛋白酶(NE)进行检测。结果与空白对照组比较,模型组、加味麻杏石甘汤组、克拉霉素组第0.3秒用力呼气容积与用力肺活量的比值(FEV_(0.3)/FVC)明显下降,杯状细胞数、MUC5AC、NE表达均显著升高,具有统计学差异;加味麻杏石甘汤组与模型组比较,FEV_(0.3)/FVC明显上升,杯状细胞数、MUC5AC、NE表达显著降低,具有统计学差异;加味麻杏石甘汤组与克拉霉素组比较,FEV_(0.3)/FVC、杯状细胞数、NE、MUC5AC均无统计学差异。结论加味麻杏石甘汤可显著提升AECOPD模型大鼠FEV_(0.3)/FVC,抑制杯状细胞增生,降低肺组织中NE及MUC5AC的表达,从而减轻AECOPD气道黏液高分泌,这可能是加味麻杏石甘汤治疗AECOPD的机制之一。     

NDRG2基因通过去泛素化酶Zc3h12d在NF-κB转导通路中的调控机制

目的：研究NDRG2基因与去泛素化酶Zc3h12d在NF-κB转导通路中的调控机制。方法：采用LPS刺激人支气管气道上皮16HBE细胞株来构建炎性刺激时气道黏液高分泌模型，用瞬时转染过表达NDRG2基因和Zc3h12d siRNA干扰下调16HBE细胞，将细胞分成LPS+瞬时转染过表达NDRG2组(A组)、LPS+瞬时转染过表达NDRG2+Zc3h12d siRNA组(B组)、LPS+瞬时转染Zc3h12d siRNA组(C组)、LPS+转染空白载体组(D组)，以未做任何处理的16HBE细胞作为对照组(E组)。反转录PCR检测气道黏蛋白(mucin，MUC)5AC mRNA的表达水平，ELISA法检测IL-1β、IL-6等炎症因子及MUC5AC的分泌水平，Western印迹检测Zc3h12d和NF-κB p65的分泌水平，激光共聚焦法检测16HBE细胞内MUC5AC表达。结果：与B组比较，A组炎症因子及MUC5AC表达明显减少，差异有统计学意义(P<0.05)；而B组与C组比较，差异无统计学意义(P>0.05)。与D组比较，A组炎症因子及MUC5AC的表达明显降低，差异均有统计学意义(均P<0.05)。D组与E组比较，LPS刺激后炎症因子及MUC5AC分泌增多，差异均有统计学意义(均P<0.05)。结论：NDRG2基因可以通过Zc3h12d而实现对NF-κB转导通路的调控。