【作者】 李琪；

【导师】 周向东；

【作者基本信息】 重庆医科大学 ， 内科学， 2010， 博士

【摘要】 目的:比较单纯尼古丁与烟草抽提物对培养气道上皮细胞的效应特点,并进一步了解经香烟和尼古丁诱导后的改变规律,以及相关炎性因子表达的诱导效应差异,明确尼古丁在致炎抗炎及黏液高分泌方面的功过地位;同时明确尼古丁作用的膜受体在正常气道的分布规律及表型;并进一步了解尼古丁可能存在的抗炎效应的分子机制及相关信号转导途经。为香烟成份改良提供努力方向,为尼古丁替代疗法提供理论依据,同时帮助完善对尼古丁外周作用的认识,有助于进一步拓展尼古丁的有益使用范围。方法:(1)体外培养人气道上皮细胞系HBE16细胞,予以不同浓度的香烟氯仿抽提物(CE)、尼古丁刺激,选取最佳作用浓度。观察刺激前后细胞的生长和增殖情况,采用ELISA技术测定各组细胞经CE、尼古丁、脂多糖(LPS)刺激后培养上清中前炎性因子肿瘤坏死因子(TNF)-α、白介素(IL)-8、IL-6及黏蛋白(MUC)5AC蛋白相对含量,real-time PCR检测细胞中上述因子的转录水平变化情况,免疫荧光化学法观察细胞中MUC5AC的表达情况。(2)以人脑胶质瘤细胞株U251细胞为阳性对照,分别设立CE组、尼古丁组,采用Western印迹分析及real-time PCR检测各细胞组中烟碱型乙酰胆碱受体(nAChRs)各亚型的表达情况,并采用相关亚型特异性siRNA转染细胞,ELISA检测转染前后细胞培养上清中TNF-α、IL-8、IL-6及MUC5AC蛋白水平的变化,real-time PCR检测上述因子在基因转录水平的变化情况。(3)进一步将细胞分为CE组、LPS组、尼古丁+CE组、尼古丁+LPS组,尼古丁+CE+α-BTX组,尼古丁+LPS+α-BTX组,以Western印迹分析各处理组细胞中磷酸化I-κBα、I-κBα蛋白的表达情况、细胞核中核转录因子(NF)-κBp65的蛋白含量;并采用pNF-κB-Luc荧光素酶报告基因质粒转染法以检测各组中NF-κB的活性;检测加入NF-κB抑制剂PDTC后对细胞TNF-α、IL-8、IL-6的蛋白及基因表达水平的影响情况;同时检测了各组细胞中细胞外信号激酶1/2(ERK1/2)、c-Jun氨基末端激酶(JNK)及p38丝裂原活化蛋白激酶(MAPK)的磷酸化蛋白表达情况,并观察细胞经单纯CE及尼古丁刺激后不同时点上述MAPK信号因子的表达及变化情况。结果:(1)分别选取100μg/mL的CE及20μM的尼古丁为最适作用浓度,LPS的作用浓度为10μg/mL,分为不同组孵育细胞。检测到CE、LPS刺激后细胞中TNF-α、IL-8、IL-6、MUC5ACmRNA相对含量及培养液上清中TNF-α、IL-8、IL-6、MUC5AC蛋白相对含量均有明显增加,与对照组相比,差别有统计学意义;而给予尼古丁孵育的细胞上述指标与正常对照组相比,并未见明显升高,差别无统计学意义;免疫荧光检测到MUC5AC在胞浆中的表达也较CE、LPS组弱;当尼古丁分别与CE、LPS共同作用细胞后,检测到两组细胞培养上清液中TNF-α、IL-8、IL-6蛋白及细胞中mRNA的相对含量较CE、LPS单独孵育组均有显著下降,P<0.05;尼古丁与CE、尼古丁与LPS共同孵育组中上清MUC5AC的蛋白分泌水平也有明显下降,P<0.05,差别有统计学意义,但两组细胞中MUC5AC mRNA表达与单独CE组、LPS组相比,并未有明显下降。(2)培养的人HBE16气道上皮细胞中,α1、α5、α7、β2 nAChR mRNA均有明显表达,尼古丁刺激组中上述指标的表达高于正常对照组及CE刺激组;正常组HBE16细胞、经CE及尼古丁刺激后的HBE16细胞,均未见α2、α4、β1 nAChR mRNA表达;经尼古丁刺激后,见少量α3 nAChR mRNA表达。细胞中α1、α5、α7、β2 nAChR蛋白均有明显表达,尼古丁刺激组表达的量多于未予刺激的HBE16细胞组;未予尼古丁刺激的HBE16细胞组见微弱的α2、α3、α4蛋白表达,而经尼古丁刺激后,上述的蛋白表达并不明显,两组细胞均未见有明确的β1蛋白表达。细胞转染α1 nAchR siRNA、α5nAchR siRNA后再予以尼古丁及LPS处理,检测到培养上清中TNF-α、IL-8、IL-6蛋白相对含量及细胞中mRNA表达水平与尼古丁及LPS共同孵育组中相比,差别无统计学意义,而α7 nAchR siRNA转染的细胞组经尼古丁及LPS刺激后,培养上清中上述蛋白的相对含量及细胞中mRNA相对含量与LPS、尼古丁共同刺激组相比,有明显升高。(3)CE及LPS单独刺激后,磷酸化-I-κBα蛋白及细胞核中NF-κBp65蛋白均有显著增加,与正常对照组相比,P<0.01;加入尼古丁与CE及LPS共同孵育的细胞组中,磷酸化-I-κBα及NF-κBp65均出现明显降低,与单独CE、LPS孵育组相比,差别有统计学意义,P<0.01;I-κBα蛋白的含量未见明显变化;尼古丁+CE+α-BTX组、尼古丁+LPS+α-BTX组中磷酸化-I-κBα蛋白及NF-κBp65蛋白的含量与尼古丁+CE组、尼古丁+LPS组相比有明显增加。转染荧光素酶报告质粒pNF-kB-luc后观察到,尼古丁+CE组、尼古丁+LPS组中的荧光强度值小于CE组、LPS组,差别有统计学意义,P<0.01,尼古丁+CE+α-BTX组、尼古丁+LPS+α-BTX组中的荧光强度值出现增加,与尼古丁+CE组、尼古丁+LPS组相比,P<0.01。进一步PDTC预处理细胞30min后,再分别予以CE、LPS刺激,细胞培养上清中TNF-α、IL-8、IL-6蛋白相对含量及细胞mRAN水平较与单纯CE刺激组、单纯LPS刺激组相比均有明显下降,P<0.01;尼古丁+CE组、尼古丁+LPS组中上述因子的蛋白相对含量及mRNA相对含量也较单纯CE组、单纯LPS组有明显下降;将细胞以PDTC预处理后,再分别施以尼古丁+CE、尼古丁+LPS刺激,细胞培养上清中的TNF-α、IL-8、IL-6蛋白相对含量下降更为明显,与单纯CE刺激组、单纯LPS刺激组相比,P<0.01。细胞经CE及LPS刺激后,可观察到细胞中磷酸化p38 MAPK、ERK1/2、JNK蛋白表达增多,与对照组相比,P<0.01;加入尼古丁孵育的细胞,再分别经CE及LPS刺激,检测到细胞中磷酸化ERK1/2蛋白的相对含量显著降低,分别为0.34±0.07、0.39±0.08,与单纯CE组(0.74±0.12)、LPS组(0.79±0.13)相比,差别有统计学意义,P<0.01;而p38及JNK磷酸化蛋白未见有明显减少。加入α7 nAChR特异性抑制剂α-BTX的尼古丁+CE及尼古丁+LPS组中,磷酸化ERK1/2蛋白的相对含量分别为0.59±0.11,0.63±0.13,与未加α-BTX刺激的尼古丁+CE组、尼古丁+ LPS组相比,差别有统计学意义,P<0.01;但同样对p38及JNK磷酸化蛋白的影响不大。进一步观察到HBE16细胞经CE处理后,p38MAPK、pERK1/2及pJNK蛋白的表达随时间延长而增强,6h时间点的表达水平明显高于30min时的表达水平,P<0.01;经尼古丁孵育的细胞,p38MAPK在作用30min后有表达,随着时间延长表达逐渐降低,pERK1/2在各个时间点均未见明显表达,pJNK在第30min开始表达,2h作用表达最强,在第6h表达开始降低,与2h时间点的表达量相比,差别有统计学意义,P<0.01。结论:(1)尼古丁在香烟所致气道黏液高分泌环节中并无过多正向效应,相反,其可能通过减少该过程中致炎因子的产生,抑制后续过度炎症反应。(2)尼古丁能减少前炎性因子及重要炎性趋化因子TNF-α、IL-8、IL-6蛋白等的产生,具有一定的抗炎功能,且证实系通过与α7 nAChR结合起降低I-κBα磷酸化的作用,从而抑制核转录因子NF-κB核转位,发挥抑制上述因子表达的效应;该过程也可能系尼古丁通过减少ERK1/2活化水平而实现。(3)尼古丁不增加黏蛋白MUC5AC的基因转录及合成,且能使炎性刺激所生成的多余黏液外分泌相对减少,从而维持气道在慢性炎性刺激下黏液的高潴留状态,形成气道黏液生成与高分泌的相对稳态。更多还原

【Abstract】 Objective To compare the different effects of nicotine and cigarette smoke extract to the airway epithelial cells, to further explore their different expression of proinflammatory factors and mucins, to know the potency of nicotine in anti-inflammation and mucus hypersecretion in chronic airway inflammatory diseases. To identify the expression of nicotine receptor nicotinic acetylcholine receptor(nAChRs) in airway epithelial cells, to study the role of nAChRs and signal pathway in nicotine-participated events, to learn more about the effection of nicotine in the periphery system, and to provide the possibility and evidence for improvement of cigarette components, to progress the beneficial and serviceable range of nicotine.Methods (1)The well cultured HBE16 airway epithelial cells were exposed to serial dilutions of cigarette smoke chloroform extract(CE)、Lipopolysaccharide(LPS)、and nicotine, respectively for 24 hours, to choose the most suitable concentration for cells incubation. To measure tumor necrosis factor(TNF)-α、Interleukin(IL)-8、IL-6、and mucin(MUC)5AC protein secreted in culture medium by enzyme linked immunosorbent assay(ELISA), the levels of TNF-α、IL-8、IL-6、and MUC5AC mRNA in each cells group were detected by real-time polymerase chain reaction(PCR). Also MUC5AC prtein in cells were observed by immunofluorescence.(2)Using human neurospongioma cells line U251 as positive control, to detect the protein and mRNA expression of nAChRs and its subtypes in HBE16 airway epithelial cells before and after CE、nicotine stimulation. To measure TNF-α、IL-8、IL-6、and MUC5AC protein secretion and mRNA expression in HBE16 cells after tranfected withα1nAChR siRNA、α5nAChR siRNA、andα7nAChR siRNA by ELISA and real-time PCR, respectively.(3)HBE16 airway epithelial cells line were divided into CE group、LPS group、nicotine plus CE group、nicotine plus LPS group、nicotine, CE plusα7nAChR inhibitorα-bungarotoxin(α-BTX) group,and nicotine, LPS plusα-BTX group. Phospho-IκBα、I-κBα、and nuclear factor(NF)-κBp65 protein production in each group were assayed by western blot; The activity of NF-κB in cells were measured by transient transfection of pNF-κB-Luc reporter vector; Also the protein production and mRNA expression of TNF-α、IL-8、and IL-6 were assayed after cells pretreaed with NF-κB inhibitor Pyrrolidinedithiocarbamic acid ammonium salt(PDTC); Then the phosphosrylation of extracelluar signal-regulated kinase(ERK) protein, c-Jun N-terminal kinase(JNK) protein, and p38 mitogen activated protein kinase(MAPK) protein were assayed by western blot. The phosphoralytion of the ERK1/2、JNK、and p38MAPK protein were obsereved in different time spot after treated with CE and nicotine,respectively.Results (1)100μg/mL CE、20μM nicotine、and 10μg/mL LPS were choosed as the suitable concentration for incubation. We found that 100μg/mL CE and 10μg/mL LPS could induce the TNF-α、IL-8、IL-6、MUC5AC protein and mRNA expression of the HBE16 airway epithelial cell line, P<0.05, compared with normal cells. Whereas nicotine did not cause significant TNF-α、IL-8、IL-6、MUC5AC protein and mRNA expression, there are no statistically difference when compared with CE、LPS treated cells. When cells pretreated with nicotine before CE or LPS stimulation, the expression of TNF-α、IL-8、IL-6 protein and mRNA were decreased obviously, compared with CE、LPS treated group alone; The MUC5AC protein secretion in culture supernatants in nicotine plus CE group、nicotine plus LPS group were also reduced, compared with CE or LPS treated group alone, but the mRNA expression did not showed significant difference, compared with CE or LPS treated group alone.(2)We deteced expression ofα1、α5、α7、β2 nAChR mRNA in HBE16 cells, and were higher in nicotine stimulated cells than that of in normal control HBE16 cells and CE stimulated cells,α2、α4、β1 nAChR mRNA were not found in any normal HBE16 cell、CE or nicotine treated cells. Nicotine caused a feeble expression ofα3 nAChR mRNA. Western blot assayed showedα1、α5、α7、β2 nAChR protein expression in HBE16 cells in both control or nicotine groups, and nicotine-treated cells was higher than normal control HBE16 cells, feeble expression ofα2、α3、α4 protein were detected in normal HBE16 cells, however, after nicotine stimulation, the expression were decreased. Noβ1nAChR protein was found in any HBE16 cell. Transfection of HBE16 cells withα1nAchR siRNA、α5nAchR siRNA before nicotine and LPS incubation, results showed that TNF-α、IL-8、IL-6 protein and mRNA expression had no obvious change when compared with no siRNA transfected cells. Whereas,α7 nAchR siRNA trasfected cells showed a significant upregulation of TNF-α、IL-8、IL-6 protein and mRNA expression, compared with non transfected cells in the stimulation of nicotine and LPS(.3)The phosphorylation of I-κBαprotein and NF-κBp65 protein were increased in both CE and LPS treated groups, P<0.01, compared with normal control group. Nicotine preincubation reduced the activation of phospho-I-κBαand NF-κBp65 protein by CE and LPS stimulation. The activity of NF-κB in nicotine-treated cell was lower than CE、LPS treated cells.α7 nAChR specific inhibitorα-BTX could reverse the effect of nicotine. NF-κB potent inhibitor PDTC caused a significant decrease of TNF-α、IL-8、IL-6 protein and mRNA expression. It was also showed that the phospho-ERK1/2、phospho-JNK、and phospho-p38MAPK protein in CE、LPS treated cells were increased, nicotine-treated cell did not cuase significant increase of phospho-JNK and phospho-p38MAPK, moreover, the activation of ERK1/2 were much inhibited by nicotine. We further found that nicotine-treated cells showed an increasing expression of phospho-p38MAPK in 30 min incubation, and was began to decrease during the following period, phosph-ERK1/2 did not found in whole process, phospho-JNK expressin began at 30min, the highest expression was detected at 2h, and decreased at 6h. CE-treated cell showed an increasing activation of all the three kinase in the 6h exposure.Conclusions (1)Nicotine did not cause excessive production and expression of mucin MUC5AC, on the contrary, it could reduce the activation of proinflammatory factors and cytokines caused by CE and LPS assault, to refrain the following more serious inflammation to a certain extent which may complicated the diseases.(2)Nicotine could decrease the expression of TNF-α、IL-8、IL-6,α7 nAChR is participated in the process, which could downregulate the phosphorylation of I-κBα, to further inhibit the nuclear translocation of NF-κB, and to affect the expression of inflammatory factors. Also , suppression of ERK1/2 phosphoralytion may play a part in it.(3)Nicotine could decrease the mucus secretion in supernatants of the cultured cells which stimulated by CE、LPS. This distinctive effect may provide the potency of nicotine to maintain the mucus over retention in airway secretory cells, then probably to form a relative stable stasis between mucus hyperproduction and hypersecretion in airways.更多还原