尼古丁在吸烟导致气道慢性炎症中的独特效应

发布时间：2018-01-06 15:01

本文关键词：尼古丁在吸烟导致气道慢性炎症中的独特效应　出处：《重庆医科大学》2010年博士论文　论文类型：学位论文

【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,P0.05;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,P0.05,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,P0.01;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,P0.01;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,P0.01,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,P0.01。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,P0.01;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,P0.01。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,P0.01;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,P0.01;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,P0.01;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,P0.01;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,P0.01。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。
[Abstract]:Objective: To compare effect of nicotine and tobacco extract on cultured airway epithelial cells, and further understand the changes of cigarettes and nicotine induced, and the expression of inflammatory factors induced by different effects, clear nicotine in inflammation and mucus secretion of anti-inflammatory and high status; and clear the distribution and phenotype of membrane effect of nicotine receptors in the normal airway; and further understanding of the molecular mechanisms underlying the anti-inflammatory effects of nicotine may exist and the related signal transduction pathway. To provide direction for improvement of cigarette components, to provide a theoretical basis for nicotine replacement therapy, while helping to improve the understanding of the peripheral effects of nicotine, help to further expand the scope of beneficial use of nicotine.
Methods: (1) HBE16 cells in human airway epithelial cells cultured in vitro, the cigarette to the chloroform extract of different concentration (CE), nicotine stimulation, selection of optimal concentration. The observation before and after stimulation of cell growth and proliferation, determination of nicotine by CE, cells were used ELISA technology, lipopolysaccharide (LPS) of the tumor in the supernatant of cultured inflammatory necrosis factor stimulation (TNF) - alpha, interleukin (IL) -8, IL-6 and mucin5ac (MUC) the relative content of 5AC protein, changes in transcription of the real-time factor of PCR were detected by immunofluorescence, photochemical method to observe the expression of MUC5AC in cells. (2) in human brain glioma tumor cell line U251 cells were set as positive control, CE group, nicotine group, by Western blot analysis and real-time PCR were detected by groups of nicotinic acetylcholine receptor (nAChRs) expression of the subtypes, and the subtype specific SiRNA transfected cells, and ELISA detection of transfected cell culture supernatant of TNF- alpha, IL-8, changes of IL-6 and MUC5AC protein levels, real-time PCR to detect the changes in gene transcription factor. (3) the cells were divided into CE group, LPS group, +CE group, +LPS group, nicotine, nicotine, nicotine +CE+ alpha -BTX group, -BTX group with nicotine +LPS+ alpha, Western blot analysis of each group of cells in phosphate I- kappa B alpha, expression of I- kappa B alpha protein, cell nuclear transcription factor (NF) - kappa Bp65 protein content; and the use of pNF- kappa B-Luc luciferase reporter gene plasmid transfection method was used to detect NF- kappa B activity detection; adding NF- kappa B inhibitor PDTC on the expression of TNF- alpha, IL-8, gene and protein expression level of IL-6 effect; also detected while cell in extracellular signal kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 mitogen The phosphorylation protein expression of activated protein kinase (MAPK) was observed, and the expression and change of the above MAPK signal factors were observed at different times after CE and nicotine stimulation.
Results: (1) nicotine were selected 100 g/mL CE and 20 M is the most suitable concentration, the concentration of LPS is 10 g/mL, divided into different groups. The cells were incubated to detect CE, TNF- alpha, LPS stimulation in cells after IL-8, IL-6, MUC5ACmRNA relative content and culture of TNF- alpha supernatant, IL-8, IL-6, MUC5AC protein content was increased significantly, compared with the control group, the difference was statistically significant; and given the above indexes nicotine incubation cells compared with normal control group, and there was no obvious increase, the difference was not statistically significant; immunofluorescence detected the expression of MUC5AC in cytoplasm was CE, group LPS and CE were weak; when nicotine, LPS cells, detected two groups of cell culture supernatant of TNF- alpha, IL-8, mRNA and relative content of IL-6 protein in cells with CE, LPS alone incubation group were significantly decreased, P0.05 and CE; nicotine, nicotine and LPS were incubated together Group in the supernatant of MUC5AC protein secretion level was decreased, P0.05, the difference was statistically significant, but the two groups of cells in the MUC5AC expression of mRNA and CE alone group, LPS group, did not significantly decrease. (2) in cultured human airway epithelial cells HBE16, alpha 1, alpha 5, alpha 7 beta. 2 nAChR mRNA showed obvious expression, expression of the index of nicotine stimulation was higher than in the normal control group and CE stimulation group; normal group of HBE16 cells by CE and nicotine stimulated HBE16 cells showed no expression of alpha 2, alpha 4, beta 1 nAChR mRNA; after nicotine stimulation, a few expression of 3 nAChR mRNA. Cells in alpha 1, alpha 5, alpha 7 beta 2, nAChR protein expression was evident in HBE16 cell group nicotine stimulation group was much higher than the expression does not stimulate the cells in HBE16 group; not the weak nicotine stimulation of alpha 2, alpha 3, alpha 4 expression, but after nicotine stimulation, the the expression is not obvious, two There is no clear cell beta 1 protein expression in cells transfected with nAchR siRNA. Alpha 1, alpha 5nAchR after siRNA and LPS treatment to nicotine, detected in culture supernatant of IL-8, TNF- alpha, compared with nicotine and LPS levels were incubated in group expression of mRNA relative content and cell IL-6 protein, no difference statistical significance, and the cell group alpha 7 nAchR siRNA transfected by LPS and nicotine stimulation, LPS and training of the relative content of the relative content of mRNA and the cell protein in the supernatant, nicotine stimulation groups had significantly increased. (3) CE and LPS alone stimulated the phosphorylation of both NF- kappa Bp65 protein -I- kappa B alpha protein and the nucleus increased significantly, compared with normal control group, P0.01 group and CE cells; adding nicotine and LPS were incubated in the phosphorylation of -I- kappa B alpha and NF- K Bp65 were significantly reduced, and CE alone, LPS incubation group compared, there was statistical difference Significance of P0.01; no significant changes in the content of I- kappa B alpha protein +CE+ alpha -BTX group; nicotine, nicotine +LPS+ alpha -BTX group phosphorylated -I- kappa B alpha kappa Bp65 protein and NF- protein content of nicotine and nicotine in +CE group, compared with the +LPS group increased significantly. Transfection of luciferase reporter plasmid pNF-kB-luc was observed after nicotine in group +CE, the fluorescence intensity of nicotine in the +LPS group was lower than CE group, LPS group, a statistically significant difference between P0.01 and nicotine +CE+ alpha -BTX group, the fluorescence intensity of nicotine +LPS+ alpha in -BTX group increased along with nicotine nicotine compared to +CE group, + LPS group, P0.01. PDTC further pretreatment of cells after 30min. Then respectively CE, LPS stimulation, cell culture supernatant of TNF- alpha, IL-8, IL-6 protein content and cell level of mRAN compared with pure CE stimulation group, simple LPS stimulation group were significantly decreased compared with P0.01 group, +CE; nicotine, the nicotine in the +LPS group The relative content of mRNA and factor protein compared with CE group, LPS group decreased significantly; the cells pre treated with PDTC, then were treated with nicotine +CE +LPS nicotine stimulation, supernatants of TNF- alpha, IL-8, the relative content of IL-6 protein decreased obviously, and the simple CE stimulation group simple, LPS stimulation group compared to P0.01. cells by CE and LPS after stimulation were observed in the phosphorylation of p38 MAPK, ERK1/2, JNK protein expression increased, compared with control group P0.01; adding the cells incubated with nicotine, respectively by CE and LPS stimulation, the relative content of phosphorylated ERK1/2 into cells detection of proteins was significantly decreased, respectively, 0.34 + 0.07,0.39 + 0.08, and CE group (0.74 + 0.12), LPS group (0.79 + 0.13) compared to a statistically significant difference between P0.01 and p38; and the phosphorylation of JNK was decreased significantly. With alpha 7 nAChR specific inhibitors of alpha -BTX Nicotine and nicotine +CE in the +LPS group, the relative content of phosphorylated ERK1/2 protein were 0.59 + 0.11,0.63 + 0.13, +CE group and nicotine without alpha -BTX stimulation, compared with nicotine + LPS group, the difference was statistically significant, P0.01; but the same effect on p38 and phosphorylation of JNK protein is further observed in HBE16. Cells treated with CE, p38MAPK, pERK1/2 and the expression of pJNK protein increased with time, the expression, the expression level of 6h was significantly higher than that of 30min at the time point of P0.01; by nicotine incubation of the cells, the expression of p38MAPK in 30min, with the prolonged expression gradually decreased, pERK1/2 at each time point were there was no obvious expression, pJNK was expressed in 30min, 2h was the strongest, began to decrease in 6h expression, compared with the expression of 2h, the difference was statistically significant, P0.01.
Conclusion: (1) the nicotine in cigarettes caused by airway mucus hypersecretion process does not have too many positive effects, on the contrary, it can reduce the inflammatory cytokines in this process, inhibition of subsequent excessive inflammatory reaction. (2) nicotine can reduce proinflammatory cytokine and chemokine TNF- alpha, IL-8, IL-6 protein production has certain anti-inflammatory function, and confirmed by nAChR and 7 with low I- and alpha kappa B alpha phosphorylation, thereby inhibiting nuclear factor kappa B nuclear translocation of NF-, expression of the inhibitory effect factor; the process may be by reducing the level of nicotine ERK1/2 activation. (3) nicotine does not increase the synthesis of mucin gene transcription and MUC5AC, and can make the inflammatory stimulus generated excess mucus exocrine is relatively reduced, so as to maintain the airway in chronic inflammatory stimulation of mucus high retention of airway mucus, students A relatively steady and hypersecretion.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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