The Distinct Effects Of Nicotine In Chronic Airway Inflammation Induced By Cigarette Smoking

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.