The Effect Of Nuclear Transcription Factor Nrf2in Cigarette Smoke-induced MUC5AC Mucin Expression

BackgroundCigarette smoke contains a lot of oxidants, radicals and organics, some of which deposit in the small airway for long time. Sustained oxidant irritations in the lungs from cigarette smoke make it obvious oxidant stress. The typical pathological changes in the airways of those patients with long time smoking are goblet cell metaplasia and hyperplasia which lead to mucous incensement.Airway mucous is a mixture of different secretions. It could be divided into gel forming and membrane-spanning mucin according to their basic structural characteristics. The four gel-forming mucin muc2, muc5ac, muc5b and mucl9mainly expressed in large airways and secreted by epithelial goblet cells and submucosal mucous cells. The mucous in the airways could absorb some particles and pathogens, and cleaned through cilia movement of the epithelial cilia cells. The hypersecretion of MUC5AC and MUC5B by epithelial cells is the main cause of cough and sputum production. Airway diseases like cystic fibrosis, chronic obstructive pulmonary disease and asthma complicated with hypersecretion in the airways make the incidence and mortality even worse.Nuclear factor erythroid2related factor2(Nrf2) is the most strongest transcription factor in the CNC transcription family which is expressed widely in liver, kidneys, lungs and many other tissues. Nrf2-mediated oxidant reaction is a major cellular defense mechanism.Nrf2deletion or inactivity could aggravate the cytotoxicity of oxidant leading to cellular dysfunction, apoptosis and death.There is no report indicates the role of Nrf2in MUC5AC expression induced by cigarette smoke. We hope to prove the fact that Nrf2plays an important role in the MUC5AC expression induced by cigarette smoke and inflammation in the airways, there for a new medication for airway inflammation and hypersecretion caused by cigarette smoke.ContentsPart Ⅰ The influence of cigarette smoke in MUC5AC mucin and transcriptor Nrf2of A549cellsObjectives:1,To prepare stable cigartte smoke solution;2,To find a proper concentration of cigarette smoke solution(CSS) through MTT;3,To detect the expression of Nrf2and MUC5AC mRNA after treatment of cigarette smoke.Methods:1. to prepare cigarette somke solution and evaluated with a spectrophotometer;2. to evaluated the cytotoxicity of cigarette smoke through MTT;3. To detect the Nrf2and MUC5AC mRNA change after treated with cigarette for different time with the method of Q-PCR and WB.Results:1,The average OD320of CSSs during the continuous was0.7394, the standard deviation is0.05804, the95%confidential interval were0.7213-0.7575, one-way ANOVA analysis showed there were no statistic difference between them(P>0.05), which means that method to prepare CSS is quite standard and normative.2,Along with the increase of the solutions'concentrations and the time of incubation, the inhibitory rates of cigarette somke solutions to A549cells increased by the method of MTT. When the concentration was lower than10%, the inhibitory rates were significantly lower than those when the concentrations were higher than25%. For the concentration of10%, the inhibitory rate after48hour-incubation was the highest and there were no obvious difference after24hour-incubation.3,10%of CSS could induce the Nrf2and MUC5AC mRNA and the proteins' expression. The concentration of Nrf2and MUC5AC mRNA and the proteins increased along with the incubation time and reached the highest level after24hours of incubation.Conclusions:1, The method we used to prepare CSS is standard and normative. Those CSS had time-and concentration-dependent cytotoxicity to A549cells in vitro.2, The CSS could induce the expression of MUC5AC mucin in A549. Within24hours the effect was time dependent.3,The CSS could induce the expression of transcription factor Nrf2in A549.Part Ⅱ.The effect of transcription factor Nrf2in the induction of MUC5AC with CSS in A549cellsObjectives:1. to verify the enhancement effect in the induction of MUC5AC with CSS in vitro of Nrf2knock-down;2,to verify the decrease effect in the induction of MUC5AC with CSS in vitro of Nrf2over-expression;3,to verify the protective effect of N-acetyl cysteine (NAC) in the induction of MUC5AC with CSS in vitro.Methods:1,To determine the expression of Nrf2and MUC5AC mRNA and proteins of A549cells by the method of Q-PCR and WB after stimulation of CSS on the basis of Nrf2knock-down with siRNA.2,To determine the expression of Nrf2and MUC5AC mRNA and proteins of A549cells by the method of Q-PCR and WB after stimulation of CSS on the basis of Nrf2over-expression with Pcdna3-Myc3-Nrf23. To determine the Nrf2and MUC5AC mRNA of A549cells by the method of Q-PCR after stimulation of CSS with N-acetylcysteine.Results:1,The siRNA interference could effectively decrease the level of Nrf2. Theincrease of MUC5AC after stimulation of CSS in the negative control group is 64.33%while that in the interference group is195.33%. The latter is almost significantly twice higher than the former (P<0.001)。2,The pCDNA3-Myc3-Nrf2interference could effectively promote the expression of Nrf2. The increase of MUC5AC after stimulation of CSS in the negative control group is47.44%while that in the over-expression group is25.74%. Over expression of Nrf2could significantly decrease the MUC5AC level by45.73%(P<0.001)。3,20mM NAC could promote the expression of Nrf2mRNA and then decrease the MUC5AC after stimulation of CSS in A549cells.Conclusions:1,The MUC5AC mucin in A549cells increased significantly after stimulation of CSS with Nrf2knock-down by the method of siRNA interference.2. The increase of MUC5AC mucin in A549cells reduced significantly after stimulation of CSS with Nrf2over-expression by the method of pCDNA3-myc3-Nrf2interference.3.NAC could promote the expression of Nrf2and then reduced the MUC5AC mucin in A549after stimulation of CSS.Part Ⅲ. The effect of transcription factor Nrf2in the induction of MUC5ACproduction and airway inflammation with CSS in miceObjectivs:1,To verify the protective effect of Nrf2in the airway inflammation induced by cigarette smoke(CS)。2,To verify the protective effect of Nrf2in the induction of MUC5AC production in mice.Methods:1,To verify the protective effect of Nrf2in airway inflammation by the method of non-invasive airway hyperreaction determination, pathological evaluation of HE stained lung tissues and evaluation of bronchopulmonary lavage fluid(BALF) after3,7and14days of CS stimulation in both wild type ICR mice and Nrf2knockout mice.2.To verify the protective effect of Nrf2in pulmonary MUC5AC production by the method of pathological evaluation of PAS stained lung tissues, evaluation the MUC5AC in BALF by ELISA and determination the Nrf2protein by WB after3,7and14days of CS stimulation in both wild type ICR mice and Nrf2knockout mice. Results:1,After short time of CS stimulation the airway reaction in the mice increased, the total number of white cells in BALF increased, the proportion of neutrophiles in the BALF increased slightly and the inflammatory scores in HE stained of the lung tissues increased. Those changes became even more obvious with the increase of stimulatory days and with Nrf2knockout2. After short time of CS stimulation the PAS scores in PAS stained of the lung tissues increased in the Nrf2-/-group. At the same time the MUC5AC mucin in BLAF increased in the Nrf2-/-group and increased significantly with the longer days of CS stimulation.Conclusions::1,Short time CS stimulation could induce more significant neurophile-prominent airway inflammation and airway hyperresponsiveness in Nrf2-/-mice than in wild mice.2. Short time CS stimulation could induce more significant increase of pulmonary MUC5AC Nrf2-/-mice than in wild mice..Conclutions1. Cigarette smoke could induce MUC5AC expression, which is partially mediated with transcription factor Nrf2.2,Cigarette smoke could induce the inflammation in mice airways, which is protected partially with Nrf2.