The Effect Of Carbocysteine On Airway Bacterial Load And Its Mechanism In Rats Chronically Exposed To Cigarette Smoke

Chronic obstructive pulmonary disease (COPD) is often associated with the progression of acute, accelerated decline in lung function, increased economic burden and mortality.69.6% of the acute attack and bacterial infection.Host local inflammatory response increases proportionally with airway bacterial load. Miravitlles raised " bacterial threshold value theory"of COPD in 2002.In his view, There is a minimum threshold above which the inflammatory reaction is severe enough to elicit acute exacerbation. The factors which affect the bacterial level of aute exacerbation of COPD are internal factors (lung function impairment, heavy smoking, airway hyperresponsiveness, airway mucus hypersecretion and impaired defense mechanisms) and external factors (bacterial species, drying temperature environment, air pollution and stable and acute exacerbation of mishandling). Mucociliary clearance of airway function is non-specific defense mechanisms, can be removed by mucociliary clearance system of pathogenic bacteria, to reduce airway bacterial load.Carbocysteine is a mucolytic agent that was recently shown to have the antioxidant and anti-inflammatory properties in the latest vitro studies. It has reactive oxygen species (ROS) scavenging properties and reduce neutrophil elastase-induced mucin production and rhinovirus infection in vitro. It can reduce the rate of bacterial adhesion which is associated with mucin.Our Chinese PEACE study demonstrated a clinically and statistically significant reduction in exacerbation rates in patients with moderate-to-severe COPD receiving carbocysteine over a 12-month period compared with placebo. However, the precise mechanism of carbocysteine remains uncertain. The present in vivo study was performed to examine the effect of carbocysteine on the airway Hi load in rats exposed to cigarette smoke, as well as its related mechanism in airway mucus hypersecretion and mucociliary clearance. The purpose of present study was to provide an experimental basis for the clinical application of carbocysteine.The local inflammatory response of the host parallels the increase of airway bacterial load.There is a threshold above which the inflammatory reaction is severe enough to elicit acute exacerbation. Our previous Chinese PEACE study has demonstrated a clinically and statistically significant reduction in exacerbation rates in patients with moderate-to-severe chronic obstructive pulmonary disease(COPD) receiving carbocisteine over a 12-month period compared with placebo.However, the precise mechanism is uncertain.The present study was performed to examine the effect of carbocisteine on airway Haemophilus influenzae (Hi)'s load in rats exposed to cigarette smoking, and its related mechanism about airway mucus hypersecretion and mucocialiary clearance associated with Hi's adherence and clearance.Methods1.Ninty-six rats were randomly divided into four groups, twenty-four rats per group. Group A:Normal control group, normally fed for twelve weeks; Group B: COPD group, exposed in a self-made glass box (0.8×0.6×0.6m3) with a vent hole (2x2cm2) on top,30 cigarettes/d, twice/d,2h/d, at a smoke concentration of 5%v/v,5d/w, continuouslysmoke concentration of about 5% in the 0.5 hour,2 times daily,5 days a week for 12 weeks, group C carbocysteine prevention group:In addition to cigarette smoking, carbocysteine was administered intragastrically 30 minutes before exposure to CS from the first day of the first week; group D carbocisteine treatment group:In addition to smoking addition, carbocysteine was administered intragastrically 30 minutes before exposure to CS from the first day of the sixth week.2. Pulmonary function testAfter anesthesia via intraperitoneal administration of sodium pentobarbital anesthesia, the rates of 0.3 seconds of rats in each group was detected by AniRes2005 small animal lung function analysis system (Beijing Bei Lanbo Technology).3. Lung biopsy and BALF sacrificing solutionThe lung tissue was formalin-fixed, paraffin-embedded, and sectioned for histochemistry and immunohistochemistry. The rest of the right lung was stored at-80℃for later quantitative polymerase chain reaction (qPCR) and western blot analysis.4. Alcian blue-periodic acid Schiff staining (AB-PAS) Paraffin sections were deparaffinized and stained with Alcian blue/periodic acid-schiff for mucin glycoproteins and goblet cell hyperplasia5. Immunohistochemistry SABC immunohistochemical method for the detectation of the numbers of Muc5ac positive cells and Muc5ac expression. 6. Real-time quantitative reverse transcription polymerase chain (RT-PCR) method Detect the expression of rat airway Muc5ac mRNA.7. Western blot test (Western Blot)Detection of airway Muc5ac protein expression. 8. Scintillation camera technology measured mucociliary clearance in rats Oropharyngeal instillation of 99mTc-Sc measured clearance process, as the evaluation of mucociliary clearance in rats 9. Rat lungs after vaccination Hi Determination of bacterial load After 12 weeks, the rats inoculated with quantitative endotracheal Haemophilus influenzae.Bronchoalveolar lavage fluid (B ALF) and lungs was aseptically collected for quantitative bacterial cultures at 3h. Meanwhile, mucociliary clearance (MCC) was measured by quantifying the clearance of 99mTc-Sc dropped from the oropharynx.Results1.Established COPD mouse model:detection of lung histopathology showed a number of inflammatory cell infiltration and alveolar wall rupture associated with alveolar fusion, alveolar expansion in group B, pulmonary function testing FEV0.3/FVC<70%, consistent with COPD diagnosis. a few inflammatory cell infiltration, only part of alveolar wall rupture and integration of alveolar expansion in group C and group D, FEV0.3/FVC also improved.2. Goblet cell metaplasia and expression of Muc5ac in rat airway:there is increased mucus hypersecretion, that is goblet cell metaplasia and upregulation of Muc5ac expression in group B, P<0.05. 3. Rat airway bacterial load:increased airway bacterial load in group B compared with group A, bacterial load decreased in group C and group D after the carbocysteine intervention, P<0.05.4. Mucociliary clearance determination Compared with the control group A, mucociliary clearance aggravated in COPD group. And there is improved mucociliary clearance in group C and group D, P<0.05.Conclusion1. It can replicate COPD rat models by exposed to cigarette smking for 12 weeks.2. Carbocysteine decreaced the airway Hi's load in COPD rats and there is a decreaced mucus hypersecretion and improved MCC by carbocisteine administration.3.There is improved airway mucus hypersecretion and mucocialiary clearance associated with Hi's adherence and clearance. This maybe partly related to decreaced airway Hi's load.4.Furthermore there was a better effect in carbocisteine prevention group, maybe related to its sufficient anti-oxidation effect. ObjectiveCarbocysteine is a mucolytic agent that was recently shown to have anti-inflammatory and antioxidant properties in the latest study in vitro. Carbocysteine can effectively scavenge reactive oxygen species, hydroxyl radicals and glutathione, and reduce bacterial adhesion rate which is highly associated with mucin. Our previous studies in vivo confirmed carbocysteine can reduce COPD airway bacterial load and reduced the mucus hypersecretion, and smoking can cause airway mucus hypersecretion by oxidative stress mechanism.We speculated that can inhibit mucus hypersecretion by anti-oxidative mechanism, thereby lower airway bacterial load via reducing bacterial adhesion.This study examined The effect of carbocysteine on mucin production in NCI-H292 cells induced by cigarette smoke and the amount of production of ROS.MethodNCI-H292 cells, a human lung mucoepidermoid carcinoma cell line, treatment factors are smoking and carbocisteine. EGFR and ERK specific inhibitor AG1478 and PD98059 for the intervention factor. MUC5ACmRNA, MUC5AC protein was measured by using RT-PCR, ELISA and Western-blotting. And intracellular ROS production and phosphorylation of EGFR and the amount of EGFR and ERK.ResultCarbocysteine reduce smoking-induced MUC5ACmRNA and protein expression, carbocysteine also reduce smoking-induced intracellular ROS production. Carbocysteine reduced p-EGFR and p-ERK protein.ConclusionCarbocysteine inhibit mucin production in smoking-induced NCI-H292 cells which maybe partly related to reduced ROS production. The effect of carbocysteine maybe partly related to p-EGFR and p-ERK pathway.