The Effect Of An'ningpai And Eucalyptol On Airway Mucociliary Clearance And Inflammatory Mechanism In Rats Exposed To Cigarette Smoke

Objective: Chronic obstructive pulmonary disease(COPD)is a kind of chronic inflammatory diseases,and smoking is the definite risk factor.There were many scientific research findings published about that chronically cigarette smoke-exposed rats were used as COPD models.The pathophysiology of COPD includes airway chronic inflammation,mucous hypersecretion,oxidative stress,airway smooth muscle contraction and airway remodeling.Mucociliary Clearance(MCC)dysfunction is considered as main pathological change of chronic respiratory diseases such as COPD.With increasing researchs on MCC function,the roles of mucous regulator in patients with COPD were gradually paid more attention.That long-term application of mucous regulator could obviously reduce the occurrence of acute exacerbation of COPD patients had been confirmed in evidence-based studies.Therefore,myrtol standardized(Gelomyrtol forte),as the representative of eucalyptol preparation,had been widely applied in clinical.However,myrtol standardized was the mixture of eucalyptol,limonene and alpha pinene,there was no research related contrasting the effects between mixture and its main single composition-eucalyptol.And studies about eucalyptol preparation were more focused on clinical.Though early clinical studies had pointed out that eucalyptol preparation contributed to reduce airway mucus secretion and inflammation,the mechanism was not clear.Based on the above reasons,this study contrasted the effects between An'ningpai(the synthetic agents,main ingredient containing eucalyptol,limonene and alpha pinene,similar to myrtol standardized ingredient)and its main component eucalypto in chronic cigarette smoke-exposed rats,further explored its mechanism on airway MCC function and inflammation.Methods:1.The effects of An'ningpai and eucalyptol on airway MCC function in rats exposed to cigarette smokeThe models was Established by continuously exposeing cigarette smoke for 12 weeks.An'ningpai and eucalyptol,which intervened at the beginning of smoke exposed,were compared with after intervention treatment between different groups on rats general morphology,lung HE structure,airway bacterial load,airway scavenging activity on exogenous Haemophilus influenzae,microstructure change under TEM(Transmission Electron Microscope),glycosaminoglycan properties secreted by goblet cells in AB-PAS staining,airway mucin MUC5 AC production and expression.The rats was divided into 5 gourps: normal control group,smoke exposed group,smoke+An'ningpai intervention group,smoke+eucalyptol intervention group,smoke+glycerin intervention group.Glycerin was the solvent of An'ningpai and eucalyptol.2.The effects of An'ningpai and eucalyptol on airway inflammation and its mechanism in rats exposed to cigarette smokeTo further compare An'ningpai and eucalyptol on airway inflammation level in cigarette smoked-exposed rats and to explore its mechanism,the indicators were compared between groups on rats airway inflammation,including bronchoalveolar lavage fluid(BALF)cell count,the expression levels of ICAM-1,TNF alpha,and IL-6,and detection of p38 MAPK and p-p38 MAPK protein levels.And combining with the MUC5 AC expression level,the correlation between indicators were evaluated.To explore whether the effect of An'ningpai and eucalyptol on inflammatory mediators were related to p38 MAPK phosphorylation.Data analysis was performed using the statistical software SPSS16.0.All data were expressed as mean±SD.Differences between the two groups using independent sample T test.Differences between groups were analyzed using the one-way ANOVA(homogeneity of variance with LSD and S-N-K test,heterogeneity of variance with Tamhane's T2 analysis).Correlation comparison using Pearson correlation analysis.A P value of less than 0.05 was considered a statistically difference,less than 0.01 was considered statistically significant difference.Results: 1.The effects of An'ningpai and eucalyptol on airway MCC function in rats exposed to cigarette smokeGeneral state change and weight loss was existed in smoke exposed group,the changing of lung function and pathological structure was conformed to patients with chronic obstructive pulmonary disease,that prompted model preparation was success.Compared with normal control group,the airway bacterial load was increased and scavenging activity on exogenous Haemophilus influenzae was slower in smoke exposed group rats.TEM results that airway epithelium cilia lacked even exfoliated and cell organelles seriously damaged were shown,which prompted cilia function was impaired.And also the goblet cells structure damaged was founded in TEM.Glycosaminoglycan composition secreted by airway goblet cells also changed in AB-PAS staining results,which prompted secreted mucous properties affected.Immunohistochemistry and PCR results showed MUC5 AC expression increased in airway.Compared with smoke exposed group,the pathological structure was relatively improved in An'ningpai intervention group,though obvious structural changes still exited compared with normal control group.Bacterial load was reduced in the rat airway of An'ningpai intervention group,and scavenging activity on exogenous Haemophilus influenzae was accelerated.TEM results showed airway epithelial cilia structure was relatively intact,that prompted medical intervention for cilia played a protective role.But for the damage of the cell structures and organelles the improvement was not obvious.The effect of An'ningpai on mucous secretion was manifested in regulating acid mucopolysaccharide proportion secreted by airway epithelium goblet cells,affecting the properties of mucus,and reducing airway MUC5 AC expression level.The therapeutic effect of eucalyptol intervention group was similar to that of An'ningpai intervention group,no significant statistical difference showed.Glycerol as a drug control had no influence to the detection indexes.2.The effects of An'ningpai and eucalyptol on airway inflammation and its mechanism in rats exposed to cigarette smokeThe anti-inflammatory effects was also found in An'ningpai intervention group,and the inflammation index improved levels were significantly related with mucin improved levels.Compared with normal control group,increased BALF inflammatory cell count and expression of TNF-?,IL-6,ICAM-1 were shown in smoke exposed group rats,accompaning by higher p38 MAPK and p-p38 MAPK protein levels.Compared with smoke exposed group,BALF inflammatory cell ratio and expression of TNF-?,IL-6,ICAM-1 were obviously reduced by An'ningpai intervention,accompanied with higher p38 MAPK and p-p38 MAPK protein levels.And the p38 MAPK and p-p38 MAPK expression decreased,more obviously drop of p-p38 MAPK suggested that An'ningpai had the effect of p38 MAPK dephosphorylation.The anti-inflammatory effect of eucalyptol intervention group was similar to that of An'ningpai intervention group,no significant statistical difference showed.Conclusions: 1.An'ningpai has the effects on reducing airway bacterial load in rats exposed to cigarette smoke,increasing bactiria scavenging activity by improving airway mucociliary clearance function.The effects are mainly performed by protecting airway epithelial cilia structure,regulating acid mucopolysaccharide ratio and mucin MUC5 AC level secreted in goblet cells.2.In An'ningpai regulating airway mucociliary clearance function,the effects on inflammatory mediators were involved in at the same time,which mainly manifested the lower expression of inflammatory indexes such as TNF-?,IL-6,ICAM-1 and reducing of p38 MAPK phosphorylated ratio.The induction dephosphorylated of p38 MAPK may be the common mechanism of An'ningpai effect on regulation of mucin secretion and anti-inflammation,in it,eucalyptol played a main role.