Pinellia Ternata Treatment Attenuates Corticosteroid Withdrawal-induced Rebound Of Mucus Secretion And Airway Inflammation In A Rat Chronic Obstructive Pulmonary Disease Model

Chronic obstructive pulmonary disease(COPD)ranks the third leading cause of death worldwide.COPD is a common disease characterized by irreversible airflow obstruction and persistent inflammation to harmful environmental stimuli,usually cigarette smoke.Most COPD patients show characteristics of both chronic bronchitis(CB)and emphysema.COPD patients often experience exacerbations,which can be defined as acute events that lead to an accelerated decline in lung function and health status.COPD exacerbations are generally associated with accelerated loss of lung function,impaired life quality,and enormous health-care costs.Thus,prevention of exacerbations is a major therapeutic aim in COPD management.Inhaled corticosteroids are extremely effective in reducing inflammation and improving symptoms and lung functionin the treatment of COPD.The combination of inhaled glucocorticoids and long-acting ?2 agonist significantly reduces the exacerbation rate of COPD,thus is recommended in patients with frequent exacerbations.It is estimated that up to 80%of people with COPD use inhaled corticosteroids either alone or in combination with a long-acting ?2 agonist nowadays.However,emerging evidences has linked inhaled corticosteroids with increased risk of pneumonia in COPD.The occurrence of pneumonia and other side effects in COPD patients might prompt clinicians to consider discontinuation of inhaled corticosteroids.Paradoxically,withdrawal of inhaled corticosteroids is associated with an increased frequency of adverse effects,and patients experienced inhaled corticosteroid withdrawal showed much higher exacerbation rate than those not on inhaled corticosteroid treatment,as reported in the Inhaled Steroids in Obstructive Lung Disease in Europe(ISOLDE)study.This finding was subsequently supported by other studies in addition to other adverse effects,including deterioration in lung function,increased symptoms,and impaired life quality.In a randomised study,even with the maintenance of long-acting[32 agonist,inhaled corticosteroid withdrawal was associated with deterioration in lung function and dyspnoea,and increased mild exacerbations;moreover,a great decrease was observed in lung function during the final step of glucocorticoid withdrawal.To avoid this paradoxical situation,attention must be paid to carefully monitor the corticosteroid withdrawal process and alternative treatments are needed.Pinellia ternata(Araceae)is a monocotyledonous perennial herbaceous plant,the tuber of which has been used since ancient time(and is still widely in use)in China as an important herb in traditional Chinese medicine,for therapy of certain chronic respiratory diseases.Empirically,its pharmacological effects include sedative,relieving cough,promoting phlegm excretion,and anti-inflammatory and anti-emetic effects.It is used for treating airway inflammatory diseases in Korea and Japan as well.Based on the clinical effects of Pinellia ternata on chronic respiratory diseases,we hypothesize that it may have potential protective effects on the side effects caused by corticosteroid withdrawal.Objective:Mucus hypersecretion and airway inflammation are fundamental features of COPD,and can serve as measurements for evaluating the severity of COPD.We therefore investigated the effects of withdrawal of budesonide,the most widely used inhaled corticosteroids in COPD patients,on production of mucus and key inflammatory cytokines in the airway in a cigarette smoke-LPS combination-induced COPD rat model,and explored the potential protective effects and the underlying mechanism of Pinellia ternata during budesonide withdrawal.MethodsFirstly,The rat COPD model was established by intratracheal instillation of lipopolysaccharide twice and passive cigarette smoking for 4 weeks.Briefly,rats were instilled with 0.2 ml LPS(1 mg/ml)on the first day;and from the second day,the rats were placed in a 0.8x0.5x0.5 m3 glass box,where existed cigarette smoke(Red Flag Canal,Henan Tobacco Ltd.,China;tar 11 mg,Nicotine 0.7 mg,CO 13 mg),for 30 min at 24 h intervals till the fourteenth day.From the fifteenth day,intratracheal instillation of LPS and passive smoking were repeated.And then,seventy SPF healthy SD adult male rats were randomly divided into seven groups(a,b,c,d,e,f,g),with 10 rats in each group.Groups b,c,d,e,f,g were subjected to COPD modelling,while group a was normal control,which was only treated with 4 ml atomization saline inhalation for 4 weeks.After COPD modelling,group b was treated with4 ml saline inhalation for 4 weeks;group c was treated with 4 ml budesonide inhalation for 4 weeks;group d was treated with budesonide for 2 weeks and then was sacrificed;group e was changed to atomization saline inhalation for 2 weeks after 2 weeks of budesonide;group f was changed to Pinellia ternata gavage for 2 weeks after 2 weeks of budesonide inhalation;and group g was treated with Pinellia ternata gavage for 4 weeks.All the inhalations were performed for 30 min daily.At the end all the rats were sacrificed for subsequent experiments.Finally,goblet cells of the airway were stained with Alcian Blue/Periodic acid-Schiff(AB/PAS)method.MUC5AC protein was determined by immunohistochemical method.Expression of JNK,ERK and p38 were determined by Western blot method.IL-1? and TNF-? in bronchoalveolar lavage fluid(BALF)were determined by ELISA method.ResultsPinellia ternata treatment reduces budesonide withdrawal-induced increase of goblet cells in rat COPD modelWe firstly checked the number of the goblet cells in the airway,which is one of the key features of chronic airway inflammation,of both control and COPD groups.The numbers of the goblet cells in COPD group(COPD+Saline 4w)were much higher than those in the control group(Normal Saline 4w),and budesonide treatment(COPD+ Bude 4w/2w)blocked this increase.However,2w after the withdrawal of budesonide(COPD+Bude 2w Saline 2w),goblet cells showed a significant rebound increase.Strikingly,Pinellia ternata treatment(COPD+Bude 2w Pine 2w)depleted budesonide withdrawal-induced increase of goblet cells,suggesting a possible protective effect of Pinellia ternata on corticosteroid withdrawal-induced side effects.Pinellia ternata treatment attenuates budesonide withdrawal-induced mucin secretionAs budesonide withdrawal induced increase of the number of goblet cells,and Pinellia ternata treatment blocked this increase,we also wanted to know their effects on mucin secretionin the airway.We chose MUC5AC for immunohistochemistry staining.We found the expression of MUC5AC was greatly enhanced in the COPD group,and budesonide treatment blocked this enhancement.Similar to the number of goblet cells,a rebound of MUC5AC level was induced by budesonide withdrawal.Pinellia ternata treatment inhibited this rebound(Fig.3),which further supported the possible protective effect of Pinellia ternata.Pinellia ternata treatment blocks budesonide withdrawal-induced airway IL-1?and TNF-? secretionBoth the goblet cell number and theMUC5AC level indicated that there was mucus hypersecretion in the rats of the COPD group and the budesonide withdrawal group,we then measured two major pro-inflammatory cytokines,IL-1? and TNF-?,in the bronchoalveolar lavage fluid,to explore if there is any airway inflammation,the other feature of COPD.The levels of IL-1? and TNF-? were enhanced in the COPD group as compared to those of the control group;budesonide treatment blocked this enhancement,and its withdrawal led to a rebound.Importantly,Pinellia ternata treatment after budesonide withdrawal prevented the rebound of IL-1? and TNF-a release.These results suggest a protective effect of Pinellia ternata treatment on the airway from corticosteroid withdrawal-mediated inflammatory damage.Budesonide withdrawal-induced airway inflammation is mediated by ERK activationAs COPD modelling and budesonide withdrawal both induced airway inflammation,we further explored which pro-inflammatory signaling pathway was responsible for this.We studied the activation of three different mitogen-activated protein kinases(MAPKs),i.e.,ERK,p38,and JNK,and found that ERK,but neither p38 nor JNK,was strongly activated in COPD group than the control group.This activation was blocked by budesonide treatment.Moreover,the activation of ERK showed a rebound 2w after budesonide withdrawal,suggesting that budesonide withdrawal-induced airway inflammation is mediated by ERK activation.Pinellia ternate treatment blocks budesonide withdrawal-induced ERK activationGiven that budesonide withdrawal-induced airway inflammation was mediated by ERK activation,and Pinellia ternate treatment blocked budesonide withdrawal-induced airway inflammation,we wondered if the effect of Pinellia ternate treatment on airway inflammation was through the blockade of ERK activation.Indeed,Pinellia ternate treatment blocked budesonide withdrawal-induced ERK activation(Fig.5),suggesting Pinellia ternate treatment blocks budesonide withdrawal-induced airway inflammation by inhibiting ERK activation.MUC5AC expression correlates to goblet cell hyperplasia,airway inflammation,and ERK activityAs budesonide withdrawal caused rebound responses which included increased goblet cell number,enhanced MUC5AC secretion,elevated levels of key inflammatory cytokines,and activation of ERK,we wanted to explore the relationships among these effects.To address this question,we performed Pearson correlation analyses between MUC5AC expression and other individual effects,with measurements from different groups all together.As shown the level of MUC5AC positively correlated to all the other individual effects.These results suggest that MUC5AC expression is related to goblet cell hyperplasia,airway inflammation,and ERK activity.Conclusion1.Pinellia ternata,a traditional Chinese medicine,attenuates budesonide withdrawal-induced rebound increase of goblet cell number,MUC5AC expression,IL-1? and TNF-? release,and ERK activity,in a rat COPD animal model.2.The functional pathway of ERK activation-proinflammatory cytokines release-MUC5 AC expression is responsible for these rebound responses.3.Pinellia ternata treatment might be a promising therapeutic strategy for the management of inhaled corticosteroid withdrawal in COPD patients.