Investigation On The Role Of Neutrophils In AECOPD And The Protective Effect Of Ginsenoside Rg3

Chronic Obstructive Pulmonary Disease?COPD?is characterized by persistent respiratory symptoms and airflow limitation,being the fourth leading cause of death worldwide.Acute Exacerbation of COPD?AECOPD?is the leading cause of high morbidity and mortality in COPD patients,manifested by increased purulent sputum,dyspnea,cough and wheezing.Respiratory infection,including viral and bacterial infection,is the main cause of AECOPD,among which bacterial infection accounts for50%of AECOPD pathogenesis.Nontypeable haemophilus infuenza?NTHi?,Catamora,Streptococcus pneumoniae and Staphylococcus aureus are common bacteria strains isolated from AECOPD patients,with NTHi being the most common one in sputum.Therefore,clarifying the mechanism of NTHi-induced AECOPD can help to gain insight into the disease process and to prevent the disease effectively.Neutrophil is the first defense against exogenous infection,rapidly recruited to the infection site in the presence of chemokines to exert phagocytosis and anti-inflammatory effects.However,excessive neutrophil migration could lead to redundant secretion of neutrophil elastase,metalloproteinase and oxidatively active substances,which could in turn cause damage to local tissue.In the process of neutrophil migration,formation of pseudopods and remodeling of cytoskeletal actin play an important role,mainly regulated by the phosphatidylinositol?PtdIns?3-kinase?PI3K?signaling pathway.This procedure is initiated upon the stimulation of neutrophil surface G protein coupled receptors?GPCRs?by chemo-attractants ligands,such as N-formylmethionyl-leucyl-phenyl-alanine?fMLP?.Dissociated G??subunit could activate PI3K and phosphorylate protein kinase B?AKT?,which help promote the polarization and aggregation of F-actin in cell edge?mainly the pseudopod region?,thus participating in neutrophil migration.Neutrophil is considered to be one of the most common inflammatory cells in the airways of COPD patients,closely related to NTHi infection.NTHi infection could induce inflammatory response in endothelial cells and produce a series of chemokines??Interleukin,IL?-1?,IL-8,etc.?recruiting a large number of neutrophils infiltrated in the airways,this severe airway neutrophil inflammation leads to decreased lung function and airway dysfunction.Hence,further investigation on role of neutrophils in NTHi-induced AECOPD,and reducing airway neutrophil inflammation via the inhibition of neutrophil migration are key scientific issues for AECOPD treatment.Current clinical treatment of AECOPD includes inhaled bronchodilators,corticosteroids and antibiotics,however,these therapies are limited by their adverse effects and host resistance,leading to a fade effect in controlling the disease.Therefore,there is an urgent need for safer and more effective novel therapeutic agents and methods for AECOPD.Plant-derived drugs,like ginseng,have been shown having anti-inflammatory effects and no serious side effects.More than 100 monomers have been identified from ginseng and ginsenosides are one of the most active ingredients,among them,ginsenoside Rg3 has various pharmacological activities such as anti-inflammatory,anti-tumor and anti-fatigue effects.Recently,studies have been keen on the therapeutic role of ginsenoside Rg3 in respiratory diseases,such as the inhibition on epithelial-mesenchymal transformation and lung cancer invasion through regulating lithotrophic glycosylation and Mitogen-activated protein kinase?MAPK?and nuclear factor kappa-B?NF-?B?signaling pathways.Ginsenoside Rg3 could also inhibit NF-?B activity,reduce NF-?B-induced inflammatory cytokine and attenuate LPS-induced acute lung injury by regulating PI3K/AKT/mTOR pathway.The purpose of this study is to investigate the specific mechanism of neutrophil inflammation in in vivo and in vitro NTHi-induced AECOPD models,and to clarify whether ginsenoside Rg3 could help protect against the disease by inhibiting neutrophil migration and thus modulating airway inflammation.?.NTHi-induced AECOPD and inflammatory response in miceMETHODS:8-week-old female BALB/C mice were randomly divided into 4groups?n=16?:normal control?NC?group,NTHi group,cigarette smoke?CS?group and co-infection?NTHi+CS?group.Among them,mice in CS and NTHi+CS groups were exposed to cigarette smoke nasally for 14 weeks,establishing murine COPD model,NC and NTHi groups mice were exposed to normal air.After 14 weeks,mice in NTHi and NTHi+CS groups were inoculated with NTHi via nasal dripping,establishing the murine AECOPD model,NC and CS groups mice were inoculated with equal amounts of saline.24h later,each mouse was anesthetized and fixed.Spirometer was used to detect lung function in mice;hematoxylin-eosin staining?HE?was used to observe structural changes in lung tissue;cell Diff staining and flow cytometry were used to analyze the number and proportion of neutrophils and macrophages in mice bronchoalveolar lavage fluid?BALF?;enzyme linked immunosorbent assay?ELISA?was used to detect expression of inflammatory cytokines in mice BALF.RESULTS:Compared with NC group,mice in NTHi group had decreased forced expiratory volume in 100 ms/forced vital capacity?FEV0.1/FVC?and increased neutrophil infiltration in airway.After 14 weeks of cigarette smoke exposure,mice in CS group showed significant weight loss,increased functional residual capacity?FRC?and airway resistance?RI?,decreased FEV0.1/FVC and dynamic compliance?Cydn?,lung tissue damage,increased mean linear alveolar intercept,increased infiltrated inflammatory cells and cytokines IL-6 and Keratinocyte-derived Cytokine?KC,homologous IL-8?in BALF.After inoculation of NTHi in smoke exposed mice,the above symptoms were exacerbated with decreased weight,lower Cydn and higher RI and mean alveolar intercept in comparison with CS group.Neutrophils and macrophages infiltration,and IL-6 and KC secretion were also significantly increased in BALF in NTHi+CS group.?.The role of neutrophils in NTHi-induced AECOPDMETHODS:Firstly,neutrophil transepithelial migration model was established through the co-culture of purified neutrophils and BEAS-2B cells.After the stimulation of NTHi and cigarette smoke on BEAS-2B,epithelial cells were divided into NC group,NTHi group,CS group and NTHi+CS group.IL-6 and IL-8 expression in cell supernatants were detected via ELISA.Diff staining was used to observe the number and morphology of migrated neutrophils.Then NTHi and cigarette smoke were used for neutrophils stimulation and neutrophils were divided into NC group,NTHi group,CS group and NTHi+CS group.Expression of lipid PtdIns?4,5?P2?PIP2?and PtdIns?3,4,5?P3?PIP3?in neutrophils were detected via ELISA,PI3K signaling pathway-related protein expressions were detected via Western blot,the expression and distribution of F-actin were detected by immunofluorescence.RESULTS:After stimulation on epithelial cells,the expressions of IL-6 and IL-8in supernatant were significantly elevated and the number of trans-epithelial migrated neutrophils was increased in the model groups,compared with NC group.The expression of IL-6 and IL-8 and the number of migrated neutrophils were significantly higher in NTHi+CS group than in CS groups.After stimulation on neutrophils,PI3K-related signaling was significantly activated,the phosphorylation levels of PIP3 and AKT proteins were significantly increased,F-actin was polymerized and located on the edge of neutrophils in model groups.Changes were most significant in NTHi+CS group.?.Protective effect of ginsenoside Rg3 on AECOPD mice1.Protective effect of ginsenoside Rg3 on AECOPD neutrophil inflammationMETHODS:8-week-old female BALB/C mice were randomly divided into 9groups?n=16?:NC group,CS group,CS exposure with Rg3 treatment?CS+10 mg/kg Rg3,CS+20 mg/kg Rg3 and CS+40 mg/kg Rg3?group,?NTHi+CS+10 mg/kg Rg3,NTHi+CS+20 mg/kg Rg3 and NTHi+CS+40 mg/kg Rg3?group.After 12 weeks of CS exposure,mice in Rg3 treated groups were administered with ginsenoside Rg3intragastrically daily for 2 weeks.NTHi drip inoculation was carried out in NTHi+CS and NTHi+CS+Rg3 groups on the 14^th week,24h before anaesthetization.Spirometer was used to detect the lung function,HE staining was used to observe changes in lung structure,flow cytometry was used to analyze the proportion of neutrophils in BALF and ELISA was used to detect the expression of IL-6 and KC.RESULTS:Compared with model groups,40 mg/kg Rg3 could significantly increase body weight,20 mg/kg and 40 mg/kg Rg3 could improve mice lung function,lung pathological damage and reduce inflammatory cells infiltration,neutrophils ratio and the expression of IL-6 and IL-8 in BALF of mice,which showed statistically significant differences compared with the CS and NTHi+CS groups.2.Inhibitory effect of ginsenoside Rg3 on neutrophil migrationMETHODS:Ginsenoside Rg3 was administered to neutrophils that were isolated and purified from human peripheral blood,cells were divided into six groups:NC group,fMLP-positive control?fMLP?group,fMLP+Rg3?10?M?group,fMLP+Rg3?20?M?group,fMLP+Rg3?40?M?group and inhibitor?LY294002?group.The effect ginsenoside Rg3 on neutrophil migration was first examined based on the neutrophil transepithelial migration model.Then fMLP,ginsenoside Rg3 and LY294002 were administered to neutrophils respectively.Contents of lipid PIP2 and PIP3 were detected by ELISA,expression of PI3K signaling pathway-related proteins were detected by Western blot,distribution and expression of F-actin in neutrophils were detected by immunofluorescence.RESULTS:Compared with blank group,trans-epithelial migrated neutrophils were significantly reduced in all Rg3 groups.After the administration fMLP,ginsenoside Rg3 and LY294002,PI3K-associated signaling pathway in fMLP group was significantly activated,with lower PIP2 and higher PIP3 expressions,increased PI3K and AKT protein phosphorylation levels and F-actin protein polarization than that of the NC group.Compared with fMLP group,PI3K signaling pathway was inhibited to some extent in all fMLP+Rg3 groups,in which PIP3 expression,PIP3 and AKT protein phosphorylation levels and F-actin protein polarization expression were significantly reduced in f MLP+Rg3?40?M?group,similar to the results in LY294002group.3.Metabolomic study of ginsenoside Rg3 in AECOPD miceMETHODS:Mice urine from NC,NTHi+CS and NTHi+CS+40 mg/kg groups were first collected and renamed into NC,AECOPD and Rg3 groups?n=6?.The samples were detected via ultra performance liquid chromatography combined with quadrupole time of flight mass spectrometry?UPLC-QTOF-MS?.Data acquisition and analysis were performed using the MassLynx V4.1 workstation.RESULTS:Compared with the NC group,many endogenous metabolites were significantly altered in the urine of AECOPD model mice.Ginsenoside Rg3significantly back-regulated the levels of 19 metabolites,including L-tryptophan,arachidonic acid,linoleic acid,leukotriene A4,riboflavin and cortisol,which inferred that ginsenoside Rg3 might participate in the regulation of linoleic acid metabolism,riboflavin metabolism,arachidonic acid metabolism,tryptophan metabolism,steroid hormones biosynthesis and sphingolipid metabolism pathways,playing a protective role in AECOPD.Conclusions:1.Our AECOPD model established by cigarette smoking and NTHi infection was characterized by the neutrophilic respiratory inflammation caused by redundant neutrophil migration..2.The mechanism of AECOPD neutrophilia might be the phosphorylation of AKT, thus the activation of PI3K signaling pathway.3.Ginsenoside Rg3 could reduce neutrophil migration by inhibiting the PI3K signaling pathway inside neutrophils,improving airway neutrophil infiltration and inflammation,thus having a protective effect against AECOPD.4.Ginsenoside Rg3 participated in the regulation of six metabolic pathways in AECOPD mice:linoleic acid metabolism,riboflavin metabolism,arachidonic acid metabolism,tryptophan metabolism,steroid hormone biosynthesis and sphingolipid metabolism.Innovations:1.Our study found that the activation of PI3K signaling pathway is the main mechanism of the airway neutrophilic inflammation in NTHi induced AECOPD models,suggesting that PI3K might be one of the therapeutic targets of AECOPD.2.We found that ginsenoside Rg3 could reduce the AECOPD neutrophilic inflammation by inhibiting the activation of PI3K signaling pathway inside neutrophil,suggesting that ginsenoside Rg3 had the potential to be developed as a clinical drug to prevent and treat AECOPD.