Molecular Mechanism And Treatment Of Influenza-induced Exacerbations Of Chronic Obstructive Pulmonary Disease

BackgroundChronic obstructive pulmonary disease(COPD)is the most common Chronic respiratory disease worldwide,characterized by persistent respiratory symptoms and airflow limitations.The progressive development of airflow restriction and the decline of lung function seriously impair the patient’s labor force and quality of life,resulting in a huge social,economic and medical burden.Acute exacerbation of chronic obstructive pulmonary disease(AECOPD)is an important event in the course of patients with chronic obstructive pulmonary disease,and it is an important cause of repeated hospitalization and death of patients.The most common predisposing factor for acute exacerbation is respiratory infections,especially respiratory viral infections The previous research results of our research group and related literature suggest that influenza A virus(IAV)(seasonal influenza virus subtype H3N2)is the most common inducing factor in the acute exacerbation of COPD.Therefore,it is important to fully understand the molecular mechanism of how IAV induces acute exacerbation of COPD and how to participate in the pathogenesis of acute exacerbation.Oseltamivir phosphate,a neuraminidase inhibitor against IAV,is currently commonly used in clinical practice However,drug-resistant strains have continued to appear,and the therapeutic effect has declined in recent years.Therefore,it is an urgent need for clinicians to develop a new target drug of IAV or combine with other antiviral drugs.Shufeng Jiedu Capsule(SFJDC)is a traditional Chinese patent medicine with broad-spectrum antibacterial and antiviral effects,can regulate the immune reaction to infection.It has little side effects in antiviral effects,no restricted treatment window and non-resistant strains.Combining with Oseltamivir as a novel antiviral treatment were included in our study.Therefore,the purpose of this research is to investigate the molecular mechanism of airway inflammation activated by IAV/H3N2 in patients with acute exacerbation of COPD and to evaluate the treatment effect of SFJDC combined with Oseltamivir phosphateMethodsThe research methods are mainly divided into four parts:(1)Human bronchial epithelial cells(HBEC)were isolated and cultured from normal(NHBE)and COPD(DHBE)bronchial tissues and co-cultured with IAV,the activation of NLRP3 inflammasome was involved in this process,as determined by RNA sequencing,and then with siRNA or MCC950(a specific small molecule inhibitor of NLPR3);confirmed at the cellular level that IAV stimulates bronchial epithelial cells in COPD patients with the activation of NLRP3 inflammation signaling pathway.(2)Rats were randomly picked out for COPD modeling with exposure to cigarette smoke.Non-smoking control rats were exposed to air for the same period.Arterial blood gas analysis and pulmonary function tests were performed immediately after the last exposure.On the last day of exposure,COPD rats were anesthetized with thiopental sodium and infected intranasally with 2.5×103 plaque forming units(PFUs)of IAV/H3N2 in 100μl.Control was sham-inoculated with saline and recorded as day 0.We recorded the body weight every day and observed their behavioral changes.MCC950 in 3%DMSO vehicle was administered intranasally in COPD rats at a dose of 10 mg/kg daily beginning on day 1 post-infection until tissue harvest,death or recovery.On the 7th day after infection,all experimental rats were euthanized,serum was collected from the rats,and the right lung tissues of the rats were lavaged to collect bronchoalveolar lavage fluid(BALF)specimens;the left upper lung tissues were dehydrated paraffin sections were fixed and HE stained to observe the degree of lung tissue damage.The left lower lung was homogenized and the mRNA and protein levels of the NLRP3 signaling pathway were detected by qRT-PCR and Western blotting.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of IL-1β and IL-18 in serum and BALF of rats.(3)Collect the serum and BALF of patients with acute exacerbation of COPD infected with IAV,and compare the expression levels of IL-1β and IL-18 with healthy controls collected at the same time.It was further confirmed that COPD patients infected with IAV activated the NLRP3 signaling pathway.(4)UHPLC/ESI Q-Orbitrap mass spectrometry was used to analyze the drug composition of SFJDC;DHBE cells were co-cultured with IAV for 24h,and then given SFJDC and/or Oseltamivir;appropriate drug concentration was determined by MTT assay;establish COPD rat models of IAV infected were treated with SFJDC and/or Oseltamivir,and the behavioral changes in rats were observed.Real-time quantitative PCR and Western blotting were used to detect changes in mRNA and protein levels of the NLRP3 inflammation pathway.The ELISA was used to measure the levels of IL-1β and IL-18 in serum and BALF to evaluate the treatment effect of SFJDC and/or OseltamivirResultsThe research results are mainly divided into four parts:(1)Primary bronchial epithelial cells of normal control and COPD patients were successfully isolated,purified,identified,and cultured.The cells were observed to be cobblestone-like with an inverted light microscope,and the characteristic cells of epithelial cells were abundantly expressed Cytokeratin 17/19;transcriptome high-throughput sequencing of primary bronchial epithelial cells and IAV co-cultured for 24h showed that the NLRP3 inflammation signaling pathway in DHBE cells was significantly higher in mRNA expression levels than NHBE;Western blotting analysis showed that the levels of NLRP3 inflammation signaling pathway increased after co-culturing IAV in DHBE for 3h.After co-culturing in NHBE for 6h,the change in expression levels can be observed However,both cells reached the peak level of NLRP3 at 12h after inoculation and maintained to 24h.ASC,Caspase-1,IL-1β and IL-18 were also elevated in a similar time-dependent pattern in these cells.Both the mRNA and protein expression of the NLRP3 inflammasome were decreased when DHBE were treated with NLRP3 siRNA and MCC950.(2)In experimental COPD rats,the NLRP3 inflammasome was elevated by IAV Treating these animals with MCC950 alleviated lung damage,improved their survival and reduced NLRP3 inflammasome expression as well as the levels of IL-1β,IL-18 in BALF.(3)Nasopharyngeal,serum and BALF from 37 AECOPD patients were collected.19 of 37 cases are IAV-negative and 18 of 37 cases are IAV-positive.The FEVi/FVC ratio declined with the severity of obstruction in the positive group compared with control.The expression levels of IL-1β and IL-18 in both serum and BALF supernatants were significantly higher in IAV-positive group than negative patients.(4)SFJDC and/or Oseltamivir have no obvious cytotoxicity in the appropriate drug concentration range.The results of in cell experiments suggest that after inoculation with IAV and bronchial epithelial cells for 24h,the expression levels of mRNA and protein associated with NLRP3 inflammation significantly increased,and mRNA of these components was treated with SFJDC and/or Oseltamivir in vitro,protein levels were significantly reduced.In vivo experiments have shown that after influx of influenza virus in the nasal cavity of COPD rats,SFJDC combined with Oseltamivir can improve the survival rate of rats,reduce clinical symptoms,increase rat weight,reduce lung injury,reduce IL-1β and IL-18 levels in serum and BALF,and significantly reduced the NLRP3 inflammatory body-related components expression and virus titersConclusionIAV activates the NLRP3 inflammation signaling pathway in the bronchial epithelial cells of AECOPD patients through in vitro and in vivo pathway,inducing an earlier and stronger airway inflammatory response,reasonably explaining why influenza viruses can cause more serious clinical cases and rapid disease development MCC950 can specifically inhibit the NLRP3 inflammation signaling pathway,significantly improve the survival rate of rats and inhibit airway inflammation,which may provide strong evidence for finding new influenza treatments in patients with AECOPD.At the same time,the combination of SFJDC and Oseltamivir may represent a new treatment strategy that can improve the treatment efficiency and quality of life of patients with IAV-induced chronic airway disease,especially those with AECOPD.