基于Nrf2/MARCO信号通路探讨六味补气方调控巨噬细胞胞葬治疗肺肾气虚证COPD的作用机制OA

Objective:To investigate the mechanisms by which Liuwei Buqi prescription(LWBQ)regulates alveolar macrophage efferocytosis and improves inflammatory responses in rats with chronic obstructive pulmonary disease(COPD)characterized by lung-kidney Qi deficiency based on the nuclear factor erythroid 2-related factor 2(Nrf2)/macrophage receptor with collagenous structure(MARCO)pathway.Methods:Successfully modeled rats were randomly divided into a model group,low-dose LWBQ group(LWBQ-L,2.25 g·kg-1·d-1),medium-dose LWBQ group(LWBQ-M,4.5 g·kg-1·d-1),high-dose LWBQ group(LWBQ-H,9 g·kg-1·d-1),and aminophylline group(AMIN,50 mg·kg-1·d-1),with 8 rats in each group.Another 8 healthy rats were included as the blank group.Except for the blank group,rats in the remaining groups were subjected to smoke exposure combined with forced swimming,intratracheal lipopolysaccharide(LPS)instillation,and subcutaneous hydrocortisone injection to establish a COPD model with lung-kidney Qi deficiency.After successful modeling,rats were administered different doses of LWBQ or AMIN by gavage.Body weight,fur condition,and oral secretions were observed.Pulmonary function was measured using an animal lung function analyzer.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of interferon-γ(IFN-γ),interleukin-6(IL-6),interleukin-1(IL-1),and tumor necrosis factor-α(TNF-α)in bronchoalveolar lavage fluid(BALF)and serum(SER).Hematoxylin-eosin(HE)staining was used to examine pathological changes in lung tissue.Giemsa staining was performed to detect eosinophils,basophils,and neutrophils in BALF.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)was used to detect apoptosis in lung tissue.Western blot and real-time polymerase chain reaction(Real-time PCR)were employed to determine the protein and mRNA expression levels of efferocytosis-related proteins growth arrest-specific gene 6(GAS6),milk fat globule-epidermal growth factor 8(MFG-E8),and pathway-related proteins Nrf2 and MARCO in lung tissue.Results:Compared with the blank group,the model group showed reduced food intake,nasal and oral secretions with sputum,and decreased body weight(P＜0.01),decreased peak expiratory flow(PEF)(P＜0.01),increased forced vital capacity(FVC)(P＜0.01),and decreased forced expiratory volume in 0.3 s/forced vital capacity[FEV0.3/FVC(％)](P＜0.01).The expression levels ofIFN-γ,IL-6,IL-1,and TNF-α in BALF and SER were increased(P＜0.01).Lung tissue exhibited structural destruction,hyperplasia,inflammatory exudation,increased apoptotic cells,and increased mean optical density(P＜0.01).The protein and mRNA expression levels of GAS6,MFG-E8,and MARCO,as well as Nrf2 mRNA expression,were increased(P＜0.01).Compared with the model group,the LWBQ groups showed increased food intake,reduced nasal and oral secretions with sputum,and increased body weight(P＜0.05,P＜0.01).PEF was increased(P＜0.01).FVC was increased in rats treated with low-and medium-dose LWBQ(P＜0.01),and FEV0.3/FVC(％)was increased in rats treated with medium-and high-dose LWBQ(P＜0.05,P＜0.01).The expression levels of IFN-γ,IL-6,IL-1,and TNF-α in BALF and SER were decreased(P＜0.01).Lung tissue structure was relatively intact,with improvement in hyperplasia and inflammatory exudation.The number of apoptotic cells in lung tissue was reduced,and mean optical density was decreased(P＜0.05,P＜0.01).The protein and mRNA expression levels of efferocytosis-related proteins GAS6 and MFG-E8 and pathway-related proteins Nrf2 and MARCO were increased(P＜0.01).Conclusion:LWBQ can alleviate pulmonary and systemic inflammation,improve lung function,and reduce lung tissue damage in rats with COPD characterized by lung-kidney Qi deficiency.The mechanism may be related to enhancement of alveolar macrophage efferocytosis through regulation of the Nrf2/MARCO pathway.