Exercise's protective role in chronic obstructive pulmonary disease via modulation of M1 macrophage phenotype through the miR-124-3p/ERN1 axis

Abstract

ObjectiveChronic obstructive pulmonary disease is a leading cause of global mortality. Although exercise training improves lung function and quality of life in chronic obstructive pulmonary disease patients, its mechanisms remain unclear. This study aims to reveal the effects of exercise on chronic obstructive pulmonary disease and elucidate the underlying molecular mechanisms involved.MethodsThis pre-post observational cohort study included 11 chronic obstructive pulmonary disease patients who underwent a 12-week exercise training program to evaluate the effects of exercise intervention. A cigarette smoke-induced chronic obstructive pulmonary disease mouse model and a cigarette smoke extract-induced bronchial endothelial cell model were utilized to investigate the protective mechanisms of exercise. Next-generation sequencing, bioinformatics analyses, luciferase reporter assays, and RT-qPCR were employed to assess mRNA and miRNA expression levels and correlation. Bioinformatics analysis and luciferase reporter assays were performed to identify direct downstream targets of miR-124-3p.ResultsExercise ameliorated lung inflammation in chronic obstructive pulmonary disease mouse lung tissue and human peripheral blood leucocytes. It inhibited M1 macrophage activation in response to cigarette smoke extract and led to the upregulation of miR-124-3p expression. Overexpression of miR-124-3p attenuated cigarette smoke extract-induced lung injury and inflammatory responses. Luciferase reporter assays identified endoplasmic reticulum to nucleus signaling 1 as a direct downstream target of miR-124-3p, and endoplasmic reticulum to nucleus signaling 1 overexpression reversed the anti-inflammatory effects mediated by miR-124-3p.ConclusionsExercise improved patient endurance and alleviated emphysema-related inflammation. It increased miR-124-3p expression, which inhibited the M1 macrophage phenotype and attenuated smoking-related lung injury by targeting endoplasmic reticulum to nucleus signaling 1.

Keywords: COPD; M1 macrophage; inflammation; miR-124-3p; pulmonary rehabilitation; smoking-related COPD.

Figure 1.

Changes of exercise endurance, circulation and metabolic function in patients with COPD after exercise training. (a–c) No significant improvement in pulmonary ventilation function before and after exercise. (d–f) A significant increase in exercise duration, endurance, and peak oxygen uptake in COPD patients after exercise. (g and h) A trend toward delayed anaerobic threshold in some patients. (i) A partial increase in oxygen uptake per stroke after exercise training. (j) Pre-exercise CT scan of a COPD patient, (k) post-exercise CT scan of the same patient. Panel j shows slightly reduced density of pulmonary vascular bronchial bundles, increased lung field radiolucency, and a slight decrease in localized cystic lucencies compared to Panel k, highlighting improvements in lung morphology and reduced inflammation after exercise.

Figure 2.

Exercise ameliorates COPD-induced lung injury by inhibiting inflammation and abnormal miRNA expression. (a–d) ELISA detection shows the expression of inflammatory factors IL-6, IL-17, TNF-α, and IL-1β. Data are expressed as means ± SD. (e) RT-qPCR detection shows the expression of miR-124-3p miRNA. Data are expressed as means ± SD. (d) Heat map detection shows that exercise results in abnormal expression of miRNAs, aPR means COPD patients after exercise, and bPR means COPD patients before exercise. (e) Gene ontology (GO) enrichment of different expressed miRNAs. Data are expressed as means ± SD.

Figure 3.

In vivo experimental detection shows that exercise ameliorates COPD-induced lung injury by inhibiting inflammation and regulating the macrophage phenotype. (a–c) ELISA detection shows the expression of inflammatory factors TNF-α, IL-6, and IL-1β. Data are expressed as means ± SD. (d) RT-qPCR detection shows the expression of miR-124-3p in lung tissues from control animals, in mice with COPD, and in exercised COPD mice. Data are expressed as means ± SD. (e and f) Immunofluorescence detection shows the macrophage phenotype as indicated by iNOS expression. Data are expressed as means ± SD. *** p < 0.001, ** p < 0.01 versus controls. ^### p < 0.001 versus COPD. Scale bar: 20 μm. (g and h) TUNEL staining shows apoptosis in mouse lung tissues. Data are expressed as means ± SD. ** p < 0.01 versus controls. ^## p < 0.01 versus COPD. Scale bar: 20 μm.

Figure 4.

ERN1 is the downstream target of miR-124-3p. (a) Bioinformatics analysis predicted binding sites in 3'-UTR-ERN1 for miR-124-3p. The mutant (Mut) version of 3'-UTR-ERN1 is shown. (b) Relative luciferase activity was determined 48 hours after transfection of RAW 264.7 cells with miR-124-3p mimic/NC or ERN1 wild-type/Mut. Data are expressed as means ± SD. ^*** p < 0.01. (c) Prediction of miR-124-3p binding sites in the 3'-UTR of mouse ERN1. The mutant version of 3'-UTR-ERN1 is shown. (d) Relative luciferase activity 48 hours after transfection of RAW cells with miR-124-3p mimic/NC or 3'-UTR-ERN1 wild-type/Mut. Data are expressed as means ± SD. ^*** p < 0.001. (e and f) RT-qPCR detection shows the expression of miR-124-3p and ERN1 in Raw264.7 cells. Data are expressed as means ± SD. ^*** p < 0.001 versus NC. ^### p < 0.001 versus mimic.

Figure 5.

Upregulation of ERN1 reverses the inhibitory effect of miR-124-3p on the M1 macrophage phenotype and inflammatory factor expression induced by CSE. (a–c) ELISA detection shows the expression of inflammatory factors TNF-α, IL-6, and IL-1β. Data are expressed as means ± SD. ^** p < 0.01, ^*** p < 0.001 versus CSE. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus CSE + mimic. (d) Western blot detection shows the expression of iNOS in Raw264.7 cells. Data are expressed as means ± SD. * p < 0.01 versus CSE. (e) RT-qPCR detection shows the expression of ERN1 in human blood leucocytes. Data are expressed as means ± SD. ^** p < 0.01, ^*** p < 0.001 versus NC. CSE represents cigarette smoke extract stimulation; CSE + mimic indicates cigarette smoke extract stimulation combined with miR-124-3p overexpression; CSE + mimic + ERN1 refers to cigarette smoke extract stimulation combined with both miR-124-3p and ERN1 overexpression; NC represents the healthy control group; COPD represents patients with chronic obstructive pulmonary disease; COPD + exercise indicates COPD patients after exercise training.