MiR-421 targeting the MPP7/EGFR/AKT axis to regulate macrophage M2 polarization and alleviating airway inflammation in asthmatic mice

doi:10.11958/20253182

Abstract

Abstract:

Objective To investigate the mechanism by which miR-421 affects macrophage M2 polarization of macrophages and improves airway inflammation in asthmatic mice. Methods BALB/c mice were randomly divided into the control group, the asthma group, the mimic NC group, the miR-421 mimic group, the miR-421 mimic + OE-NC group and the miR-421 mimic + OE-Membrane-associated Guanylate Kinase p55 Scaffold Protein 7 (MPP7) group, with 12 mice in each group. Except for the control group, all other groups were subjected to establish asthma model. After successful modeling, each intervention was administered once, and indicators were assessed after 7 days. The expression levels of serum-free miR-421 and MPP7 mRNA were detected by qRT-PCR. Airway resistance and lung compliance were measured using a dedicated system. Serum levels of immunoglobulin E (IgE), interleukin (IL)-4 and IL-13 were determined by enzyme-linked immunosorbent assay (ELISA). Lung tissue morphology was assessed by HE staining. The proportion of M2-type macrophages in lung tissue was analyzed by flow cytometry. Western blot assay was used to detect the protein levels of Arginase-1 (Arg-1), MPP7, epidermal growth factor receptor (EGFR) and p-protein kinase B (AKT)/AKT in lung tissue. The targeting relationship between miR-421 and MPP7 was verified by dual-luciferase reporter assay. Results Compared with the control group, mice of the asthma group exhibited significant pathological changes: thickened tracheal walls, fused adjacent alveoli forming cystic cavities and obvious inflammatory cell infiltration. Furthermore, the asthma group showed decreased expression of serum miR-421 and lung dynamic compliance, while serum MPP7 mRNA, inspiratory resistance, expiratory resistance, IgE, IL-4, IL-13, the proportion of M2-type macrophages in lung tissue, and the protein expression of Arg-1, MPP7, EGFR and p-AKT/AKT were significantly increased (P<0.05). Compared with the asthma group and the mimic NC group, changes in the aforementioned indicators showed the opposite trends in the miR-421 mimic group (P<0.05). OE-MPP7 reversed the improvement effect of overexpression of miR-421 on airway inflammation in asthmatic mice. Conclusion Overexpression of miR-421 may inhibit macrophage M2 polarization by suppressing the MPP7/EGFR/AKT axis, thereby alleviating airway inflammation in asthmatic mice.

Key words: asthma, microRNAs, guanylate kinases, ErbB receptors, proto-oncogene proteins c-akt, macrophages, M2 polarization, miR-421

CLC Number:

R562.25

Figures/Tables 8

References 20

[1]	TIAN C, LIU Q, ZHANG X, et al. Blocking group 2 innate lymphoid cell activation and macrophage M2 polarization:potential therapeutic mechanisms in ovalbumin-induced allergic asthma by calycosin[J]. BMC Pharmacol Toxicol, 2024, 25(1):30. doi:10.1186/s40360-024-00751-9.
[2]	ZHANG Y, YANG Y, LIANG H, et al. Nobiletin,as a novel PDE4B inhibitor,alleviates asthma symptoms by activating the cAMP-PKA-CREB signaling pathway[J]. Int J Mol Sci, 2024, 25(19):10406. doi:10.3390/ijms251910406.
[3]	BECERRA-DÍAZ M, LERNER A D, YU D H, et al. Sex differences in M2 polarization,chemokine and IL-4 receptors in monocytes and macrophages from asthmatics[J]. Cell Immunol, 2021, 360:104252. doi:10.1016/j.cellimm.2020.104252.
[4]	HOU C, YAN L, SUN K, et al. Nuclear receptor corepressor 1 deficiency exacerbates asthma by modulating macrophage polarization[J]. Cell Death Discov, 2023, 9(1):429. doi:10.1038/s41420-023-01724-3.
[5]	LIU H, SUN J, GAO L, et al. MicroRNA-421 attenuates macrophage-mediated inflammation by inhibiting PDCD4 in vitro[J]. Mol Med Rep, 2021, 24(1):527. doi:10.3892/mmr.2021.12166.
[6]	VERMA M, MICHALEC L, SRIPADA A, et al. The molecular and epigenetic mechanisms of innate lymphoid cell (ILC) memory and its relevance for asthma[J]. J Exp Med, 2021, 218(7):e20201354. doi:10.1084/jem.20201354.
[7]	LIAO W, FAN L, LI M, et al. MPP7 promotes the migration and invasion of breast cancer cells via EGFR/AKT signaling[J]. Cell Biol Int, 2021, 45(5):948-956. doi:10.1002/cbin.11538.
[8]	PEI L, LV X, JIA G, et al. CircSCNN1A is a tumor suppressor in renal cell carcinoma via inducing the upregulation of MPP7 by the sponge effect on miR-421[J]. Transpl Immunol, 2022, 75:101736. doi:10.1016/j.trim.2022.101736.
[9]	李雅琼, 黄静. 芦竹碱对哮喘小鼠气道炎症和重塑的保护作用及机制[J]. 中国药理学通报, 2025, 41(4):718-725.
	LI Y Q, HUANG J. Protective effect of gramine on airway inflammation and remodeling in asthmatic mice and its mechanism[J]. Chin Pharmacol Bull, 2025, 41(4):718-725. doi:10.12360/CPB202409088.
[10]	HUANG Q, LI Y, LI C, et al. Cigarette smoke aggravates asthma via altering airways inflammation phenotypes and remodelling[J]. Clin Respir J, 2023, 17(12):1316-1327. doi:10.1111/crj.13718.
[11]	WANG Z, REN Y, LI Y, et al. MiR-186-5p carried by M2 macrophage-derived exosomes downregulates TRPP2 expression in airway smooth muscle to alleviate asthma progression[J]. Int Immunopharmacol, 2025, 148:114107. doi:10.1016/j.intimp.2025.114107.
[12]	李俊杰. 呼出气一氧化氮、外周血嗜酸性粒细胞计数、免疫球蛋白E水平与哮喘患者肺功能指标水平的相关性[J]. 中国民康医学, 2025, 37(4):138-140.
	LI J J. Correlations among levels of exhaled nitric oxide,peripheral blood eosinophil count,immunoglobulin E and lung function index levels in patients with asthma[J]. Chin J Med Health, 2025, 37(4):138-140. doi:10.3969/j.issn.1672-0369.2025.04.041.
[13]	WU Y, NI Z, WANG S, et al. The mechanism of Sanzi Yangqin decoction for asthma treatment based on network pharmacology and experimental verification[J]. BMC Complement Med Ther, 2023, 23(1):452. doi:10.1186/s12906-023-04272-6.
[14]	JUNG C J, PARK S M, LEE D G, et al. Adenophora stricta root extract alleviates airway inflammation in mice with ovalbumin-induced allergic asthma[J]. Antioxidants (Basel), 2023, 12(4):922. doi:10.3390/antiox12040922.
[15]	HAN X, LIU L, HUANG S, et al. RNA m6A methylation modulates airway inflammation in allergic asthma via PTX3-dependent macrophage homeostasis[J]. Nat Commun, 2023, 14(1):7328. doi:10.1038/s41467-023-43219-w.
[16]	ZHU X, HE L, LI X, et al. LncRNA AK089514/miR-125b-5p/TRAF6 axis mediates macrophage polarization in allergic asthma[J]. BMC Pulm Med, 2023, 23(1):45. doi:10.1186/s12890-023-02339-1.
[17]	高风丽, 孙丽丽, 田春燕, 等. 平喘颗粒对卵清蛋白致敏诱导的哮喘小鼠巨噬细胞M2极化及补体因子D的影响[J]. 时珍国医国药, 2024, 35(13):2912-2918.
	GAO F L, SUN L L, TIAN C Y, et al. Effects of pingchuan granules on M2 polarization,complement factor D expression and air-way remodeling of macrophages in asthmatic mice[J]. Lishizhen Med Mater Med Res, 2024, 35(13):2912-2918. doi:10.3969/j.issn.1008-0805.2024.13.02.
[18]	YANG L, LIU S, HE Y, et al. Exosomes regulate SIRT3-related autophagy by delivering miR-421 to regulate macrophage polarization and participate in OSA-related NAFLD[J]. J Transl Med, 2024, 22(1):475. doi:10.1186/s12967-024-05283-8.
[19]	LIN Y J, YANG C C, LEE I T, et al. Reactive oxygen species-dependent activation of EGFR/Akt/p38 mitogen-activated protein kinase and JNK1/2/FoxO1 and AP-1 pathways in human pulmonary alveolar epithelial cells leads to up-regulation of COX-2/PGE2 induced by silica nanoparticles[J]. Biomedicines, 2023, 11(10):2628. doi:10.3390/biomedicines11102628.
[20]	WANG J, ZHU M, WANG L, et al. Amphiregulin potentiates airway inflammation and mucus hypersecretion induced by urban particulate matter via the EGFR-PI3Kα-AKT/ERK pathway[J]. Cell Signal, 2019, 53:122-131. doi:10.1016/j.cellsig.2018.10.002.

组别	miR-421	MPP7 mRNA
对照组	1.00±0.00	1.00±0.00
哮喘组	0.23±0.03^a	2.23±0.26^a
mimic NC组	0.25±0.02	2.25±0.21
miR-421 mimic组	0.89±0.08^bc	1.51±0.16^bc
miR-421 mimic+OE-NC组	0.91±0.09	1.53±0.18
miR-421 mimic+OE-MPP7组	0.90±0.08	1.91±0.19^de
F	410.900^＊＊	82.120^＊＊

组别	miR-421	MPP7 mRNA
对照组	1.00±0.00	1.00±0.00
哮喘组	0.23±0.03^a	2.23±0.26^a
mimic NC组	0.25±0.02	2.25±0.21
miR-421 mimic组	0.89±0.08^bc	1.51±0.16^bc
miR-421 mimic+OE-NC组	0.91±0.09	1.53±0.18
miR-421 mimic+OE-MPP7组	0.90±0.08	1.91±0.19^de
F	410.900^＊＊	82.120^＊＊

组别	吸气阻力/[cmH₂O/（s·mL）]	肺通气顺应性/（cmH₂O/mL）	呼气阻力/[cmH₂O/（s·mL）]
对照组	1.12±0.10	0.28±0.02	1.41±0.10
哮喘组	3.29±0.13^a	0.04±0.01^a	4.28±0.21^a
mimic NC组	3.31±0.12	0.05±0.01	4.30±0.22
miR-421 mimic组	1.46±0.11^bc	0.19±0.02^bc	2.05±0.12^bc
miR-421 mimic+OE-NC组	1.48±0.12	0.20±0.02	2.08±0.14
miR-421 mimic+OE-MPP7组	2.09±0.14^de	0.14±0.02^de	3.15±0.12^de
F	763.000^＊＊	345.600^＊＊	719.400^＊＊

组别	吸气阻力/[cmH₂O/（s·mL）]	肺通气顺应性/（cmH₂O/mL）	呼气阻力/[cmH₂O/（s·mL）]
对照组	1.12±0.10	0.28±0.02	1.41±0.10
哮喘组	3.29±0.13^a	0.04±0.01^a	4.28±0.21^a
mimic NC组	3.31±0.12	0.05±0.01	4.30±0.22
miR-421 mimic组	1.46±0.11^bc	0.19±0.02^bc	2.05±0.12^bc
miR-421 mimic+OE-NC组	1.48±0.12	0.20±0.02	2.08±0.14
miR-421 mimic+OE-MPP7组	2.09±0.14^de	0.14±0.02^de	3.15±0.12^de
F	763.000^＊＊	345.600^＊＊	719.400^＊＊

组别	IgE/（ng/L）	IL-4/（ng/L）
对照组	2.06±0.11	13.67±0.72
哮喘组	8.54±0.43^a	45.56±2.93^a
mimic NC组	8.59±0.44	45.51±2.88
miR-421 mimic组	3.57±0.19^bc	20.18±1.09^bc
miR-421 mimic+OE-NC组	3.59±0.22	20.21±1.07
miR-421 mimic+OE-MPP7组	5.69±0.31^de	30.67±1.68^de
F	954.900^＊＊	599.600^＊＊
组别	IL-13/ （ng/L）	M2型巨噬细胞占比/%
对照组	6.15±0.34	6.13±0.35
哮喘组	30.23±1.78^a	21.15±1.08^a
mimic NC组	29.98±1.63	21.11±1.06
miR-421 mimic组	8.89±0.51^bc	9.23±0.50^bc
miR-421 mimic+OE-NC组	8.92±0.53	9.25±0.51
miR-421 mimic+OE-MPP7组	15.58±0.83^de	16.63±0.87^de
F	1 192.000^＊＊	422.300^＊＊