The effect and mechanism of exosomes from umbilical cord mesenchymal stem cells on pulmonary inflammation in chronic obstructive pulmonary disease rats

doi:10.11958/20230708

Abstract

Abstract:

Objective To investigate the effect and mechanism of human umbilical cord mesenchymal stem cell exosomes (Exo) on pulmonary inflammation in chronic obstructive pulmonary disease (COPD) rats. Methods A total of 18 male SD rats were randomly divided into the control group, the COPD group and the COPD + Exo group with 6 rats in each group. COPD rat model was estalished by giving cigarette smoke and lipopolysaccharide (LPS) inhalation in the COPD group and the COPD + Exo group. Rats in the COPD + Exo group were injected with 100 μL of human umbilical mesenchymal stem cell exosome diluent (containing 2 × 10⁷ exosomes) via tail vein. The control group and the COPD group were given equal amounts of phosphate buffered solution. One week after intervention, rats were killed and the peripheral blood, alveolar lavage fluid (BALF) and lung tissue samples were collected. The histopathological changes in lung tissue of each group were observed using Hematoxylin and Eosin (HE) staining. The number of inflammatory cells in peripheral blood was counted by fully automated hematology analyzer. The serum levels of IL-1β, IL-6 and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of IL-1β, IL-6 and TNF-α mRNA in lung tissue were detected by qRT-PCR. Western blot assay was used to detect the protein expression levels of TLR4, p-NF-κB p65 and IκBα in lung tissue. Results In the control group, the structure of pulmonary alveolus was intact and regular, while in the COPD group, the structure of alveolus was disordered, some alveolus expanded irregularly, fused into bullae and even ruptured. The alveolar septa were widened and filled with a large number of inflammatory cells. Compared with the COPD group, the COPD + Exo group had more intact alveolar structure and less infiltration of inflammatory cells in the alveolar septum. The inflammatory cells in peripheral blood decreased. The expression levels of IL-1β, IL-6 and TNF-α in serum and lung tissue were decreased (P < 0.05). The expression of TLR4 and IκBα protein in lung tissue were increased, while the expression of p-NF-κb p65 protein decreased (P < 0.05). Conclusion Human umbilical cord mesenchymal stem cell exosomes can alleviate pulmonary inflammation in COPD rats by modulating TLR4/NF-κB signaling pathway, thereby playing a lung-protective effect.

Key words: pulmonary disease, chronic obstructive, mesenchymal stem cells, exosomes, inflammation, Toll-like receptor 4, NF-kappa B

CLC Number:

R563

Figures/Tables 8

References 37

[1]	CHRISTENSON S A, SMITH B M, BAFADHEL M, et al. Chronic obstructive pulmonary disease[J]. Lancet, 2022, 399(10342):2227-2242. doi:10.1016/S0140-6736(22)00470-6.
[2]	SONG Q, CHEN P, LIU X M. The role of cigarette smoke-induced pulmonary vascular endothelial cell apoptosis in COPD[J]. Respir Res, 2021, 22(1):39. doi:10.1186/s12931-021-01630-1.
[3]	中华医学会呼吸病学分会慢性阻塞性肺疾病学组, 中国医师协会呼吸医师分会慢性阻塞性肺疾病工作委员会, 陈荣昌, 等. 慢性阻塞性肺疾病诊治指南(2021年修订版)[J]. 中华结核和呼吸杂志, 2021, 44(3):170-205.
	Group of Chronic Obstructive Pulmonary Disease, Respiratory Branch of Chinese Medical Association, Working Committee of Chronic Obstructive Pulmonary Disease, Respiratory Branch of Chinese Medical Association, CHEN R C, et al. Guidelines for the diagnosis and treatment of chronic obstructive pulmonary disease (2021 revision)[J]. Chinese Journal of Tuberculosis and Respiration, 2021, 44(3):170-205. doi:10.3760/cma.j.cn112147-20210109-00031.
[4]	LIU J, GAO J, LIANG Z, et al. Mesenchymal stem cells and their microenvironment[J]. Stem Cell Res Ther, 2022, 13(1):429. doi:10.1186/s13287-022-02985-y.
[5]	ZOU J X, YANG W N, CUI W S, et al. Therapeutic potential and mechanisms of mesenchymal stem cell-derived exosomes as bioactive materials in tendon-bone healing[J]. J Nanobiotechnology, 2023, 21(1):14. doi:10.1186/s12951-023-01778-6.
[6]	MOHAN A, AGARWAL S, CLAUSS M, et al. Extracellular vesicles:novel communicators in lung diseases[J]. Respir Res, 2020, 21(1):175. doi:10.1186/s12931-020-01423-y.
[7]	HARRELL C R, JOVICIC N, DJONOV V, et al. Mesenchymal stem cell-derived exosomes and other extracellular vesicles as new remedies in the therapy of inflammatory diseases[J]. Cells, 2019, 8(12):1605. doi:10.3390/cells8121605.
[8]	SHEN Z W, HUANG W, LIU J, et al. Effects of mesenchymal stem cell-derived exosomes on autoimmune diseases[J]. Front Immunol, 2021, 12:749192. doi:10.3389/fimmu.2021.749192.
[9]	LIN Z J, WU Y L, XU Y T, et al. Mesenchymal stem cell-derived exosomes in cancer therapy resistance:recent advances and therapeutic potential[J]. Mol Cancer, 2022, 21(1):179. doi:10.1186/s12943-022-01650-5.
[10]	ZHOU L, LUO H, LEE J W. Role of extracellular vesicles in lung diseases[J]. Chin Med J, 2022, 135(15):1765-1780. doi:10.1097/CM9.0000000000002118.
[11]	ABBASZADEH H, GHORBANI F, ABBASPOUR-AGHDAM S, et al. Chronic obstructive pulmonary disease and asthma:mesenchymal stem cells and their extracellular vesicles as potential therapeutic tools[J]. Stem Cell Res Ther, 2022, 13(1):262. doi:10.1186/s13287-022-02938-5.
[12]	SHU J Z, LI D F, OUYANG H P, et al. Comparison and evaluation of two different methods to establish the cigarette smoke exposure mouse model of COPD[J]. Sci Rep, 2017, 7(1):15454. doi:10.1038/s41598-017-15685-y.
[13]	KORSGREN M, LINDEN M, ENTWISTLE N, et al. Inhalation of LPS induces inflammatory airway responses mimicking characteristics of chronic obstructive pulmonary disease[J]. Clin Physiol Funct Imaging, 2012, 32(1):71-79. doi:10.1111/j.1475-097X.2011.01058.x.
[14]	SHI M M, YANG Q Y, MONSEL A, et al. Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles[J]. J Extracell Vesicles, 2021, 10(10):e12134. doi:10.1002/jev2.12134.
[15]	CHURG A, COSIO M, WRIGHT J L. Mechanisms of cigarette smoke-induced COPD:insights from animal models[J]. Am J Physiol Lung Cell Mol Physiol, 2008, 294(4):L612-L631. doi:10.1152/ajplung.00390.2007.
[16]	AGHAPOUR M, RAEE P, MOGHADDAM S J, et al. Airway epithelial barrier dysfunction in chronic obstructive pulmonary disease:role of cigarette smoke exposure[J]. Am J Respir Cell Mol Biol, 2018, 58(2):157-169. doi:10.1165/rcmb.2017-0200TR.
[17]	JONES B, DONOVAN C, LIU G, et al. Animal models of COPD:what do they tell us?[J]. Respirology, 2017, 22(1):21-32. doi:10.1111/resp.12908.
[18]	LIANG G B, HE Z H. Animal models of emphysema[J]. Chin Med J(Engl), 2019, 132(20):2465-2475. doi:10.1097/CM9.0000000000000469.
[19]	SMITH K R, LEONARD D, MCDONALD J D, et al. Inflammation,mucous cell metaplasia,and Bcl-2 expression in response to inhaled lipopolysaccharide aerosol and effect of rolipram[J]. Toxicol Appl Pharmacol, 2011, 253(3):253-260. doi:10.1016/j.taap.2011.04.001.
[20]	DE SOUZA XAVIER COSTA N, RIBEIRO JÚNIOR G,DOS SANTOS ALEMANY A A, et al. Early and late pulmonary effects of nebulized LPS in mice:An acute lung injury model[J]. PLoS One, 2017, 12(9):e0185474. doi:10.1371/journal.pone.0185474.
[21]	FONCECA A M, ZOSKY G R, BOZANICH E M, et al. Accumulation mode particles and LPS exposure induce TLR-4 dependent and independent inflammatory responses in the lung[J]. Respir Res, 2018, 19(1):15. doi:10.1186/s12931-017-0701-z.
[22]	何永鸿, 强丽, 王宋平. 阿托伐他汀钙对COPD模型大鼠肺血管重塑的影响及机制探讨[J]. 天津医药, 2021, 49(6):598-602.
	HE Y H, QIANG L, WANG S P. The effect of atorvastatin calcium on pulmonary vascular remodeling in chronic obstructive pulmonary disease rats[J]. Tianjin Med J, 2021, 49(6):598-602. doi:10.11958/202013011.
[23]	ZHU W T, LI C H, DAI T T, et al. Effect of allyl isothiocyanate on oxidative stress in COPD via the AhR/CYP1A1 and Nrf2/NQO1 pathways and the underlying mechanism[J]. Phytomedicine, 2023, 114:154774. doi:10.1016/j.phymed.2023.154774.
[24]	JU J, LI Z, SHI Q. Baicalin inhibits inflammation in rats with chronic obstructive pulmonary disease by the TLR2/MYD88/NF-κBp65 signaling pathway[J]. Evid Based Complement Alternat Med, 2022, 2022:7273387. doi:10.1155/2022/7273387.
[25]	ARMITAGE J, TAN D, MOODLEY Y, et al. Mesenchymal stem cell infusion modulates systemic inflammation in patients with chronic obstructive pulmonary disease(COPD)[J]. Respirology, 2016, 21(Suppl 2):133.
[26]	GAO J, LIANG Y, CHEN J, et al. CXCR4 enhances the inhibitory effects of bone mesenchymal stem cells on lung cell apoptosis in a rat model of smoking-induced COPD[J]. Apoptosis, 2023, 28(3/4):639-652. doi:10.1007/s10495-022-01800-6.
[27]	WEISS D J, SEGAL K, CASABURI R, et al. Effect of mesenchymal stromal cell infusions on lung function in COPD patients with high CRP levels[J]. Respir Res, 2021, 22(1):142. doi:10.1186/s12931-021-01734-8.
[28]	ALVARENGA-NASCIMENTO C R, AADB LEIA, SANTOS T G, et al. Immunotherapeutic strategy with mesenchymal stem cells modulating inflammation in an experimental model of COPD[J]. European Respiratory Journal, 2020, 56(Suppl 64):314.
[29]	VOLAREVIC V, MARKOVIC B S, GAZDIC M, et al. Ethical and safety issues of stem cell-based therapy[J]. Int J Med Sci, 2018, 15(1):36-45. doi:10.7150/ijms.21666.
[30]	TANG Y Y, ZHOU Y, LI H J. Advances in mesenchymal stem cell exosomes:a review[J]. Stem Cell Res Ther, 2021, 12(1):71. doi:10.1186/s13287-021-02138-7.
[31]	CHAN A M L, SAMPASIVAM Y, LOKANATHAN Y. Biodistribution of mesenchymal stem cells (MSCs) in animal models and implied role of exosomes following systemic delivery of MSCs:a systematic review[J]. Am J Transl Res, 2022, 14(4):2147-2161.
[32]	XIA L J, ZHANG C L, LV N Y, et al. AdMSC-derived exosomes alleviate acute lung injury via transferring mitochondrial component to improve homeostasis of alveolar macrophages[J]. Theranostics, 2022, 12(6):2928-2947. doi:10.7150/thno.69533.
[33]	WAN X, CHEN S, FANG Y, et al. Mesenchymal stem cell-derived extracellular vesicles suppress the fibroblast proliferation by downregulating FZD6 expression in fibroblasts via micrRNA-29b-3p in idiopathic pulmonary fibrosis[J]. J Cell Physiol, 2020, 235(11):8613-8625. doi:10.1002/jcp.29706.
[34]	CHEN Q, LIN J, DENG Z Q, et al. Exosomes derived from human umbilical cord mesenchymal stem cells protect against papain-induced emphysema by preventing apoptosis through activating VEGF-VEGFR2-mediated AKT and MEK/ERK pathways in rats[J]. Regen Ther, 2022, 21:216-224. doi:10.1016/j.reth.2022.07.002.
[35]	YANG L, WEN M, LIU X, et al. Feikang granules ameliorate pulmonary inflammation in the rat model of chronic obstructive pulmonary disease via TLR2/4-mediated NF-κB pathway[J]. BMC Complement Med Ther, 2020, 20(1):170. doi:10.1186/s12906-020-02964-x.
[36]	ROY A, SRIVASTAVA M, SAQIB U, et al. Potential therapeutic targets for inflammation in toll-like receptor 4 (TLR4)-mediated signaling pathways[J]. Int Immunopharmacol, 2016, 40:79-89. doi:10.1016/j.intimp.2016.08.026.
[37]	LI Y, ZHAO J, SHAO H, et al. Preventive effect of total flavonoids of Trollius altaicus on a chronic obstructive pulmonary disease rat model based on the TLR4/NF-κB pathway[J]. Ann Transl Med, 2022, 10(4):222. doi:10.21037/atm-22-331.

基因名称	引物（5′→3′）	产物大小/bp
IL-1β	上游：GCACAGTTCCCCAACTGGTA	109
	下游：ACACGGGTTCCATGGTGAAG	109
IL-6	上游：CTCTCCGCAAGAGACTTCCA	92
	下游：TCTCCTCTCCGGACTTGTGAA	92
TNF-α	上游：CCACGCTCTTCTGTCTACTG	145
	下游：GCTACGGGCTTGTCACTC	145
GAPDH	上游：CAGGAGGCATTGCTGATGAT	138
	下游：GAAGGCTGGGGCTCATTT	138

基因名称	引物（5′→3′）	产物大小/bp
IL-1β	上游：GCACAGTTCCCCAACTGGTA	109
	下游：ACACGGGTTCCATGGTGAAG	109
IL-6	上游：CTCTCCGCAAGAGACTTCCA	92
	下游：TCTCCTCTCCGGACTTGTGAA	92
TNF-α	上游：CCACGCTCTTCTGTCTACTG	145
	下游：GCTACGGGCTTGTCACTC	145
GAPDH	上游：CAGGAGGCATTGCTGATGAT	138
	下游：GAAGGCTGGGGCTCATTT	138

组别	WBC	NEU	MON	LYM
Control组	0.48±0.07	0.77±0.24	0.03±0.00	0.25±0.00
COPD组	3.10±0.27^a	2.01±0.20^a	0.15±0.01^a	2.63±0.19^a
COPD+Exo组	1.59±0.55^ab	0.35±0.15^b	0.06±0.02^b	1.20±0.47^ab
F	27.197^＊	39.738^＊	51.100^＊	33.116^＊

组别	WBC	NEU	MON	LYM
Control组	0.48±0.07	0.77±0.24	0.03±0.00	0.25±0.00
COPD组	3.10±0.27^a	2.01±0.20^a	0.15±0.01^a	2.63±0.19^a
COPD+Exo组	1.59±0.55^ab	0.35±0.15^b	0.06±0.02^b	1.20±0.47^ab
F	27.197^＊	39.738^＊	51.100^＊	33.116^＊

组别	BALF
组别	IL-1β		IL-6		TNF-α
Control组	55.05±11.49		71.58±8.77		290.51±28.28
COPD组	85.23±13.44^a		104.16±7.92^a		513.71±59.00^a
COPD+Exo组	61.02±6.42^b		84.66±12.84^b		406.74±52.32^ab
F	10.827^＊		13.234^＊		26.634^＊
组别		血清
组别		IL-1β		IL-6		TNF-α
Control组	64.02±6.84		72.00±4.45		309.18±5.25
COPD组	88.54±8.44^a		90.99±0.97^a		483.74±9.74^a
COPD+Exo组	73.22±4.92^b		73.41±7.93^b		364.64±2.40^b
F	9.708^＊		8.036^＊		11.869^＊