Impacts of miR-141-3p on pulmonary fibrosis in rats with acute respiratory distress syndrome by regulating Keap1-NRF2/ARE signaling pathway

doi:10.11958/20230636

Abstract

Abstract:

Objective To investigate the effect and mechanism of miR-141-3p on pulmonary fibrosis in rats with acute respiratory distress syndrome (ARDS). Methods Rats were divided into the control group, the model group, the agomir negative control group and the miR-141-3p agomir group according to random number table, with 10 rats in each group. In addition to the control group, the ARDS rat model was established by lipopolysaccharide (LPS) infusion. Rat alveolar type Ⅱ epithelial cells RLE-6TN cells were divided into the NC group, the LPS group, the miR-NC group, the miR-141-3p mimics group, the miR-141-3p mimics+pcDNA group and the miR-141-3p mimics+NRF2 and Kelch-like ring associated protein 1 (Keap1) group. LPS cell model was established in all groups except the NC group. The mRNA expression levels of miR-141-3p and Keap1 in lung tissue and cells were detected by qPCR. Western blot assay was used to analyze lung tissue and cell epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), microtubule associated protein light chain 3B (LC3B), autophagy associated gene Beclin-1, α-smooth muscle actin (α-SMA), type I collagen (Col-Ⅰ), Keap1 and nuclear factors E2 related factor 2 (NRF2) and heme oxygenase 1 (HO-1). HE staining and Masson staining were used to observe pathological changes of lung tissue and to estimate the area of lung tissue injury and pulmonary fibrosis. Hydroxyproline (Hyp) in lung tissue was detected by the kit. Levels of inflammatory factor interleukin-1β, tumor necrosis factor (TNF-α) and oxidative stress index malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by ELISA. Dual luciferase reporting experiment was used to verify the targeting relationship between miR-141-3p and Keap1. Results The expression of miR-141-3p was down-regulated and the expression of Keap1 was up-regulated in lung tissue and cells (P<0.05). Overexpression of miR-141-3p can reduce the degree of pathological damage and fibrosis of lung tissue in rats, Hyp content, and up-regulate expression levels of SOD, E-cadherin, LC3B, Beclin-1, NRF2 and HO-1 in lung tissue and cells, and down-regulate the expression levels of IL-1β, TNF-α, MDA, N-cadherin, α-SMA, Col-I and Keap1 (P<0.05). Overexpression of Keap1 was able to reverse the improvement effect of overexpression of miR-141-3p on alveolar epithelial cell damage in ARDS rats (P<0.05). Double Luciferase reporter gene experiment confirmed that miR-141-3p and Keap1 may have a targeted regulatory relationship. Conclusion Overexpression of miR-141-3p may activate the Keap1-NRF2/ARE signaling pathway, activate autophagy, inhibit inflammatory response, oxidative stress, and EMT progression, and improve pulmonary fibrosis in ARDS rats.

Key words: respiratory distress syndrome, pulmonary fibrosis, miR-141-3p, Keap1-NRF2/ARE signaling pathway

CLC Number:

R563.8

Figures/Tables 14

References 21

[1]	MEYER N J, GATTINONI L, CALFEE C S. Acute respiratory distress syndrome[J]. Lancet, 2021, 398(10300):622-637. doi:10.1016/S0140-6736(21)00439-6.
[2]	ZHANG R, TAN Y, YONG C, et al. Pirfenidone ameliorates early pulmonary fibrosis in LPS-induced acute respiratory distress syndrome by inhibiting endothelial-to-mesenchymal transition via the Hedgehog signaling pathway[J]. Int Immunopharmacol, 2022, 109:108805. doi:10.1016/j.intimp.2022.108805.
[3]	LIANG Y, XU Y, LU B, et al. Inositol alleviates pulmonary fibrosis by promoting autophagy via inhibiting the HIF-1α-SLUG axis in acute respiratory distress syndrome[J]. Oxid Med Cell Longev, 2022, 2022:1030238. doi:10.1155/2022/1030238.
[4]	XIA L, ZHU G, HUANG H, et al. LncRNA small nucleolar RNA host gene 16 (SNHG16) silencing protects lipopolysaccharide(LPS)-induced cell injury in human lung fibroblasts WI-38 through acting as miR-141-3p sponge[J]. Biosci Biotechnol Biochem, 2021, 85(5):1077-1087. doi:10.1093/bbb/zbab016.
[5]	LIU S, PI J, ZHANG Q. Signal amplification in the KEAP1-NRF2-ARE antioxidant response pathway[J]. Redox Biol, 2022, 54:102389. doi:10.1016/j.redox.2022.102389.
[6]	LI J, LU K, SUN F, et al. Panaxydol attenuates ferroptosis against LPS-induced acute lung injury in mice by Keap1-Nrf2/HO-1 pathway[J]. J Transl Med, 2021, 19(1):96. doi:10.1186/s12967-021-02745-1.
[7]	袁静, 从人愿, 夏金婵, 等. 黄芩苷调节巨噬细胞极化减轻脂多糖诱导的大鼠急性肺损伤[J]. 细胞与分子免疫学杂志, 2022, 38(1):9-15.
	YUAN J, CONG R Y, XIA J C, et al. Baicalin alleviates LPS-induced acute lung injury in rats by regulating macrophage polarization[J]. Chin J Cell Mol Immunol, 2022, 38(1):9-15. doi:10.13423/j.cnki.cjcmi.009281.
[8]	杨贵霞, 李想, 沈锋, 等. 穿心莲内酯对脂多糖刺激下大鼠Ⅱ型肺泡上皮细胞促凝和纤溶抑制相关因子表达的影响研究[J]. 中华危重病急救医学, 2021, 33(2):155-160.
	YANG G X, LI X, SHEN F, et al. Effect of andrographolide on the expression of procoagulant and fibrinolytic inhibition related factors in rat type Ⅱ alveolar epithelial cells stimulated by lipopolysaccharide[J]. Chin Crit Care Med, 2021, 33(2):155-160. doi:10.3760/cma.j.cn121430-20200923-00647.
[9]	MU X, WANG H, LI H. Silencing of long noncoding RNA H19 alleviates pulmonary injury, inflammation, and fibrosis of acute respiratory distress syndrome through regulating the microRNA-423-5p/FOXA1 axis[J]. Exp Lung Res, 2021, 47(4):183-197. doi:10.1080/01902148.2021.1887967.
[10]	ZHANG X, YE L, TANG W, et al. Wnt/β-catenin participates in the repair of acute respiratory distress syndrome-associated early pulmonary fibrosis via mesenchymal stem cell microvesicles[J]. Drug Des Devel Ther, 2022, 16:237-247. doi:10.2147/DDDT.S344309.
[11]	BAO X, LIU X, LIU N, et al. Inhibition of EZH2 prevents acute respiratory distress syndrome(ARDS)-associated pulmonary fibrosis by regulating the macrophage polarization phenotype[J]. Respir Res, 2021, 22(1):194. doi:10.1186/s12931-021-01785-x.
[12]	WANG X, LIU F, XU M, et al. Penehyclidine hydrochloride alleviates lipopolysaccharide-induced acute respiratory distress syndrome in cells via regulating autophagy-related pathway[J]. Mol Med Rep, 2021, 23(2):100. doi:10.3892/mmr.2020.11739.
[13]	XIE T, XU Q, WAN H, et al. Lipopolysaccharide promotes lung fibroblast proliferation through autophagy inhibition via activation of the PI3K-Akt-mTOR pathway[J]. Lab Invest, 2019, 99(5):625-633. doi:10.1038/s41374-018-0160-2.
[14]	HILL C, LI J, LIU D, et al. Autophagy inhibition-mediated epithelial-mesenchymal transition augments local myofibroblast differentiation in pulmonary fibrosis[J]. Cell Death Dis, 2019, 10(8):591. doi:10.1038/s41419-019-1820-x.
[15]	ZHANG B, ZHAO C, HOU L, et al. Silencing of the lncRNA TUG1 attenuates the epithelial-mesenchymal transition of renal tubular epithelial cells by sponging miR-141-3p via regulating β-catenin[J]. Am J Physiol Renal Physiol, 2020, 319(6):F1125-F1134. doi:10.1152/ajprenal.00321.2020.
[16]	ZHU L, CHEN M, WANG W, et al. microRNA-141-3p mediates epithelial cell proliferation,apoptosis,and epithelial-mesenchymal transition and alleviates pulmonary fibrosis in mice via Spred2[J]. Histol Histopathol, 2023:18585. doi:10.14670/HH-18-585.
[17]	QIAN W, CAI X, QIAN Q, et al. lncRNA ZEB1-AS1 promotes pulmonary fibrosis through ZEB1-mediated epithelial-mesenchymal transition by competitively binding miR-141-3p[J]. Cell Death Dis, 2019, 10(2):129. doi:10.1038/s41419-019-1339-1.
[18]	HUANG C Y, DENG J S, HUANG W C, et al. Attenuation of lipopolysaccharide-induced acute lung injury by hispolon in mice, through regulating the TLR4/PI3K/Akt/mTOR and Keap1/Nrf2/HO-1 pathways, and suppressing oxidative stress-mediated ER stress-induced apoptosis and autophagy[J]. Nutrients, 2020, 12(6):1742. doi:10.3390/nu12061742.
[19]	ZHENG F, WU X, ZHANG J, et al. Sevoflurane reduces lipopolysaccharide-induced apoptosis and pulmonary fibrosis in the RAW264.7 cells and mice models to ameliorate acute lung injury by eliminating oxidative damages[J]. Redox Rep, 2022, 27(1):139-149. doi:10.1080/13510002.2022.2096339.
[20]	DONG Z, YIN E G, YANG M, et al. Role and mechanism of Keap1/Nrf2 signaling pathway in the regulation of autophagy in alleviating pulmonary fibrosis[J]. Comput Intell Neurosci, 2022, 2022:3564871. doi:10.1155/2022/3564871.
[21]	ZHANG C, KONG X, MA D. miR-141-3p inhibits vascular smooth muscle cell proliferation and migration via regulating Keap1/Nrf2/HO-1 pathway[J]. IUBMB Life, 2020, 72(10):2167-2179. doi:10.1002/iub.2374.

基因名称	引物序列（5'→3'）	产物大小/bp
miR-141- 3p	上游：TAGGTTTGGGTGCCAGGTTC 下游：AGATACCAGAAGGGCCCAGG	78
Keap1	上游：TCCAGCTCCAGCTCCAAAAAC 下游：AGGACTGCCGATAGTAGCCC	174
U6	上游：CTCGCTTCGGCAGCACA 下游：AACGCTTCACGAATTTGCGT	71
GAPDH	上游：CCCCATACACAGTGTTAGCC 下游：GAGTGATTTTCCCGTCC	96

基因名称	引物序列（5'→3'）	产物大小/bp
miR-141- 3p	上游：TAGGTTTGGGTGCCAGGTTC 下游：AGATACCAGAAGGGCCCAGG	78
Keap1	上游：TCCAGCTCCAGCTCCAAAAAC 下游：AGGACTGCCGATAGTAGCCC	174
U6	上游：CTCGCTTCGGCAGCACA 下游：AACGCTTCACGAATTTGCGT	71
GAPDH	上游：CCCCATACACAGTGTTAGCC 下游：GAGTGATTTTCCCGTCC	96

组别	miR-141-3p	Keap1 mRNA	Keap1	NRF2	HO-1
对照组	1.03±0.10	1.00±0.09	0.46±0.05	0.88±0.10	0.78±0.09
模型组	0.41±0.04^a	1.59±0.16^a	0.94±0.09^a	0.44±0.06^a	0.37±0.04^a
agomir-NC组	0.45±0.06^a	1.61±0.14^a	0.95±0.10^a	0.45±0.07^a	0.35±0.05^a
miR-141-3p agomir组	0.88±0.10^b	1.18±0.13^b	0.52±0.06^b	0.81±0.09^b	0.62±0.08^b
F	152.209^＊＊	52.517^＊＊	115.083^＊＊	81.454^＊＊	92.186^＊＊

组别	miR-141-3p	Keap1 mRNA	Keap1	NRF2	HO-1
对照组	1.03±0.10	1.00±0.09	0.46±0.05	0.88±0.10	0.78±0.09
模型组	0.41±0.04^a	1.59±0.16^a	0.94±0.09^a	0.44±0.06^a	0.37±0.04^a
agomir-NC组	0.45±0.06^a	1.61±0.14^a	0.95±0.10^a	0.45±0.07^a	0.35±0.05^a
miR-141-3p agomir组	0.88±0.10^b	1.18±0.13^b	0.52±0.06^b	0.81±0.09^b	0.62±0.08^b
F	152.209^＊＊	52.517^＊＊	115.083^＊＊	81.454^＊＊	92.186^＊＊

组别	肺损伤评分	肺纤维化面积评分
对照组	0.00±0.00	0.00±0.00
模型组	3.08±0.36^a	3.29±0.46^a
agomir-NC组	3.12±0.34^a	3.32±0.49^a
miR-141-3p agomir组	1.24±0.26^b	1.70±0.35^b
F	110.639^＊＊	44.875^＊＊