miR-141-3p通过调节Keap1-NRF2/ARE信号通路对急性呼吸窘迫综合征大鼠肺纤维化的影响

doi:10.11958/20230636

摘要/Abstract

摘要：

目的探讨miR-141-3p对急性呼吸窘迫综合征（ARDS）大鼠肺纤维化的影响及作用机制。方法将大鼠按随机数字表法分为对照组、模型组、agomir-NC组、miR-141-3p agomir组，每组10只；除对照组外，其余大鼠采用脂多糖（LPS）滴注法构建ARDS模型；将大鼠肺泡Ⅱ型上皮细胞RLE-6TN细胞分为NC组、LPS组、miR-NC组、miR-141-3p mimics组、miR-141-3p mimics+pcDNA组、miR-141-3p mimics+NRF2与Kelch样环相关蛋白1（Keap1）组，除NC组外，其余各组建立LPS细胞模型；qPCR检测肺组织和细胞中miR-141-3p、Keap1 mRNA表达；Western blot检测肺组织和细胞上皮型钙黏附素（E-cadherin）、神经型钙黏附素（N-cadherin）、微管相关蛋白轻链3B（LC3B）、自噬相关基因Beclin-1、α平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原（Col-Ⅰ）、Keap1、核因子E2相关因子2（NRF2）、血红素氧合酶1（HO-1）表达；HE和Masson染色观察肺组织病理变化并进行肺损伤评分和肺纤维化面积评分；试剂盒检测肺组织羟脯氨酸（Hyp）；酶联免疫吸附试验（ELISA）检测炎性因子白细胞介素（IL）-1β、肿瘤坏死因子（TNF）-α和氧化应激指标丙二醛（MDA）、超氧化物歧化酶（SOD）水平；双萤光素酶报告实验验证miR-141-3p与Keap1靶向关系。结果 ARDS大鼠肺组织和细胞中miR-141-3p表达下调，Keap1表达上调（P<0.05）；过表达miR-141-3p可降低大鼠肺组织病理损伤和纤维化程度、Hyp含量，上调肺组织和细胞中SOD、E-cadherin、LC3B、Beclin-1、NRF2、HO-1表达，下调IL-1β、TNF-α、MDA、N-cadherin、α-SMA、Col-Ⅰ、Keap1表达（P<0.05）；过表达Keap1可逆转过表达miR-141-3p对ARDS大鼠肺泡上皮细胞损伤的改善作用（P<0.05）；双萤光素酶报告基因实验证实miR-141-3p与Keap1可能存在靶向调控关系。结论过表达miR-141-3p可能激活Keap1-NRF2/ARE信号通路，激活自噬，抑制炎症反应、氧化应激和上皮-间充质转化进展，改善ARDS大鼠肺纤维化。

关键词: 呼吸窘迫综合征, 肺纤维化, miR-141-3p, Keap1-NRF2/ARE信号通路

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

中图分类号:

R563.8

图/表 14

参考文献 21

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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^＊＊