miR-146b对急性呼吸窘迫综合征大鼠肺组织中ICAM-1表达的调控作用

doi:10.13481/j.1671-587X.20210307

摘要/Abstract

摘要： 目的

探讨miR-146b对急性呼吸窘迫综合征（ARDS）大鼠肺组织中细胞间黏附分子1（ICAM-1）表达的影响，阐明miR-146b治疗ARDS的分子机制。

方法

采用尾静脉注射油酸法建立ARDS大鼠模型，并将建模成功的大鼠分为模型组（只给予油酸）、agomir阴性对照组和miR-146b agomir组，每组15只，另选15只大鼠作为假手术组（只给予生理盐水，不建模）。术前1 h，miR-146b agomir组大鼠给予miR-146b agomir进行干预，agomir阴性对照组大鼠给予miR-146b agomir阴性对照试剂进行干预。造模成功24 h后，采用血气分析仪检测各组大鼠血氧分压（PaO₂）和氧合指数（OI），检测各组大鼠肺组织湿/干重（W/D）比值，ELISA法检测各组大鼠肺泡灌洗液（BALF）中白细胞介素1β（IL-1β）、白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）水平，HE染色观察各组大鼠肺组织病理形态表现，实时荧光定量PCR（RT-qPCR）法检测各组大鼠肺组织中miR-146b和ICAM-1 mRNA表达水平，Western blotting法检测各组大鼠肺组织中ICAM-1蛋白表达水平，双荧光素酶报告系统检测miR-146b和ICAM-1的靶向关系。

结果

与模型组和agomir阴性对照组比较，miR-146b agomir组大鼠动脉血PaO₂和OI及肺组织W/D比值升高（P<0.01），BALF中IL-1β、IL-6和TNF-α水平升高（P<0.01）。HE染色，假手术组大鼠肺组织结构正常，无明显炎症病理改变；模型组和agomir阴性对照组大鼠肺组织呈现肺泡腔出血、肺泡壁增厚和红细胞渗出等病理变化；miR-146b agomir组大鼠肺组织上述病理形态变化减轻。与模型组和agomir阴性对照组比较，miR-146b agomir组大鼠肺组织中ICAM-1 mRNA和蛋白表达水平明显降低（P<0.01），而miR-146b表达水平明显升高（P<0.01）。双荧光素酶报告系统实验证实miR-146b靶向调控ICAM-1基因。

结论

miR-146b通过靶向抑制ICAM-1表达水平，降低ARDS大鼠肺组织炎症水平，缓解肺组织功能损伤，对ARDS肺组织起到保护作用。

关键词: miR-146b, 细胞间黏附分子1, 急性呼吸窘迫综合征, 靶向调控, 炎症因子

Abstract: Objective

To observe the effect of miR-146b on the expression of intercellular adhesion molecule-1 （ICAM-1） in lung tissue of the rats with acute respiratory distress syndrome （ARDS）， and to clarify the molecular mechanism of miR-146b in the treatment of ARDS.

Methods

The ARDS rat models were established by injecting oleic acid into the tail vein， and the successfully modeled rats were divided into model group（only given oleic acid）， agomir negative control group and miR-146b agomir group， with 15 rats in each group； another 15 rats were selected as sham operation group（only given saline）. One hour before operation， the rats in miR-146b agomir group were given miR-146b agonist for intervention， and the rats in agomir negative control group were given miR-146b agonist negative control reagent for intervention.Twenty-four hours after successful modeling， the partial pressure of oxygen （PaO₂） and oxygenation index （OI） of the rats in various groups were detected by the blood gas system； the wet/dry weight ratios （W/D） of lung tissue of the rats in various groups were detected； the levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in bronchoalveolar lavage fluid （BALF） of the rats in various groups were detected by ELISA assay； the pathomorphology of lung tissue of the rats in various groups was observed by HE staining； the expression levels of miR-146b and ICAM-1 mRNA in lung tissue of the rats in various groups were detected by Real-time fluorescence quantitative PCR（RT-qPCR） method； the expression levels of ICAM-1 protein in lung tissue of the rats in various groups were detected by Western blotting method； the targeting relationship between miR-146b and ICAM-1 was detected by dual luciferase reporter system.

Results

Compared with model group and agomir negative control group， the PaO₂ and OI， and the ratio of W/D of lung tissue of the rats in miR-146b agomir group were increased （P<0.01）， and the levels of IL-1β， IL-6， and TNF-α in BALF of the rats in miR-146b agomir group were increased （P<0.01）.The HE staining results showed that the structure of lung tissue of the rats in sham operation group was normal， there was no obvious inflammatory pathological changes， while the pathological changes such as alveolar hemorrhage， alveolar wall thickening and red blood cell exudation in the lung tissue were found in model group and agomir negative control group， and the above pathomorphological changes of lung tissue of the rats in miR-146b agomir group were alleviated. The expression levels of ICAM-1 mRNA and protein of the rats in miR-146b agomir group were significantly lower than those in model group and agomir negative control group （P<0.01）， while the expression level of miR-146b was significantly higher than those in model group and agomir negative control group （P<0.01）.The dual luciferase reporter system experiment results confirmed that the miR-146b could targetedly regulate the ICAM-1 gene.

Conclusion

MiR-146b can reduce the inflammation level of lung tissue of the ARDS rats， relieve the lung tissue function damage， and protect the lung tissue from ARDS by targeted down-regulation of the expression level of ICAM-1.

Key words: miR-146b, intercellular adhesion molecule 1, acute respiratory distress syndrome, targeted regulation, inflammatory factor

中图分类号:

龙光文, 张谦, 杨秀林, 吉春玲, 董裕康. miR-146b对急性呼吸窘迫综合征大鼠肺组织中ICAM-1表达的调控作用[J]. 吉林大学学报(医学版), 2021, 47(3): 587-594.

Guangwen LONG, Qian ZHANG, Xiulin YANG, Chunling JI, Yukang DONG. Regulation effect of miR-146b on expression of intercellular adhesion molecule-1 in lung tissue of rats with acute respiratory distress syndrome[J]. Journal of Jilin University(Medicine Edition), 2021, 47(3): 587-594.

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Group	PaO₂（P/mmHg）	OI（P/mmHg）	Ratio of W/D
Sham operation	102.00±10.00	533.00±39.00	3.82±0.41
Model	58.00±7.00^*	312.00±25.00^*	6.35±0.28^*
Agomir negative control	61.00±6.00^*	337.00±41.00^*	6.47±0.52^*
MiR-146b agomir	75.00±9.00^△#	465.00±27.00^△#	4.56±0.65^△#

Group	IL-1β	IL-6	TNF-α
Sham operation	35.06±7.54	41.28±4.92	60.12±6.83
Model	71.32±8.43^*	63.07±5.88^*	113.58±10.20^*
Agomir negative control	71.60±6.11^*	62.84±6.01^*	109.64±11.36^*
MiR-146b agomir	50.08±5.29^△##	53.93±5.14^△#	81.05±7.55^△##

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