吡非尼酮通过miRNA-425-5p调控TGF-β/Smad通路对大鼠心肌纤维化的影响

doi:10.11958/20212788

天津医药 ›› 2022, Vol. 50 ›› Issue (10): 1037-1042.doi: 10.11958/20212788

吡非尼酮通过miRNA-425-5p调控TGF-β/Smad通路对大鼠心肌纤维化的影响

邹琳(), 张昕(), 李丽

内蒙古科技大学包头医学院第一附属医院心功能科（邮编014000）

收稿日期:2022-01-05 修回日期:2022-06-02 出版日期:2022-10-15 发布日期:2022-10-20
通讯作者: 张昕 E-mail:454395752@qq.com;zhangxinwdq@sina.com
作者简介:邹琳（1994）,男,硕士在读,主要从事心力衰竭方面研究。E-mail： 454395752@qq.com
基金资助:
包头市卫生健康科技计划项目(wsjkkj001)

Effects of pirfenidone on myocardial fibrosis in rats by regulating TGF-β/Smad pathway through miRNA-425-5p

ZOU Lin(), ZHANG Xin(), LI Li

Department of Cardiac Function, the First Affiliated Hospital of Baotou Medical College, Inner Mongolia University of Science and Technology, Baotou 014000, China

Received:2022-01-05 Revised:2022-06-02 Published:2022-10-15 Online:2022-10-20
Contact: ZHANG Xin E-mail:454395752@qq.com;zhangxinwdq@sina.com

邹琳, 张昕, 李丽. 吡非尼酮通过miRNA-425-5p调控TGF-β/Smad通路对大鼠心肌纤维化的影响[J]. 天津医药, 2022, 50(10): 1037-1042.

ZOU Lin, ZHANG Xin, LI Li. Effects of pirfenidone on myocardial fibrosis in rats by regulating TGF-β/Smad pathway through miRNA-425-5p[J]. Tianjin Medical Journal, 2022, 50(10): 1037-1042.

摘要/Abstract

摘要：

目的探讨吡非尼酮通过调控miRNA-425-5p及转化生长因子（TGF）-β/Smad通路改善心肌梗死大鼠心肌纤维化程度的作用。方法将60只雄性SD大鼠分为3组：假手术组（不结扎冠状动脉）、模型组、吡非尼酮组（吡非尼酮0.3 g/kg灌胃）。造模4周后采用心脏多普勒超声评价各组大鼠心脏功能; 酶联免疫吸附试验检测血浆中白细胞介素6（IL-6）、Ⅰ型胶原蛋白α2（COL1α2）、Ⅲ型胶原蛋白α1（COL3α1）的水平;采用Masson染色法评价大鼠心肌纤维化程度,使用Image-Pro Plus 6.0分析各组心肌胶原容积分数;Western blot检测心肌组织中TGF-β1、Smad2、Smad3蛋白表达水平;实时荧光定量PCR（qPCR）检测心肌组织中TGF-β1和miRNA-425-5p表达水平。同时在体外分离、培养乳鼠心肌成纤维细胞,分为对照组（不加任何物质）、miRNA-425-5p mimic组（加入转染miRNA-425-5p mimic）、吡非尼酮组（加入1.5 g/L的吡非尼酮）。采用qPCR检测各组miRNA-425-5p和TGF-β1 mRNA表达水平。结果模型组大鼠与假手术组相比心脏左心室舒张末期内径（LVEDd）、左心室收缩末期内径（LVESd）增大,左心室射血分数（LVEF）、左心室短轴缩短率（FS）降低（P<0.05）;Masson染色及定量分析结果显示心肌纤维化加重;炎性因子IL-6、COL3α1、COL1α2水平明显升高（P<0.05）;心肌组织TGF-β1、Smad2、Smad3蛋白表达升高,miRNA-425-5p表达水平降低（均P<0.05）。吡非尼酮组与模型组相比,LVEDd、LVESd缩小,LVEF、FS升高（P<0.05）,Masson染色及定量分析结果示心肌纤维化程度减轻,血浆炎性因子IL-6、COL3α1、COL1α2水平明显降低（P<0.05）;心肌组织TGF-β1、Smad2、Smad3蛋白表达水平降低,miRNA-425-5p表达水平升高（均P<0.05）。体外细胞实验结果表明,miRNA-425-5p mimic组与对照组相比,miRNA-425-5p表达水平升高,TGF-β1 mRNA表达水平降低（P<0.05）。吡非尼酮组与对照组相比,miRNA-425-5p表达水平升高,TGF-β1 mRNA表达水平降低（P<0.05）。结论吡非尼酮可通过调节miRNA-425-5p表达水平调控TGF-β/Smad信号通路,减轻心肌纤维化,改善心肌梗死后心力衰竭大鼠心脏功能。

关键词: 纤维化, 微RNA, 转化生长因子β1, Smad蛋白质类, 受体作用蛋白丝氨酸苏氨酸激酶类, 吡非尼酮, 微小RNA-425-5p

Abstract:

Objective To investigate the effects of pirfenidone (PFD) on myocardial fibrosis in rats with myocardial infarction by regulating miRNA-425-5p and transforming growth factor (TGF)-β/Smad pathways. Methods Sixty male SD rats were divided into three groups: the sham operation group (no coronary artery ligation), the myocardial infarction model group (coronary artery was ligated) and the PFD group (coronary artery ligation + PFD 0.3 g /kg by gavage). Cardiac function of rats in each group was evaluated by cardiac Doppler ultrasonography 4 weeks after modeling. The levels of interleukin-6 (IL-6), type Ⅰ collagen α2 (COL1α2) and type Ⅲ collagen α1 (COL3α1) were detected by ELISA. Masson staining was used to evaluate the degree of myocardial fibrosis in rats. Image-pro Plus 6.0 was used to analyze myocardial collagen volume fraction in each group. The protein expression levels of TGF-β1, Smad2 and Smad3 in myocardial tissue were detected by Western blot assay. The expression level of miRNA-425-5p in myocardial tissue was detected by quantitative real-time PCR (qPCR). Meanwhile, cardiac fibroblasts from suckling rats were isolated and cultured in vitro and divided into 3 groups: the control group (without any substance), the miRNA-425-5p mimic group (the control group was transfected with miRNA-425-5p mimic), the PFD group (the control group was added with PFD at the final concentration of 1.5 g/L). The mRNA expression levels of miRNA-425-5p and TGF-β1 in each group were detected by qPCR. Results Compared with the sham operation group, cardiac LVESd and LVEDd were increased in the model group, LVEF and FS% decreased (P<0.05). Masson staining and quantitative analysis showed that myocardial fibrosis was aggravated. The serum levels of IL-6, COL3α1 and COL1α2 were significantly increased (P<0.05). The protein expression levels of TGF-β1, Smad2 and Smad3 were significantly increased, and the expression of miRNA-425-5p was significantly decreased (P<0.05). Compared with the model group, LVESd and LVEDd were decreased in the PFD group, while LVEF and FS% were increased (P<0.05). Masson staining and quantitative analysis showed that the degree of myocardial fibrosis was alleviated, and serum levels of inflammatory factors IL-6, COL3α1 and COL1α2 were significantly decreased (P<0.05). The protein expression levels of TGF-β1, Smad2 and Smad3 in myocardial tissue were significantly decreased, while miRNA-425-5p was significantly increased (P<0.05). In vitro cell assay results showed that compared with the control group, the miRNA-425-5p expression level increased in the miRNA-425-5p mimic group, while TGF-β1 mRNA expression level decreased (P<0.05). Compared with the control group, the expression level of miRNA-425-5p was increased, and the expression level of TGF-β1 mRNA was decreased in the PFD group (P<0.05). Conclusion Pirfenidone can regulate TGF-β/Smad signaling pathway by regulating miRNA-425-5p expression level, alleviate myocardial fibrosis and improve cardiac function in rats with heart failure after myocardial infarction.

Key words: fibrosis, microRNAs, transforming growth factor beta1, Smad proteins, receptor-interacting protein serine-threonine kinases, Pirfenidone, miRNA-425-5p

中图分类号:

R542.23

图/表 8

表1 引物序列

Tab. 1 Sequences of the primers

基因名称	引物序列（5'→3'）	产物大小（bp）
TGF-β1	上游：GGACTACTACGCCAAAGAAG 下游：TGTTGCTCCACAGTTGAC	198
miRNA-425-5p	上游：GGGTAGTAGTAGT 下游：TGGGTCTGGGTC	83
GAPDH	上游：GTCGGTGTGAACGGATTTG	181
	下游：TCCCATTCTCAGCCTTGAC	181
U6	上游：CTCGCTTCGGCAGCACA	94
	下游：AACGCTTCACGAATTTGCGT	94

图1 3组大鼠心脏彩超结果

Fig.1 Results of cardiac color ultrasound in three groups of rats

表2 3组大鼠心脏结构及功能比较 ($\bar{x} \pm s$)

Tab. 2 Comparison of the heart structure and function between the three groups of rats

组别	n	LVEF	FS	LVEDd（mm）	LVESd（mm）
假手术组	10	0.815±0.015	0.482 ±0.061	6.61±0.18	1.22±0.10
模型组	17	0.608±0.021^a	0.286±0.062^a	7.75±0.42^a	2.99±0.23^a
吡非尼酮组	18	0.723±0.022^ab	0.397±0.046^ab	7.09±0.25^ab	1.96±0.20^ab
F		348.388^＊＊	41.636^＊＊	44.271^＊＊	290.946^＊＊

图2 3组大鼠心脏组织病理学变化（Masson染色,×100）

Fig.2 Heart histopathological changes in the three groups of rats (Masson, ×100)

表3 3组大鼠血浆IL-6、COL1α2、COL3α1水平比较（n=10,ng/L,$\bar{x} \pm s$）

Tab. 3 Comparison of plasma levels of IL-6, COL1α2 and COL3α1 between the three groups of rats

组别	IL-6	COL1α2	COL3α1
假手术组	60.99±5.89	15.16±1.31	15.09±1.70
模型组	101.80±3.04a	27.24±1.03a	28.25±1.23a
吡非尼酮组	80.71±4.80ab	20.99±2.21ab	22.28±1.71ab
F	186.376＊＊	143.790＊＊	179.023＊＊

3 Western blot检测3组大鼠心肌组织TGF-β1、Smad2、Smad3蛋白表达 A：假手术组;B：模型组;C：吡非尼酮组

Fig.3 The protein expression levels of TGF-β1, Smad2 and Smad3 in myocardial tissues of rats detected by Western blot assay

表4 3组大鼠心肌组织TGF-β1、Smad2、Smad3蛋白表达水平比较（n=4,$\bar{x} \pm s$）

Tab. 4 Comparison of protein expression levels of myocardial tissue TGF-β1, Smad2 and Smad3 between the three groups of rats

组别	TGF-β1	Smad2	Smad3
假手术组	0.40±0.02	0.58±0.01	0.56±0.07
模型组	0.97±0.07^a	1.09±0.02^a	1.12±0.04^a
吡非尼酮组	0.71±0.03^ab	0.76±0.03^ab	0.86±0.02^ab
F	155.789^＊＊	689.734^＊＊	127.620^＊＊

表1 3组大鼠心肌成纤维细胞 TGF-β1、miRNA-425-5p mRNA相对表达量比较（n=3,$\bar{x} \pm s$）

Tab. 1 Comparison of mRNA relative expression levels of TGF-β1 and miRNA-425-5p between the three groups of rats

组别	TGF-β1	miRNA-425-5p
对照组	2.08±0.07	1.31±0.03
miRNA-425-5p mimic组	0.93±0.03^a	3.78±0.20^a
吡非尼酮组	1.30±0.02^ab	2.16±0.02^ab
F	515.251^＊＊	356.347^＊＊

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吡非尼酮通过miRNA-425-5p调控TGF-β/Smad通路对大鼠心肌纤维化的影响

Effects of pirfenidone on myocardial fibrosis in rats by regulating TGF-β/Smad pathway through miRNA-425-5p

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