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

doi:10.11958/20212788

Abstract

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

CLC Number:

R542.23

Figures/Tables 8

References 16

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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

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

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^＊＊

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^＊＊

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＊＊