Naringenin promotes angiogenesis of ischemic myocardium after myocardial infarction through miR-223-3p/IGF1R axis

Abstract

Introduction: Naringenin exerts a protective effect on myocardial ischemia and reperfusion. It has been reported that miR-223-3p is a potential target for the treatment of myocardial infarction (MI). In view of the unreported correlation between Naringenin and miR-223-3p, this study was designed to confirm that the ameliorative effects of Naringenin on MI is directly related to the regulation of miR-223-3p.

Methods: Through electrocardiogram detection, Masson pathological staining and immunohistochemistry of angiogenesis-related factors, alleviative effects of Naringenin on heart function, myocardial injury and angiogenesis in MI mice were observed individually. Hypoxic HUVECs were selected in the in vitro experimental model. The cell viability, angiogenesis and migration ability were analyzed to fathom out the pro-angiogenesis potential of Naringenin. The effect of Naringenin on miR-223-3p, as well as the downstream molecular mechanism was verified through bioinformatics analysis and rescue experiments.

Results: Naringenin improved heart functions of MI mice, reduced degree of myocardial fibrosis, stimulated expressions of angiogenic factors and down-regulated level of miR-223-3p in myocardial tissue. In in vitro experiments, Naringenin increased the viability of hypoxic HUVECs, as well as the abilities of tube formation and migration, and further inhibited the expression of miR-223-3p. In the rescue trial, miR-223-3p mimic reversed the therapeutic effect of Naringenin. Type 1 insulin-like growth factor receptor (IGF1R), as a downstream target gene of miR-223-3p, partially offset the cellular regulatory effects of miR-223-3p after overexpression of IGF1R.

Conclusions: Naringenin improves the angiogenesis of hypoxic HUVECs by regulating the miR-223-3p/IGF1R axis, and has the potential to promote myocardial angiogenesis in MI mice.

Keywords: MiR-223-3p; Myocardial infarction; Naringenin; Revascularization; Type 1 insulin-like growth factor receptor.

Fig. 1

Naringenin improved heart function in mice with MI (A) The chemical structure of Naringenin (B–C) The cardiac function of mice was evaluated by detecting the values of EF and FS (n = 10 mice/group). All experiments were repeated three times to average. ∗∗∗p < 0.001 vs Sham; ^###p < 0.001 vs Model. Abbreviations: MI, myocardial infarction; Nar, Naringenin; Nar-L, low-dose Naringenin; Nar-H, high-dose Naringenin; EF, Ejection Fractions; FS, Fraction Shortening.

Fig. 2

Naringenin reduced the degree of myocardial fibrosis in model mice, stimulated the expression of angiogenic factors and down-regulated miR-223-3p expression (A and C) The degree of myocardial fibrosis in mice was observed by Masson staining and the fibrosis percentage was calculated (B, D and E) The expressions of CD34 and CD31 in mouse myocardial tissue were measured by immunohistochemistry and the relative positive cell rates were calculated (F) The expression of miR-223-3p in mouse myocardial tissue was detected by qRT-PCR. U6 was an internal reference. All experiments were repeated three times to average. ∗p < 0.05, ∗∗∗p < 0.001 vs Sham; ^###p < 0.001 vs Model. Abbreviations: MI, myocardial infarction; Nar, Naringenin; Nar-L, low-dose Naringenin; Nar-H, high-dose Naringenin.

Fig. 3

Naringenin promoted the viability, tube formation and migration of hypoxic HUVECs, and further inhibited the expression of miR-223-3p (A) The effect of Naringenin on the viability of HUVECs in normoxic or hypoxic condition was tested by CCK-8 experiment (B and E) The effect of Naringenin on tube formation ability of HUVECs in normoxic or hypoxic condition was determined by tube formation experiments. The image was enlarged 100 times (C and F) The effect of Naringenin on the migration ability of HUVECs in normoxic or hypoxic condition was tested by scratch test. The image was enlarged 100 times (D) The regulation of Naringenin on miR-223-3p expression in HUVECs in normoxic or hypoxic condition was quantified by qRT-PCR. U6 was the internal reference. All experiments were repeated three times to average. ∗∗p < 0.01, ∗∗∗p < 0.001 vs Control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs Hypoxia. Abbreviations: Nar, Naringenin; Nar-L, low-dose Naringenin; Nar-H, high-dose Naringenin.

Fig. 4

MiR-223-3p reversed the regulation of Naringenin in hypoxic HUVECs (A) The transfection efficiency of miR-223-3p mimic was tested by qRT-PCR. U6 was the internal reference (B) The effects of miR-223-3p mimic and Naringenin on the expression of miR-223-3p were quantified by qRT-PCR. U6 was the internal reference (C and E) The effect of Naringenin on the tube-forming ability of hypoxic HUVECs was measured by tube formation experiments. The image was enlarged 100 times (D and F) The effect of Naringenin on the migration ability of hypoxic HUVECs was detected by scratch test. The image was enlarged 100 times. All experiments were repeated three times to average. ⁺⁺⁺p < 0.001 vs MC; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs Control; ^##p < 0.01, ^###p < 0.001 vs Nar-H + MC; ^ˆˆˆp < 0.001 vs M. Abbreviations: MC, mimic control; M, miR-223-3p mimic; Nar, Naringenin; Nar-H, high-dose Naringenin.

Fig. 5

IGF1R, a downstream target gene of miR-223-3p, was a potential mechanism for Naringenin to exert the protective effect on HUVECs (A) The target genes of miR-223-3p were screened through multiple target gene prediction websites (B–C) The binding relationship between miR-223-3p and IGF1R was predicted and verified (D) The expression of IGF1R in mouse myocardium was verified by qRT-PCR. GAPDH was an internal control (E–G) The expression of IGF1R in hypoxic HUVECs was detected by qRT-PCR and Western blot. GAPDH was an internal control. All experiments were repeated three times to average. ⁺⁺⁺p < 0.001 vs MC; ∗∗∗p < 0.001 vs Control; ^###p < 0.001 vs Nar-H + MC; ^ˆˆˆp < 0.001 vs M; ^&&&p < 0.001 vs Sham; ^△△△p < 0.001 vs Model. Abbreviations: MC, mimic control; M, miR-223-3p mimic; Nar, Naringenin; Nar-L, low-dose Naringenin; Nar-H, high-dose Naringenin; IGF1R, Type 1 insulin-like growth factor receptor.

Fig. 6

IGF1R transfection significantly up-regulated the expression of IGF1R in hypoxic HUVECs (A–B) The transfection efficiency of overexpressed IGF1R plasmid was tested by qRT-PCR and Western blot (C–D) The effects of overexpression of IGF1R and miR-223-3p mimic on the level of IGF1R were detected by qRT-PCR and Western blot. GAPDH was an internal control. All experiments were repeated three times to average. ⁺⁺⁺p < 0.001 vs MC; ∗∗∗p < 0.001 vs MC + NC; ^###p < 0.001 vs M + NC; ^ˆˆˆp < 0.001 vs MC + IGF1R. Abbreviations: MC, mimic control; M, miR-223-3p mimic; NC, negative control; IGF1R, overexpressed IGF1R.

Fig. 7

Overexpression of IGF1R reversed the regulation of miR-223-3p in hypoxic HUVECs (A and C) The effects of overexpressed IGF1R and miR-223-3p mimic on the tube formation ability of hypoxic HUVECs were tested by tube formation experiments. The image was enlarged 100 times (B and D) The effects of overexpression of IGF1R and miR-223-3p mimic on the migration ability of hypoxic HUVECs were evaluated by scratch test. The image was enlarged 100 times (E–G) The regulation of PI3K/Akt pathway in hypoxic HUVECs by overexpression of IGF1R and miR-223-3p mimic was detected by Western blot. GAPDH was an internal control. All experiments were repeated three times to average. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs MC + NC; ^##p < 0.01, ^###p < 0.001 vs M + NC; ^ˆp < 0.05, ^ˆˆˆp < 0.001 vs MC + IGF1R. Abbreviations: MC, mimic control; M, miR-223-3p mimic; NC, negative control; IGF1R, overexpressed IGF1R.