The Mechanism of miR-23 Regulating PI3K/AKT/mTOR Pathway to Improve Myocardial Angiogenesis in Hypertensive Heart Failure Rats
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摘要:
目的 探讨微小RNA-23（microRNA-23，miR-23）通过调控磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin，PI3K/AKT/mTOR）信号通路对高血压性心力衰竭（heart failure，HF）大鼠心肌重构、纤维化及血管生成的影响机制。 方法 将40只大鼠随机分为对照组、模型组、阴性对照组（antagomir-NC组）和miR-23抑制组（antagomir-23组），每组10只。采用高盐饮食诱导HF模型，并通过尾静脉注射antagomir-23进行干预。检测各组大鼠的心功能指标、心肌组织纤维化程度、细胞凋亡水平及血小板内皮细胞黏附分子-1（cluster of differentiation 31，CD31）、血管内皮生长因子（Vascular Endothelial Growth Factor，VEGF）和碱性成纤维生长因子（basic fibroblast growth factor，bFGF）等血管生成相关蛋白的表达，同时评估PI3K/AKT/mTOR信号通路活性；并通过双荧光素酶报告实验验证miR-23对PI3K的靶向调控。 结果 抑制miR-23可显著改善高血压性HF大鼠心功能，减少心肌纤维化与细胞凋亡，增强CD31、VEGF及bFGF的表达，并激活PI3K/AKT/mTOR信号通路（均P < 0.05）。双荧光素酶实验证实miR-23可负向调控PI3K的表达。 结论 抑制miR-23能够通过激活PI3K/AKT/mTOR信号通路，促进血管生成，减轻心肌损伤，从而延缓高血压性心力衰竭的进展。 Abstract:Objective To investigate the mechanisms by which miR-23 regulates the PI3K/AKT/mTOR signaling pathway to influence myocardial remodeling, fibrosis, and angiogenesis in hypertensive heart failure （HF） rats. Methods Forty rats were randomly divided into five groups （n = 10 per group）: control group, model group, antagomir-NC group, and antagomir-23 group. The HF model was established using a high-salt diet, and intervention was performed via tail vein injection of antagomir-23. Cardiac function parameters, the degree of myocardial fibrosis, cell apoptosis levels, and the expression of angiogenesis-related proteins including CD31, VEGF, and bFGF were measured in each group. Concurrently, the activity of the PI3K/AKT/mTOR signaling pathway was assessed. A dual-luciferase reporter assay was conducted to confirm that miR-23 targets PI3K. Results Inhibition of miR-23 significantly improved cardiac function in hypertensive HF rats, reduced myocardial fibrosis and apoptosis, and enhanced the expression of CD31, VEGF, bFGF, and activated the PI3K/AKT/mTOR signaling pathway in hypertensive HF rats （all P < 0.05）. The dual-luciferase assay confirmed that miR-23 negatively regulates PI3K expression. Conclusion Inhibition of miR-23 can activate the PI3K/AKT/mTOR signaling pathway, promote angiogenesis, reduce myocardial damage, thereby delaying the progression of hypertensive heart failure. -
Key words:
- Hypertension /
- Heart failure /
- miR-23 /
- Myocardial remodeling /
- Fibrosis /
- Angiogenesis
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图 1 HE染色检测miR-23抑制对高血压HF大鼠心肌损伤的影响（×200）
Figure 1. HE staining detecting the effect of miR-23 inhibition on myocardial injury in hypertensive HF rats （×200）
图 2 Masson染色检测miR-23抑制对高血压HF大鼠纤维化的影响（×20）
Figure 2. Masson staining detecting the effect of miR-23 inhibition on fibrosis in hypertensive HF rats （×20）
图 3 TUNEL染色检测miR-23抑制对高血压HF大鼠心肌细胞凋亡的影响（×200）
Figure 3. TUNEL staining detecting the effect of miR-23 inhibition on cardiomyocyte apoptosis in hypertensive HF rats （×200）
图 4 免疫荧光染色检测miR-23抑制对高血压HF大鼠心肌CD31表达的影响（×200）
Figure 4. Immunofluorescence staining to detect the effect of miR-23 inhibition on CD31 expression in myocardium of hypertensive HF rats （×200）
图 5 Western blot检测miR-23抑制对高血压HF大鼠心肌组织中VEGF、VEGF-R2、bFGF及PI3K/AKT/mTOR信号通路蛋白水平的影响
A：VEGF、VEGF-R2、bFGF及PI3K/AKT/mTOR信号通路蛋白印迹图；B：VEGF、VEGF-R2、bFGF及PI3K/AKT/mTOR信号通路蛋白表达统计图；1：对照组；2：模型组；3：antagomir-NC组；4：antagomir-23组。注：与对照组相比，aP < 0.05；与antagomir-NC组相比，bP < 0.05。
Figure 5. Western blot detection of the effects of miR-23 inhibition on protein levels of VEGF，VEGF-R2，bFGF and PI3K/AKT/mTOR signaling pathway in myocardial tissue of hypertensive HF rats
表 1 引物序列
Table 1. Primer sequences
基因 引物序列（5'-3'） miR-23 F：AAACCGUT AGGGGTTC R：TTTGGCAATCCCCAAG U6 F：AAGUGGCA ACGAACCG R：TTCACCGUTGCTTGGC PI3K F：GGAATCCAGGGAAGGAAGG R：CAGTTTGGGTGGACGGAAGA 
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表 2 各组大鼠收缩压比较（n = 6，$ \bar x \pm s $）
Table 2. Comparison of systolic blood pressure among groups （n = 6，$ \bar x \pm s $）
组别 收缩压（mmHg） 对照组 134.44 ± 11.23 模型组 180.26 ± 33.09Δ antagomir-NC组 168.04 ± 29.89Δ antagomir-23组 132.33 ± 18.04# F 5.701 P 0.006* *P < 0.05；与对照组相比，ΔP < 0.05；与antagomir-NC组相比，#P < 0.05。
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表 3 各组大鼠心功能指标比较（n = 6，$ \bar x \pm s $）
Table 3. Comparison of cardiac function indicators among groups（n = 6，$ \bar x \pm s $）
组别 LVESP（mmHg） LVEDP（mmHg） +dp/dtmax（×103 mmHg/s） -dp/dtmax（×103 mmHg/s） 对照组 123.48 ± 18.99 13.65 ± 1.40 3.57 ± 0.27 3.08 ± 0.22 模型组 93.06 ± 19.21Δ 18.97 ± 4.92Δ 2.74 ± 0.74Δ 2.42 ± 0.55Δ antagomir-NC组 94.20 ± 30.64Δ 18.51 ± 2.39Δ 2.73 ± 0.75Δ 2.44 ± 0.52Δ antagomir-23组 120.10 ± 15.87# 13.47 ± 2.64# 3.42 ± 0.49# 3.16 ± 0.31# F 3.330 5.545 3.351 5.441 P 0.040* 0.006** 0.040* 0.007** *P < 0.05，**P < 0.01；与对照组相比，ΔP < 0.05；与antagomir-NC组相比，#P < 0.05。
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表 4 各组大鼠CVF比较（n = 6，$ \bar x \pm s $）
Table 4. Comparison of CVF among groups （n = 6，$ \bar x \pm s $）
组别 CVF（%） 对照组 1.79 ± 0.25 模型组 2.30 ± 0.33Δ antagomir-NC组 2.30 ± 0.65Δ antagomir-23组 1.76 ± 0.24# F 3.413 P 0.037* *P < 0.05；与对照组相比，ΔP < 0.05；与antagomir-NC组相比，#P < 0.05。
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表 5 各组大鼠心肌细胞凋亡水平比较（n = 6，$ \bar x \pm s $）
Table 5. Comparison of cardiomyocyte apoptosis levels among groups（n = 6，$ \bar x \pm s $）
组别 凋亡指数（%） 对照组 2.28 ± 0.27 模型组 24.20 ± 4.31ΔΔΔ antagomir-NC组 23.78 ± 4.32ΔΔΔ antagomir-23组 16.07 ± 4.35# F 44.839 P <0.001*** ***P < 0.001；与对照组相比，ΔΔΔP < 0.001；与antagomir-NC组相比，#P < 0.05。
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表 6 各组大鼠心肌CD31表达水平比较（n = 6，$ \bar x \pm s $）
Table 6. Comparison of myocardial CD31 expression levels among groups（n = 6，$ \bar x \pm s $）
组别 CD31表达率（%） 对照组 3.66 ± 0.45 模型组 2.86 ± 0.39ΔΔ antagomir-NC组 2.91 ± 0.57ΔΔ antagomir-23组 3.52 ± 0.70# F 3.498 P 0.035* *P < 0.05；与对照组相比，ΔΔP < 0.01；与antagomir-NC组相比，#P < 0.05。
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表 7 各组大鼠心肌组织miR-23表达量比较（n = 6，$ \bar x \pm s $）
Table 7. Comparison of myocardial miR-23 expression levels among groups （n = 6，$ \bar x \pm s $）
组别 miR-23 对照组 1.00 ± 0.10 模型组 1.27 ± 0.28Δ antagomir-NC组 1.37 ± 0.35Δ antagomir-23组 1.03 ± 0.14# F 3.267 P 0.043* *P < 0.05；与对照组相比，ΔP < 0.05；与antagomir-NC组相比，#P < 0.05。
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表 8 荧光素酶活性比较（n = 6，$ \bar x \pm s $）
Table 8. Comparison of luciferase activity（n = 6，$ \bar x \pm s $）
组别 荧光素酶活性 miR-NC+PI3K-WT组 1.02 ± 0.08 miR-23 mimic+PI3K-WT组 0.76 ± 0.18Δ miR-NC+PI3K-MUT组 0.99 ± 0.12 miR-23 mimic+PI3K-MUT组 0.99 ± 0.20 F 3.701 P 0.029* *P < 0.05；与miR-NC+PI3K-WT组相比，ΔP < 0.05。
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