Department of Cardiology, Second Affiliated Hospital of Naval Medical University, Shanghai 200003, P.R. China.
Int J Mol Med. 2023 Apr;51(4). doi: 10.3892/ijmm.2023.5237. Epub 2023 Mar 17.
Increased levels of serum free fatty acids (FFAs) are closely associated with microvascular dysfunction. In our previous study, a coronary microvascular dysfunction (CMD) model was successfully established via lipid infusion to increase the levels of serum FFAs in mice. However, the underlying mechanisms remained poorly understood. Therefore, the aim of the present study was to explore the mechanism underlying FFA‑induced CMD. A CMD mouse model was established via lipid combined with heparin infusion for 6 h to increase the concentration of serum FFAs. Following the establishment of the model, the coronary flow reserve (CFR), extent of leukocyte activation and cardiac microvascular structures were assessed in the mice. Cardiac microvascular endothelial cells (CMECs) were treated with different concentrations of palmitic acid and cell viability was evaluated. Changes in the expression levels of AMP‑activated protein kinase (AMPK), Krüppel‑like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) were identified by immunohistochemical and western blot analyses. Experiments using AMPK activator, KLF2 overexpression plasmid, small interfering RNAs and nicorandil were subsequently designed to investigate the potential involvement of the AMPK/KLF2/eNOS signaling pathway. These experiments revealed that FFAs could induce CMD in mice, which was characterized by reduced CFR (1.89±0.37 vs. 2.74±0.30) and increased leukocyte adhesion (4,350±1,057.5 vs. 11.8±5.4 cells/mm) compared with the control mice. CD11b expression and intracellular reactive oxygen species (ROS) levels were increased in CMD model mice compared with control mice. Serum TNF‑α and IL‑6 levels were higher in the model group than in the control group. Transmission electron microscopy revealed that CMECs in heart tissues of model mice were severely swollen. In addition, palmitic acid decreased CMEC viability and increased ROS production in a dose‑dependent manner. Notably, the AMPK/KLF2/eNOS signaling pathway was demonstrated to be suppressed by FFAs both and . Activation of this axis with AMPK activator, KLF2 overexpression plasmid or nicorandil restored the CFR in CMD model mice, inhibited oxidative stress and increased CMEC viability. Taken together, the results of the present study demonstrated that FFAs could induce CMD via inhibition of the AMPK/KLF2/eNOS signaling pathway, whereas activation of this pathway led to the alleviation of FFA‑induced CMD, which may be a therapeutic option for CMD.
血清游离脂肪酸（FFAs）水平升高与微血管功能障碍密切相关。在我们之前的研究中，通过脂质输注成功建立了冠状动脉微血管功能障碍（CMD）模型，以增加小鼠血清 FFAs 水平。然而，其潜在机制仍知之甚少。因此，本研究旨在探讨 FFA 诱导的 CMD 的机制。通过脂质联合肝素输注 6 小时来增加血清 FFAs 浓度，建立 CMD 小鼠模型。在建立模型后，评估小鼠的冠状动脉血流储备（CFR）、白细胞激活程度和心脏微血管结构。用不同浓度的棕榈酸处理心脏微血管内皮细胞（CMECs）并评估细胞活力。通过免疫组织化学和 Western blot 分析鉴定 AMP 激活蛋白激酶（AMPK）、Krüppel 样因子 2（KLF2）和内皮型一氧化氮合酶（eNOS）表达水平的变化。随后设计了 AMPK 激活剂、KLF2 过表达质粒、小干扰 RNA 和尼克地尔的实验，以研究 AMPK/KLF2/eNOS 信号通路的潜在参与。这些实验表明，FFAs 可诱导小鼠发生 CMD，其特征为 CFR 降低（1.89±0.37 比 2.74±0.30）和白细胞黏附增加（4350±1057.5 比 11.8±5.4 个细胞/mm），与对照组相比。CMD 模型小鼠的 CD11b 表达和细胞内活性氧（ROS）水平升高。与对照组相比，模型组的血清 TNF-α和 IL-6 水平升高。透射电子显微镜显示心脏组织中的 CMECs 严重肿胀。此外，棕榈酸呈剂量依赖性降低 CMEC 活力并增加 ROS 生成。值得注意的是，FFAs 既抑制了 ，又抑制了 。用 AMPK 激活剂、KLF2 过表达质粒或尼克地尔激活该轴可恢复 CMD 模型小鼠的 CFR，抑制氧化应激并增加 CMEC 活力。综上所述，本研究结果表明，FFAs 可通过抑制 AMPK/KLF2/eNOS 信号通路诱导 CMD，而激活该通路可缓解 FFA 诱导的 CMD，这可能是 CMD 的一种治疗选择。
