TLR4/NF-kB/ROS与AKT/mTOR信号通路交叉对话在脂肪酸诱导心肌损伤的调控机制研究

Angiotensin -(1-7)[Ang-(1-7)] and glucagon like peptide -1 (GLP-1) are bioactive substance, which can protect the myocardium and improve myocardial injury. High concentration of fatty acid (palmitic acid) is closely related to cardiac diseases such as myocarditis and cardiomyopathy. TLR4/ NF-kB /ROS and AKT/mTOR signaling pathway, Ang- (1-7) and GLP-1 have little research on the regulation mechanism of fatty acid induced myocarditis and myocardial fibrosis. Myocardial cells, cardiac fibroblasts, heart removed from hypertension model and hyperlipidemia model rats were used in this study, through MTT experiment, Western blot and ELISA method, scratch experiment and immunofluorescence staining method, to clarify how the TLR4 / NF - kB and AKT/mTOR signaling pathways, GLP-1 agonists, Ang - (1-7), GLP-1 / TLR4 / NF - kB signaling pathways and Ang - (1-7)/Mas axis were involved to the mechanism of fatty acid and AngII induced myocardial injury. The results of this study can provide new targets and important theoretical basis for the development of drugs to effectively treatment myocarditis and myocardial fibrosis.

血管紧张素-(1-7)[Ang-(1-7)]及胰高血糖素样肽-1（GLP-1）作为生物体内活性物质，具有保护心肌并改善心肌损伤作用。高浓度脂肪酸（palmitic acid）与心肌炎、心肌病等心脏疾病密切相关。TLR4/NF-kB/ROS与AKT/mTOR信号通路、Ang-（1-7）及GLP-1在脂肪酸诱导心肌炎及心肌纤维化调控机理方面研究甚少。本研究主要是用心肌细胞、心成纤维细胞、高血压及高血脂大鼠离体心脏，通过MTT实验、Western blot、ELISA法、划痕实验及免疫荧光染色法，考察TLR4/NF-kB与AKT/mTOR信号通路、GLP-1受体激动剂、Ang-(1-7)、GLP-1/TLR4/NF-kB信号通路及Ang-(1-7)/Mas轴在脂肪酸与AngII诱导心肌损伤中的调控机制。本研究结果可为开发有效抑制心肌炎及心肌纤维化药物提供新靶点和重要理论依据。

在肥胖、高脂血症及2型糖尿病等疾病状态下，机体内游离脂肪酸浓度会提高。高浓度脂肪酸与心肌炎、心肌病、心肌纤维化等心脏疾病密切相关。本项目进行过程中，成功单离了大鼠心肌成纤维细胞，细胞呈长梭形或不规则三角形，中央有卵圆核，经荧光鉴定，本实验单离细胞为纯化的心肌成纤维细胞。研究胰高血糖素样肽-1（GLP-1）受体激动药Exendin-4对血管紧张素II诱导的心肌纤维化预防作用及其可能机制。血管紧张素II (Ang II) 能促进心肌成纤维细胞增殖，与心肌纤维化密切相关。Ang II诱导心肌成纤维细胞下调p-AKT蛋白表达，上调p-mTOR、TGF-β1、Collagen I及Collagen III蛋白表达而促进心肌纤维化。Exendin-4可通过抑制心肌成纤维细胞的增殖、下调ERK水平，减少Collagen I合成来发挥对心肌保护作用。研究钠-葡萄糖转运蛋白-2抑制剂恩格列净（EMPA）及达格列净（DAPA）对脂肪酸诱导H9c2心肌细胞损伤的保护作用及可能机制。采用荧光显微镜法和流式细胞仪检测脂肪酸诱导H9c2心肌细胞中ROS水平。脂肪酸可诱导心肌细胞上调TLR4蛋白及p-PI3K/PI3K、p- mTOR/mTOR表达、ROS水平显著增加，p-AKT蛋白表达显著降低。EMPA及DAPA对PA诱导的心肌细胞损伤有保护作用，其机制与下调TLR4、p-mTOR表达，增强AKT蛋白磷酸化，抑制ROS的生成有关。探讨石蒜碱及二氢石蒜碱对脂肪酸诱导H9c2细胞炎性损伤的保护作用及其机制。结果表明石蒜碱及二氢石蒜碱对脂肪酸诱导H9c2细胞细胞内活性氧产生均有显著的抑制作用。蛋白免疫印迹实验表明，二氢石蒜碱预处理能显著下调脂肪酸诱导H9c2细胞的TLR4、p-PI3K、p-mTOR蛋白表达，上调磷酸化AKT蛋白表达。石蒜碱对脂肪酸诱导H9c2细胞炎性损伤保护机制与抑制TLR4、p-PI3K、p-mTOR蛋白表达，促进Akt蛋白磷酸化，抑制炎性损伤H9c2细胞内ROS的产生有关。研究Ang(1-7)对H9c2细胞炎性损伤的保护机制。CCK-8实验筛选出Ang(1-7)最佳浓度为80 nM, 显著降低脂肪酸诱导H9c2细胞内ROS浓度。Extendin-4及Ang(1-7)对脂肪酸及Ang(II)诱导的心肌损伤保护作用可能与TLR4/ROS及Akt/mTOR信号通路相关。