Li Fangqin, Peng Jin, Lu Yanlin, Zhou Ming, Liang Jingwei, Le Cuiyun, Ding Jiuyang, Wang Jiawen, Dai Jialin, Wan Changwu, Wang Jie, Luo Peng, Xia Bing
School of Forensic Medicine, Guizhou Medical University, 9 Beijing Road, Guiyang 550000, Guizhou, China.
School of Forensic Medicine, Zunyi Medical University, Zunyi 563000, Guizhou, China.
Int J Cardiol. 2023 Dec 1;392:131303. doi: 10.1016/j.ijcard.2023.131303. Epub 2023 Aug 29.
Autophagy is important in regulating inflammation and cholesterol efflux, suggesting that targeting autophagy may slow down atherosclerosis (AS). Since the pathological basis of coronary artery disease (CAD) is atherosclerosis, it is crucial to investigate the role of autophagy in atherosclerosis. This study aimed to investigate the role of the chemokine CXC chemokine receptor 4 (CXCR4) in promoting macrophage autophagy through the phosphoinositide-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway to alleviate coronary artery disease.
The human left coronary artery and myocardium were collected to detect CXCR4, MAP1LC3(LC3) and SQSTM1(p62) expression. ApoE-/- mice were used to establish an atherosclerosis mice model, while human monocytes (THP-1) were used to establish a foam cell model and co-cultured with foam cells using siRNACXCR4. Western blotting was conducted to quantify CXCR4, PI3K/AKT/mTOR pathway protein, LC3, Beclin1 and p62 protein levels. The left coronary artery from humans and mouse aorta and myocardium were stained with Hematoxylin and Eosin (H&E), macrophages with Oil Red O staining and foam cells were assessed by Movat's staining. CXCR4 levels, PI3K/AKT/mTOR pathway protein, LC3 and p62 were detected by immunohistochemistry (IHC) and immunofluorescence assays. Detection of autophagosomes in macrophages using transmission electron microscopy. We further assessed whether the effect of CXCR4-mediated macrophage autophagy on the formation of atherosclerosis and structural changes in the myocardium was mediated via the PI3K/AKT/mTOR signaling pathway.
CXCR4 and p62 proteins were upregulated in human coronary lesions, mouse aorta, myocardial tissue, and foam cells, while LC3II/LC3I was downregulated. p85 (P-PI3K), Ser473 (P-AKT), and Ser2448 (P-mTOR) phosphorylated proteins associated with the PI3K/AKT/mTOR pathway were detected in AS and foam cell models. Upregulated CXCR4 inhibited autophagy of macrophages and increased the severity of atherosclerotic lesions. After specific knockdown of CXCR4 by adeno-associated virus (AAV9-CXCR4-RNAi) and siRNACXCR4, the above indicators were reversed, macrophage autophagy was promoted, the severity of atherosclerotic lesions was reduced, and the disorganized arrangement of myocardial architecture was improved.
Knockdown of CXCR4 reduces the extent of coronary artery disease by promoting macrophage autophagy through the PI3K/AKT/mTOR pathway to attenuate atherosclerosis.
自噬在调节炎症和胆固醇流出中起重要作用，提示靶向自噬可能减缓动脉粥样硬化（AS）。由于冠状动脉疾病（CAD）的病理基础是动脉粥样硬化，因此研究自噬在动脉粥样硬化中的作用至关重要。本研究旨在探讨趋化因子CXC趋化因子受体4（CXCR4）通过磷酸肌醇-3激酶/蛋白激酶B/雷帕霉素哺乳动物靶蛋白（PI3K/AKT/mTOR）途径促进巨噬细胞自噬以减轻冠状动脉疾病的作用。
收集人左冠状动脉和心肌以检测CXCR4、微管相关蛋白1轻链3（LC3）和 sequestosome 1（p62）的表达。采用载脂蛋白E基因敲除（ApoE-/-）小鼠建立动脉粥样硬化小鼠模型，用人单核细胞（THP-1）建立泡沫细胞模型，并使用小干扰RNA（siRNA）CXCR4与泡沫细胞共培养。进行蛋白质免疫印迹法以定量CXCR4、PI3K/AKT/mTOR途径蛋白、LC3、Beclin1和p62蛋白水平。用人的左冠状动脉以及小鼠主动脉和心肌进行苏木精-伊红（H&E）染色，用油红O染色巨噬细胞，并用Movat染色评估泡沫细胞。通过免疫组织化学（IHC）和免疫荧光测定法检测CXCR4水平、PI3K/AKT/mTOR途径蛋白、LC3和p62。使用透射电子显微镜检测巨噬细胞中的自噬体。我们进一步评估CXCR4介导的巨噬细胞自噬对动脉粥样硬化形成和心肌结构变化的影响是否通过PI3K/AKT/mTOR信号通路介导。
CXCR4和p62蛋白在人冠状动脉病变、小鼠主动脉、心肌组织和泡沫细胞中上调，而LC3II/LC3I下调。在AS和泡沫细胞模型中检测到与PI3K/AKT/mTOR途径相关的p85（磷酸化PI3K）、丝氨酸473（磷酸化AKT）和丝氨酸2448（磷酸化mTOR）磷酸化蛋白。CXCR4上调抑制巨噬细胞自噬并增加动脉粥样硬化病变的严重程度。通过腺相关病毒（AAV9-CXCR4-RNAi）和siRNA CXCR4特异性敲低CXCR4后，上述指标得到逆转，促进了巨噬细胞自噬，降低了动脉粥样硬化病变的严重程度，并改善了心肌结构的紊乱排列。
敲低CXCR4通过PI3K/AKT/mTOR途径促进巨噬细胞自噬以减轻动脉粥样硬化，从而降低冠状动脉疾病的程度。
