Department of Cardiology, the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang, China.
Liaoning Provincial Key Laboratory of TCM Geriatric Cardio-Cerebrovascular Diseases, Shenyang, China.
J Cell Mol Med. 2024 Apr;28(8):e18257. doi: 10.1111/jcmm.18257.
This study aims to investigate the mechanism of the anti-atherosclerosis effect of Huayu Qutan Recipe (HYQT) on the inhibition of foam cell formation. In vivo, the mice were randomly divided into three groups: CTRL group, MOD group and HYQT group. The HYQT group received HYQT oral administration twice a day (20.54 g/kg/d), and the plaque formation in ApoE mice was observed using haematoxylin-eosin (HE) staining and oil red O (ORO) staining. The co-localization of aortic macrophages and lipid droplets (LDs) was examined using fluorescent labelling of CD11b and BODIPY fluorescence probe. In vitro, RAW 264.7 cells were exposed to 50 μg/mL ox-LDL for 48 h and then treated with HYQT for 24 h. The accumulation of LDs was evaluated using ORO and BODIPY. Cell viability was assessed using the CCK-8 assay. The co-localization of LC3b and BODIPY was detected via immunofluorescence and fluorescence probe. LysoTracker Red and BODIPY 493/503 were used as markers for lysosomes and LDs, respectively. Autophagosome formation were observed via transmission electron microscopy. The levels of LC3A/B II/LC3A/B I, p-mTOR/mTOR, p-4EBP1/4EBP1, p-P70S6K/P70S6K and TFEB protein level were examined via western blotting, while SQSTM1/p62, Beclin1, ABCA1, ABCG1 and SCARB1 were examined via qRT-PCR and western blotting. The nuclear translocation of TFEB was detected using immunofluorescence. The components of HYQT medicated serum were determined using Q-Orbitrap high-resolution MS analysis. Molecular docking was employed to identify the components of HYQT medicated serum responsible for the mTOR signalling pathway. The mechanism of taurine was illustrated. HYQT has a remarkable effect on atherosclerotic plaque formation and blood lipid level in ApoE mice. HYQT decreased the co-localization of CD11b and BODIPY. HYQT (10% medicated serum) reduced the LDs accumulation in RAW 264.7 cells. HYQT and RAPA (rapamycin, a mTOR inhibitor) could promote cholesterol efflux, while chloroquine (CQ, an autophagy inhibitor) weakened the effect of HYQT. Moreover, MHY1485 (a mTOR agonist) also mitigated the effects of HYQT by reduced cholesterol efflux. qRT-PCR and WB results suggested that HYQT improved the expression of the proteins ABCA1, ABCG1 and SCARB1.HYQT regulates ABCA1 and SCARB1 protein depending on the mTORC1/TFEB signalling pathway. However, the activation of ABCG1 does not depend on this pathway. Q-Orbitrap high-resolution MS analysis results demonstrated that seven core compounds have good binding ability to the mTOR protein. Taurine may play an important role in the mechanism regulation. HYQT may reduce cardiovascular risk by promoting cholesterol efflux and degrading macrophage-derived foam cell formation. It has been observed that HYQT and ox-LDL regulate lipophagy through the mTOR/TFEB signalling pathway, rather than the mTOR/4EBP1/P70S6K pathway. Additionally, HYQT is found to regulate cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis, while taurine plays a significant role in lipophagy.
本研究旨在探讨化瘀祛痰方（HYQT）抑制泡沫细胞形成的抗动脉粥样硬化作用机制。在体内，将小鼠随机分为三组：对照组（CTRL 组）、模型组（MOD 组）和 HYQT 组。HYQT 组每天口服 HYQT 两次（20.54g/kg/d），并用苏木精-伊红（HE）染色和油红 O（ORO）染色观察 ApoE 小鼠的斑块形成。用 CD11b 和 BODIPY 荧光探针荧光标记检测主动脉巨噬细胞和脂滴（LDs）的共定位。在体外，RAW 264.7 细胞用 50μg/mL 氧化型低密度脂蛋白（ox-LDL）孵育 48 小时，然后用 HYQT 处理 24 小时。用 ORO 和 BODIPY 评估 LDs 的积累。用 CCK-8 测定细胞活力。通过免疫荧光和荧光探针检测 LC3b 和 BODIPY 的共定位。用 LysoTracker Red 和 BODIPY 493/503 分别作为溶酶体和 LDs 的标记物。用透射电子显微镜观察自噬体的形成。通过 Western blot 检测 LC3A/B II/LC3A/B I、p-mTOR/mTOR、p-4EBP1/4EBP1、p-P70S6K/P70S6K 和 TFEB 蛋白水平，同时通过 qRT-PCR 和 Western blot 检测 SQSTM1/p62、Beclin1、ABCA1、ABCG1 和 SCARB1。用免疫荧光检测 TFEB 的核转位。用 Q-Orbitrap 高分辨率 MS 分析鉴定 HYQT 血清中的成分。用分子对接鉴定 HYQT 血清中负责 mTOR 信号通路的成分。阐述了牛磺酸的作用机制。HYQT 对 ApoE 小鼠的动脉粥样硬化斑块形成和血脂水平有显著影响。HYQT 降低了 CD11b 和 BODIPY 的共定位。HYQT（10%含药血清）减少了 RAW 264.7 细胞中 LDs 的积累。HYQT 和 RAPA（雷帕霉素，一种 mTOR 抑制剂）可以促进胆固醇流出，而氯喹（CQ，一种自噬抑制剂）削弱了 HYQT 的作用。此外，MHY1485（一种 mTOR 激动剂）也通过减少胆固醇流出，减轻了 HYQT 的作用。qRT-PCR 和 WB 结果表明，HYQT 改善了 ABCA1、ABCG1 和 SCARB1 蛋白的表达。HYQT 通过 mTORC1/TFEB 信号通路调节 ABCA1 和 SCARB1 蛋白。然而，ABCG1 的激活不依赖于这条通路。Q-Orbitrap 高分辨率 MS 分析结果表明，七种核心化合物对 mTOR 蛋白具有良好的结合能力。牛磺酸可能在机制调节中发挥重要作用。HYQT 可能通过促进胆固醇流出和降解巨噬细胞来源的泡沫细胞形成来降低心血管风险。已经观察到 HYQT 和 ox-LDL 通过 mTOR/TFEB 信号通路调节脂噬，而不是 mTOR/4EBP1/P70S6K 通路。此外，发现 HYQT 通过 mTORC1/TFEB/ABCA1-SCARB1 信号轴调节胆固醇流出，而牛磺酸在脂噬中起重要作用。
