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.

中文翻译：

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化瘀祛痰方通过mTORC1/TFEB/ABCA1-SCARB1信号轴促进脂肪自噬和胆固醇外流

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的机制。在体内，将小鼠随机分为三组：CTRL组、MOD组和HYQT组。 HYQT组口服HYQT，每日2次（20.54 g/kg/d），ApoE斑块形成−/−使用苏木精-伊红（HE）染色和油红O（ORO）染色观察小鼠。使用 CD11b 荧光标记和 BODIPY 荧光探针检查主动脉巨噬细胞和脂滴 (LD) 的共定位。在体外，RAW 264.7 细胞暴露于 50 μg/mL ox-LDL 48 小时，然后用 HYQT 处理 24 小时。使用 ORO 和 BODIPY 评估 LD 的积累。使用 CCK-8 测定评估细胞活力。通过免疫荧光和荧光探针检测 LC3b 和 BODIPY 的共定位。 LysoTracker Red 和 BODIPY 493/503 分别用作溶酶体和 LD 的标记物。通过透射电子显微镜观察自噬体的形成。通过蛋白质印迹法检测LC3A/B II/LC3A/BI、p-mTOR/mTOR、p-4EBP1/4EBP1、p-P70S6K/P70S6K和TFEB蛋白水平，而SQSTM1/p62、Beclin1、ABCA1、ABCG1和通过 qRT-PCR 和蛋白质印迹法检查 SCARB1。使用免疫荧光检测TFEB的核转位。使用 Q-Orbitrap 高分辨率 MS 分析测定 HYQT 含药血清的成分。采用分子对接来鉴定 HYQT 含药血清中负责 mTOR 信号通路的成分。阐明了牛磺酸的作用机制。 HYQT对ApoE中动脉粥样硬化斑块形成和血脂水平有显着影响−/−老鼠。 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 LD 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）则削弱了HYQT的作用。此外，MHY1485（一种 mTOR 激动剂）还通过减少胆固醇流出来减轻 HYQT 的影响。 qRT-PCR和WB结果表明，HYQT提高了ABCA1、ABCG1和SCARB1蛋白的表达。HYQT根据mTORC1/TFEB信号通路调节ABCA1和SCARB1蛋白。然而，ABCG1 的激活并不依赖于该途径。 Q-Orbitrap高分辨率MS分析结果表明7种核心化合物与mTOR蛋白具有良好的结合能力。牛磺酸可能在该机制调节中发挥重要作用。 HYQT 可以通过促进胆固醇流出和降低巨噬细胞衍生的泡沫细胞形成来降低心血管风险。据观察，HYQT 和 ox-LDL 通过 mTOR/TFEB 信号通路而不是 mTOR/4EBP1/P70S6K 通路调节脂肪吞噬。此外，HYQT 通过 mTORC1/TFEB/ABCA1-SCARB1 信号轴调节胆固醇流出，而牛磺酸在脂肪自噬中发挥重要作用。