Mechanism of Huayu Qutan recipe anti-atherosclerosis mediates lipophagymammalian target of rapamycin complex 1/ transcription factor EB signaling pathway in ApoE-/-mice.

Abstract

Objective: To investigate the effects of Huayu Qutan recipe (, HYQT) on the atherosclerosis (AS) model of ApoE-/- mice with a high-fat diet and to illustrate the underlying mechanisms from modern patho-physiological conceptualizations.

Methods: High performance liquid chromatography of quadrupole time of flight-tandem mass spectrometry (HPLC-Q-TOF-MS/MS) analysis was used to identify the active compounds in the recipe. The mice were randomly allocated into 7 groups: control (CTRL) group, normal diet (ND) group, high-fat diet (HFD) group, HYQT groups (low dose, medium dose, and high dose), and simvastatin (SIM) group. Deferent doses of HYQT were gavaged twice a day, and then the protective effect of HYQT on plaque formation in ApoE-/- mice with a high-fat diet was verified viahematoxylin-eosin (HE) staining and oil red o (ORO) staining. We observed the co-localization in aortic macrophages and lipid droplets (LDs) by CD68 and the Bodipy fluorescence probe. Light chain 3 phosphoprotein class Ⅱ/light chain 3 phosphoprotein class Ⅰ (LC3Ⅱ/LC3Ⅰ) was examined by western blotting, and sequestosome 1 (SQSTM1/p62), Beclin1, Lamp1, mammalian target of rapamycin (mTOR), phosphorylated mammalian target of rapamycin (p-mTOR), and ATP-binding cassette transporter A1 (ABCA1) were examined by real-time polymerase chain reaction (RT-PCR) and Western blotting. Transcription factor EB (TFEB) nuclear translocation was determined by immunofluorescence analysis.

Results: Five active compounds were identified using HPLC-Q-TOF-MS/MS analysis: ferulic acid, chlorogenic acid, calycosin, formononetin, and 8,2'-dihydroxy-7,4'-dimethoxy-isoflavane. The effect of HYQT on atherosclerotic plaque formation in ApoE-/- mice was investigated. These findings showed that HYQT decreased the co-localization of CD68 and Bodipy and increased the co-localization of CD68 and LC3B. Medium and high doses of HYQT increased autophagosome formation and promoted the maturation of LC3Ⅱ/LC3Ⅰ. Additionally, HYQT decreased the expression of SQSTM1/p62. Medium and high doses of HYQT also increased the expression of Beclin1 and Lamp1. RT-PCR and Western blot results suggested that HYQT enhanced the expression of ABCA1 mRNA and protein and regulated the mTORC1/TFEB signaling pathway.

Conclusion: The results indicate that HYQT is an effective traditional Chinese herbal remedy for the treatment of AS. HYQT mitigates macrophage-derived foam cell formation by activating autophagy in atherosclerosis. The mTOR/TFEB signaling pathway and ABCA1 are therapeutic targets of HYQT for the treatment of AS.