Tanyu Tongzhi Formula Relieves the progression of atherosclerotic plaque through Lipid Regulation and Anti-inflammatory effects

Tingting Chen^1,2,3Lingling Xie¹Yan Zhao¹Li Wu¹Shishi Huang^1,2Wei Mao^2,3,4*

• ¹The First School of Clinical Medicine, Zhejiang Chinese Medical University, Hangzhou, Jiangsu Province, China
• ²Zhejiang Hospital (Affiliated Zhejiang Hospital, Zhejiang University School of Medicine), Zhejiang Key Laboratory of Integrative Chinese and Western Medicine for Diagnosis and Treatment of Circulatory Diseases, Hangzhou, Jiangsu Province, China
• ³Zhejiang Hospital (Affiliated Zhejiang Hospital, Zhejiang University School of Medicine), Zhejiang Engineering Research Center for Precise Diagnosis and Innovative Traditional Chinese Medicine for Cardiovascular Diseases, Hangzhou, Jiangsu Province, China
• ⁴Cardiovascular department, Zhejiang Hosipital (Affiliated Zhejiang Hospital Zhejiang University School of Medicine), Hangzhou, Jiangsu Province, China

Objective: Tanyu Tongzhi Formula (TTF), a clinically proven empirical prescription, has been utilized to treat atherosclerosis (AS) for decades. This study aimed to investigate the therapeutic mechanisms of TTF against AS by integrating bioinformatics, multi-omics, and experimental validation. Methods: The metabolites of TTF in serum were identified using Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). Bioinformatics was employed to predict drug targets and mechanisms of action. ApoE-/- C57BL/6J mice were fed a 12-week high-fat diet to establish an AS model and were treated with TTF crude extract (2.25 g/kg/d) via gavage. Interleukin 6 (IL-6) and interleukin 1β (IL-1β) were measured at weeks 6, 10, and 12. At the 12-week endpoint, aortic plaque formation and liver histopathology were evaluated. Liver transcriptomics and serum-targeted lipid metabolomics were performed to assess TTF's regulatory effects on lipid metabolism. In vitro, peritoneal macrophages (PMs) were pretreated with TTF-containing serum for 1 hour before LPS (2 µg/mL) stimulation. IL-6 and interleukin 10 (IL-10) mRNA were measured by RT-PCR, while NOD-like receptor thermal protein domain associated protein 3 (NLRP3), IL-1β, interleukin 18 (IL-18) and IL-6 expression were assessed by Western blot (WB). Results: Bioinformatics identified 28 key targets of TTF in AS treatment, primarily associated with inflammation and lipid metabolism. TTF significantly reduced aortic plaque area, attenuated hepatic steatosis, and enhanced plaque collagen content. It decreased the serum levels of lipids and pro-inflammatory mediators (IL-6 and IL-1β) in AS mice. Sphingolipids are the most significantly different lipids. In LPS-stimulated PMs, TTF suppressed IL-6 mRNA and NLRP3 inflammasome activation while upregulating IL-10 mRNA. Conclusions: TTF exerts its anti-atherosclerotic effect through inflammation reduction. These findings provide a scientific basis for its clinical application in AS treatment.