Huoxue Tongluo tablet enhances atherosclerosis efferocytosis by promoting the differentiation of Trem2 macrophages via PPARγ signaling pathway.

Background: Atherosclerosis (AS) serves as the primary pathological basis for various cardiovascular and cerebrovascular diseases. Impaired efferocytosis by macrophages within AS plaques exacerbates lipid metabolism disorders and inflammatory responses. Huoxue Tongluo Tablet (HXTL), a traditional Chinese medicine formula, has shown efficacy in treating AS and modulating macrophage function. However, its underlying mechanisms remain unclear. It is hypothesized that HXTL ameliorates AS by enhancing macrophage efferocytosis.

Purpose: To assess the efficacy and mechanisms of HXTL in treating AS at the single-cell level.

Methods: Ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was used to analyze the constituents of HXTL. HXTL was administered to ApoE⁻/⁻ mice maintained on a high-fat diet. The progression of AS was evaluated by measuring atherosclerotic plaque area, necrotic core formation, collagen depletion, lipid accumulation, lipid profiles, pro-inflammatory mediators, and oxidative stress markers. Transcriptomic analysis was performed to explore the mechanisms underlying the therapeutic effects of HXTL on AS. Efferocytosis-related marker expression was evaluated using immunohistochemistry and quantitative PCR (qPCR), and the efferocytosis index was determined by the co-localization of apoptotic cells and macrophages. Efferocytosis inhibition was induced using Cytochalasin D. Single-cell sequencing was utilized to investigate alterations in Trem2⁺ macrophages following HXTL treatment. Trem2 expression was accessed by immunohistochemistry and qPCR, while flow cytometry and immunofluorescence staining confirmed the changes in Trem2⁺ macrophages. Bioinformatic analyses were conducted to investigate the mechanism through which HXTL enhances efferocytosis by regulating Trem2⁺ macrophage subsets. Western blotting and qPCR were used to assess the expression levels of PPARγ signaling, and the regulatory role of PPARγ signaling in macrophage subpopulation generation and efferocytosis function was accessed using GW9662.

Results: UPLC-MS/MS analysis identified 99 major components in HXTL. In vivo, medium and high doses of HXTL significantly reduced atherosclerotic plaque area, improved lipid profiles, decreased pro-inflammatory mediators and reactive oxygen species (ROS), and enhanced the efferocytosis function. Inhibition of efferocytosis reversed these beneficial effects. Single-cell sequencing and in vivo validation revealed that HXTL upregulated Trem2⁺ macrophages and efferocytosis-related genes. Bioinformatics and in vivo experiments demonstrated that HXTL activated PPARγ signaling, and inhibition of PPARγ signaling negated the pro-efferocytosis effects and the upregulation of Trem2⁺ macrophage upregulation induced by HXTL.

Conclusions: HXTL activates the PPARγ pathway, upregulates Trem2⁺ macrophages, and enhances macrophage efferocytosis, thereby ameliorating AS. This study is the first to demonstrate the regulatory effects of HXTL on macrophage subpopulations and its pro-efferocytosis activity.