Zhizi-Chuanxiong Herb Pair Attenuates Ox-LDLInduced Injury in RAW264.7 Cells by Regulating Autophagy

Introduction: Atherosclerosis (AS) is a chronic inflammatory disease characterized by the accumulation of plaque in the walls of arteries. Zhizi-Chuanxiong herb pair (ZCHP) is a traditional Chinese medicine formulation whose previous studies have demonstrated its inhibitory effect on AS. However, its underlying mechanisms remain unclear. Methods: RAW264.7 cells were exposed to ox-LDL and treated with low or high-dose ZCHP, with or without the autophagy inhibitor 3-methyladenine (3-MA). We assessed lipid accumulation (Oil Red O staining, BODIPY-cholesterol assay), autophagic activity (immunofluorescence for LC3/LAMP1, transmission electron microscopy), and the expression of key autophagy and Wnt/ β-catenin pathway components (western blot, RT-PCR). Results: ZCHP significantly inhibited ox-LDL-induced cellular lipid and cholesterol accumulation. Immunofluorescence and TEM revealed that ZCHP increased autophagosome formation and promoted autophagosome-lysosome fusion, which was impaired by ox-LDL. ZCHP increased the LC3-II/I ratio and Beclin 1 expression, while decreasing p62 expression. Furthermore, ZCHP also significantly decreased the mRNA levels of Wnt1, DVL1, β-catenin, and Cyclin-D1. However, these effects were reversed by 3-MA. Discussion: The protective effects of ZCHP are primarily mediated through the promotion of autophagic flux. The coordinated downregulation of the Wnt/β-catenin pathway, which is dependent on functional autophagy, reveals a novel mechanistic insight into ZCHP's anti-atherosclerotic action. However, the study is limited by its in vitro design and the potential off-target effects of 3-- MA, necessitating further validation in animal models. Conclusion: ZCHP alleviates ox-LDL-induced macrophage injury by enhancing autophagy, an effect likely achieved through suppression of the Wnt/β-catenin signaling pathway.