Mechanisms of QingRe HuoXue Formula in atherosclerosis Treatment: An integrated approach using Bioinformatics, Machine Learning, and experimental validation

• Comprehensive bioinformatics, network pharmacology, machine learning algorithms, transcriptome sequencing, and molecular docking reveal that COL1A1 is a key target for QingRe HuoXue Formula to regulate atherosclerosis. • QingRe HuoXue Formula can promote the formation of fibrosis in atherosclerotic plaque. • QingRe HuoXue Formula can improve atherosclerosis by regulating lipid metabolism and stabilizing plaque. Atherosclerosis (AS) is the main cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. QingRe HuoXue Formula (QRHXF), a common prescription of traditional Chinese medicine, has a definite effect on the clinical treatment of AS, but its mechanism remains to be further explored. The current study aimed to demonstrate the effectiveness of the QRHXF in the treatment of AS and further reveal its potential pharmacological mechanisms. Explore the potential mechanisms of QRHXF in treating AS through network pharmacology, machine learning, transcriptome analysis, and molecular docking, then validate them through animal experiments and PCR experiments. The results indicate that through network pharmacology and machine learning methods, 10 genes including COL1A1 and CCR7 have been identified as potential candidate genes for QRHXF treatment of atherosclerosis. Molecular docking indicates that the key active compounds of QRHXF have good binding affinity with the predicted genes. Two key genes, COL1A1 and CCR7, were identified through transcriptome sequencing analysis of the aortic tissue of APOE -/- mice in the AS model. Finally, the animal and PCR experiment found that QRHXF can effectively reduce the formation of aortic plaques in APOE -/- mice of the AS model, lower blood lipid levels in mice, and upregulate the mRNA expression level of COL1A1, promoting the formation of fibrosis within plaques. We revealed the inflammatory and immune pathways underlying QRHXF treatment for AS, and verified through transcriptome sequencing and experiments that QRHXF can promote the expression of COL1A1, thereby increasing the stability of AS plaques.