Stigmasterol attenuates atherosclerosis by inhibiting inflammatory signaling and foam cell formation

Animal experiments used 8-week-old male ApoE-deficient mice randomly assigned to four groups of ten each. The control group received a standard Western diet with 0.2% (w/w) cholesterol, while the other groups were fed the same diet supplemented with 0.2 g kg-1 of phytosterol mixture (containing beta-sitosterol, campesterol, and stigmasterol), cholesterol oxidation products (COPs), or phytosterol oxidation products (POPs) for 14 weeks, with this dose corresponding to an estimated human equivalent of 2.77 mg kg-1 body weight per day. After feeding, aortic tissues were harvested, fat removed, minced, and enzymatically digested at 37 C until fully dissociated. The resulting cell suspension was filtered through a 100 um cell strainer and centrifuged at 3000 rpm for 5 min at 4 C. The pellet was resuspended in PBS with 0.04% BSA, incubated with red blood cell lysis buffer, and recentrifuged. Cells were stained for viability using LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and Hoechst 33342, sorted for nucleated cells via flow cytometry, and dead cells were removed, resulting in over 85% viability confirmed by trypan blue exclusion. Viable single-cell suspensions were counted and adjusted to 700-1200 cells/uL before loading onto the 10x Genomics Chromium Controller for single-cell capture. Subsequent cDNA synthesis and library construction followed standard protocols, with libraries sequenced on an Illumina NovaSeq 6000 using 150bp paired-end reads targeting 60,000 reads per cell. FASTQ files were processed in Seurat v3.1.1 for normalization, principal component analysis (top 10 components), t-SNE clustering, marker gene identification, and differential gene expression analysis (DEGs at p < 0.01). DEGs with log2 fold change > 0.26 were analyzed for gene ontology enrichment using DAVID, with downstream analysis conducted on the LC-Biotechnology cloud platform.