**Background:** Age-related macular degeneration (AMD) is a leading cause of blindness, with cholesterol dysregulation, oxidative stress, inflammation, and gut microbiota changes implicated in its pathogenesis. Qijudihuang pill (QP), a traditional Chinese medicine composed of eight herbs, has been used clinically for AMD but its mechanisms are unknown. This study investigated QP's effects on cholesterol metabolism, oxidative stress, inflammation, and gut microbiota in a high-fat diet (HFD) mouse model of AMD.
**Methods:** Four-week-old male C57BL/6J mice were randomly divided into three groups (n=8/group): control diet (CD), HFD (78.75% control diet + 10% lard, 10% corn oil, 1% cholesterol, 0.25% sodium cholate) for 13 weeks, and HFD+QP (same HFD for 13 weeks followed by daily oral QP decoction at 14.69 g/kg for 30 days). After treatment, cholesterol levels in serum, liver, retina, and RPE/choroid were measured using an Amplex Red Cholesterol Assay kit. Expression of cholesterol metabolism genes (Abca1, Abcg1, Cyp27a1, Cyp46a1, Nr1h3, SREBP2), antioxidant genes (Catalase, Gpx1, Sod1), and pro-inflammatory cytokines (Il-1β, Tnfα) was quantified by qRT-PCR. Gut microbiota was analyzed by 16S rRNA sequencing (V3-V4 region) and functional metagenomes predicted using PICRUSt2.
**Key Results:** HFD significantly increased cholesterol levels in serum, liver, retina, and RPE/choroid compared to CD (p<0.05 to p<0.0001). QP treatment reversed these elevations, with cholesterol levels in HFD+QP significantly lower than HFD alone (p<0.05 to p<0.0001). Expression of cholesterol efflux and metabolism genes (Abca1, Abcg1, Cyp27a1, Nr1h3) was significantly decreased in HFD liver, RPE/choroid, and retina compared to CD, and QP significantly increased their expression (p<0.05 to p<0.0001). Cyp46a1 was downregulated in HFD liver and RPE/choroid but upregulated in retina; QP restored expression in liver and RPE/choroid. SREBP2 expression was significantly increased by HFD in all tissues and reduced by QP (p<0.05 to p<0.0001). Antioxidant genes (Catalase, Gpx1, Sod1) were significantly downregulated in HFD RPE/choroid and retina versus CD, and QP significantly upregulated them (p<0.05 to p<0.0001). Pro-inflammatory cytokines Il-1β and Tnfα were significantly upregulated in HFD RPE/choroid, retina, and liver compared to CD, and QP significantly reduced their expression (p<0.05 to p<0.0001). Gut microbiota analysis showed HFD induced significant changes in beta diversity (Jaccard and Bray-Curtis metrics) and increased the Bacteroidetes/Firmicutes ratio, which was partially reversed by QP. LEfSe analysis identified 13 taxa increased and 19 decreased in HFD versus CD, while HFD+QP had only 2 increased and 13 decreased taxa compared to CD. PICRUSt2 predicted 127 MetaCyc pathways significantly different between CD and HFD; of these, 55 pathways showed no significant difference between CD and HFD+QP, indicating QP reversed HFD-induced changes. Specifically, HFD increased pathways for pro-inflammatory compounds (enterobactin, polymixin B, lipopolysaccharide) and decreased pathways for nucleoside/nucleotide biosynthesis, all reversed by QP.
**Clinical Implications:** This study provides the first mechanistic evidence that QP can counteract HFD-induced cholesterol accumulation, oxidative stress, and inflammation in ocular tissues, and partially restore gut microbiota composition and metabolic function. These findings support the traditional use of QP for AMD and suggest it may act through multiple pathways including cholesterol metabolism, antioxidant defense, anti-inflammatory effects, and gut microbiota modulation. The results highlight the potential of QP as a therapeutic strategy for AMD, though further studies are needed to identify active compounds, assess toxicity, and confirm efficacy in human patients.