Qiju Dihuang Pill modulates complement system in age-related dry eye disease: Tripartite validation through clinical-omics-animal integration

ETHNOPHARMACOLOGICAL RELEVANCE: Qiju Dihuang Pill (QJDHW) is a traditional Chinese medicine (TCM) formulation used for treating ocular diseases, and clinical evidence has demonstrated its efficacy in managing dry eye disease (DED). Age-related DED is characterized by tear film instability and ocular surface dysbiosis. Current therapeutic agents face challenges in achieving sustained efficacy. TCM offers a multi-targeted intervention strategy for age-related DED. However, the molecular mechanisms underlying QJDHW, a classical TCM formulation, remain to be systematically elucidated. AIM OF THE STUDY: This study aims to investigate the proteomic profile of age-related DED in clinical practice and further explore the specific mechanisms of QJDHW in treating age-related DED. METHODS: We established a tripartite research framework integrating clinical phenotyping, omics profiling, and herbal intervention. This study enrolled 30 participants at the First Affiliated Hospital of Hunan University of Chinese Medicine: 15 with age-related DED and 15 healthy controls. All subjects met established diagnostic, inclusion, and exclusion criteria. Morning fasting blood samples were collected, with serum separated by centrifugation for subsequent data-independent acquisition (DIA) proteomic analysis. Biomarkers and associated mechanisms were identified through bioinformatics analysis. An age-related DED rat model was established to investigate the mechanism of QJDHW (Beijing Tongrentang). Methodologies included: ocular surface assessment via tear film breakup time (BUT), Schirmer's test (SIT), and corneal fluorescein staining; spatial learning/memory evaluation using the Morris water maze (platform crossings and escape latency); histopathological examination of corneal and lacrimal gland tissues through hematoxylin-eosin (HE) staining; and multi-platform quantification of complement regulators (C3, C5, CFHR3, CD59) and inflammatory markers (IL-6) using immunohistochemistry, Western blotting, and RT-qPCR. RESULTS: Proteomic analysis revealed significant enrichment of the complement and coagulation cascades pathway, with key differential proteins including C5, CD59, and CFHR3, etc (P < 0.05). High-dose QJDHW (2.43 g/kg/day) significantly improved tear secretion, prolonged tear film stability, and repaired the morphology and function of the cornea and lacrimal gland. Mechanistically, QJDHW upregulated complement inhibitors (CFHR3, CD59) while downregulating complement activators (C3, C5) and IL-6 expression. CONCLUSION: QJDHW exerts therapeutic effects through modulation of the complement activation-inhibition system, effectively suppressing C3/C5-mediated inflammatory cascades. This study provides molecular-level evidence supporting TCM's holistic regulation strategy in age-related ocular disorders.