Modified Danzhi Xiaoyao San ameliorates depression-associated dry eye by modulating sphingolipid metabolism across the liver, hippocampus, and lacrimal gland.

ETHNOPHARMACOLOGICAL RELEVANCE: Modified Danzhi Xiaoyao San (MDZXYS) originates from Xue Ji's "Internal Medicine Abstract" (Ming Dynasty), which derived from the Song Dynasty Taiping Huimin Heji Jufang Xiao-Yao Powder by adding Moutan Cortex and Gardeniae Fructus. It is traditionally used to soothe the liver, relieve depression, and clear stagnant heat associated with liver-qi stagnation transforming into fire. Grounded in the theory that "the liver opens into the eyes," liver-soothing and heat-clearing strategy has long been applied to treat ocular diseases. Previous studies have reported beneficial effects of Danzhi Xiaoyao San and related modified formulas on depression and dry eye; however, the metabolic basis of their effects across multiple tissues remains incompletely understood.

AIM OF THE STUDY: This study evaluated the therapeutic effects of MDZXYS in a rat model of depression-associated dry eye and characterized associated changes in sphingolipid metabolism across multiple tissues.

MATERIALS AND METHODS: A rat model of depression-associated dry eye was established by combining chronic unpredictable mild stress (CUMS) with benzalkonium chloride (BAC) eye drops. low- and high-dose MDZXYS were orally administered, with venlafaxine as the positive control. Evaluations included assessments of depression-like behaviors, tear secretion, tear film break-up time (TBUT), and corneal fluorescein staining. Histopathology, ELISA for inflammatory cytokines, targeted lipidomics, and Western blotting for Sptlc2 and nSMase were performed on hippocampal, hepatic, lacrimal gland, and serum samples.

RESULTS: MDZXYS attenuated hippocampal neuronal abnormalities in the CA1, CA3, and DG regions and increased BDNF expression. It also improved pathological changes in the liver and lacrimal glands and preserved conjunctival goblet cells. MDZXYS increased tear secretion, prolonged TBUT, and reduced corneal fluorescein staining. Behavioral assessments showed increased sucrose preference and locomotor activity, together with reduced immobility time. MDZXYS also reduced the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and increased IL-4 levels in the liver, serum, and lacrimal glands. Lipidomic profiling and KEGG enrichment analysis showed that differentially altered metabolites across the hippocampus, liver, lacrimal gland, and serum were significantly enriched in the sphingolipid metabolism pathway. Among the altered sphingolipid metabolites, Cer(d18:1/20:1), Cer(d18:1/22:1), Cer(d18:1/24:1), HexCer(18:1/24:1), SM(d18:1/16:0), and SM(d18:1/20:1) showed significant associations with depressive-like behaviors and dry eye-related phenotypes. MDZXYS treatment partially reversed these disease-associated sphingolipid alterations. Western blotting further revealed tissue-specific changes in sphingolipid metabolism-related protein expression: MDZXYS increased nSMase and decreased Sptlc2 expression in the hippocampus, whereas both proteins were upregulated in the lacrimal gland.

CONCLUSIONS: MDZXYS improved inflammatory, behavioral, and ocular abnormalities and was associated with tissue-specific changes in sphingolipid metabolism across the hippocampus, liver, and lacrimal gland. MDZXYS treatment was accompanied by tissue-specific changes in Sptlc2 and nSMase expression and partial normalization of disease-associated sphingolipid alterations. These findings support the involvement of sphingolipid dysregulation in depression-associated dry eye and suggest that MDZXYS modulates sphingolipid metabolism in a tissue-specific manner. The observed cross-tissue associations provide an exploratory metabolic context for these findings but do not establish directional or causal relationships among tissues.