Zhi-Zi-Chi Decoction Alleviates Depressive-like Behaviors by Regulating Gut Microbiota and Targeting the AMPK/PI3K-TOR Pathway via Its Metabolite Protocatechuic Acid

Background: Neuroinflammation and gut–brain axis (GBX) dysregulation are key pathological drivers of stress-related neuropsychiatric disorders. Zhi-Zi-Chi Decoction (ZZCD), a classic Traditional Chinese Medicine (TCM) formula, has been clinically used to alleviate mental disturbances via the TCM principle of “clearing heat and relieving restlessness.” Still, its modern neuroprotective mechanisms, especially its links to gut microbiota and central signaling pathways, remain incompletely elucidated. Purpose: This study aimed to systematically investigate the therapeutic effects of ZZCD on chronic restraint stress (CRS)-induced neurodysfunction in mice and clarify its mechanisms from the perspectives of TCM theory, material basis, gut microbiota–metabolite axis, and central signaling pathways. Method: CRS mice were treated with ZZCD or protocatechuic acid. Behavioral tests evaluated depression- and anxiety-like behaviors. UHPLC-Q-TOF/MS identified ZZCD’s chemical constituents; 16S rRNA sequencing and untargeted metabolomics analyzed gut microbiota and metabolite changes. Western blot, immunofluorescence, and proteomics examined neuroinflammation, microglial polarization, and signaling pathway activity (PI3K/Akt/mTOR, AMPK). Results: ZZCD reversed CRS-induced depression- and anxiety-like behaviors and suppressed neuroinflammation. Mechanistically, UHPLC-Q-TOF/MS identified 424 ZZCD constituents, with prenol lipids, organooxygen compounds, and flavonoids as the most abundant. ZZCD reversed CRS-induced imbalance in gut microbiota, reducing pro-inflammatory Prevotella and enriching beneficial Lactobacillus, and mediated the enrichment of the prebiotic metabolite PCA in colonic and serum samples, which crossed the blood–brain barrier (BBB) to exert neuroprotection. Additionally, ZZCD and PCA normalized the PI3K/Akt/mTOR pathway and activated AMPK, promoting M2 microglial polarization and restoring synaptic plasticity. Conclusions: ZZCD exerts antidepressant effects by a gut-microbiota-dependent modulation of PCA-PI3K/Akt/mTOR and AMPK dual axes that converts microglia from M1 to M2, providing ethnopharmacological evidence and a mechanistic rationale for its clinical application in major depressive disorder.