Integrated metabolomics and proteomics revealed the comprehensive mechanism of Sijunzi decoction and its active components in improving multi-system dysfunction induced by spleen deficiency syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: Sijunzi decoction (SJZD) is a classic formula in traditional Chinese medicine (TCM) for the treatment of spleen deficiency syndrome (SDS). The chemical composition of SJZD mainly consists of polysaccharides (SJZDP), oligosaccharides (SJZD-OGS), and small-molecule components (SJZD-SMC) dominated by flavonoids, ginsenosides, and terpenoids. Studies have shown that SJZDP, SJZD-OGS, and SJZD-SMC form the material basis of SJZD's efficacy in ameliorating SDS, particularly by regulating gastrointestinal hormones, intestinal barrier function, and the gut microbiota. However, the underlying mechanism by which SJZD and its active components ameliorate SDS remains unclear. AIM OF THE STUDY: The present study aimed to systematically elucidate the potential mechanisms and key molecular targets of SJZD and its active components (SJZDP, SJZD-OGS, and SJZD-SMC) in alleviating SDS. MATERIALS AND METHODS: A rat model of SDS was developed using a multi-factorial approach to mimic its clinical manifestations. The in vivo pharmacodynamic differences of SJZD and its active components (SJZDP, SJZD-OGS, and SJZD-SMC) were evaluated. An integrated strategy combining untargeted metabolomics and proteomics was employed to explore the mechanisms of action of these active components. Subsequently, the Seahorse mitochondrial stress test, targeted metabolomics, immunohistochemistry, and Western blotting were used to verify the effects of SJZD and its active components on key metabolic pathways and protein targets. RESULTS: SJZD significantly restored disordered gastrointestinal hormones and impaired intestinal barrier function in SDS rats, with SJZDP showing good effects on both. Meanwhile, SJZD-OGS primarily restored intestinal barrier function, while SJZD-SMC mainly regulated gastrointestinal hormone levels. Untargeted metabolomics analysis revealed that, under the synergistic effect of active components, SJZD affected the core pathways of amino acid and energy metabolism by regulating key differential metabolites (e.g., L-ornithine, L-citrulline, and fumaric acid). Furthermore, it specifically regulated pyrimidine metabolism (via SJZDP/SJZD-OGS/SJZD-SMC), unsaturated fatty acid metabolism (via SJZDP/SJZD-OGS), and glycerophospholipid metabolism (via SJZD-SMC), thereby ameliorating hepatic metabolic disturbances in SDS rats. Proteomics analysis indicated that SJZD exerted its effects by regulating immune function, energy metabolism, glycerophospholipid metabolism, and multiple signaling pathways, including the mTOR and Notch pathways. Specifically, SJZD-OGS and SJZDP jointly modulated lipid and energy metabolism pathways, while SJZD-OGS and SJZD-SMC collaboratively regulated immune function. Meanwhile, SJZDP influenced amino acid metabolism, and SJZD-SMC promoted glycerophospholipid metabolism while also affecting multiple endocrine pathways. Collectively, the three active components alleviated SDS through a multi-target, multi-pathway mechanism. Abcg8, Ctsk, Ciapin1, and Esr1 were identified as common regulatory targets and may serve as key mediators of the therapeutic effects. Experimental validation demonstrated that SJZD and its active components restored levels of glycolytic and TCA cycle intermediates (e.g., fumaric acid) and mitochondrial respiratory capacity, thereby rectifying energy metabolism abnormalities caused by SDS. Correlation analysis further revealed that the key metabolites (such as amino acids and TCA cycle intermediates) regulated by SJZD and its components were closely associated with the differential expression of specific proteins. Activation of the ESR1/AMPK and LKB1/AMPK signaling pathways might be a key mechanism by which these active components restore energy-metabolism homeostasis. CONCLUSION: SJZDP, SJZD-OGS, and SJZD-SMC collaboratively contributed to the therapeutic efficacy of SJZD in alleviating SDS by restoring gastrointestinal function and hepatic metabolism (centered on amino acid and energy metabolism). This was achieved through the regulation of key metabolites (e.g., L-ornithine, L-citrulline, and fumaric acid) and key targets (Abcg8, Ctsk, Ciapin1, and Esr1), and activation of the ESR1/AMPK and LKB1/AMPK signaling pathways. This study comprehensively elucidated the potential mechanisms underlying SJZD's therapeutic effects on SDS and clarified the distinct contributions of its active components. It also provided a valuable reference for systematically exploring the material basis of TCM based on active components.