Xiangsha Liujunzi decoction restores mitochondrial function and ameliorates chronic ulcerative colitis by regulating the PI3K/AKT/Nrf2 and AMPK/SIRT1/PGC-1α signaling pathways

Ethnopharmacological relevance: Xiangsha Liujunzi Decoction (XS) is a classical formula traditionally employed to invigorate the spleen and treat digestive system disorders. Although XS has a therapeutic effect on chronic ulcerative colitis (UC) in clinical practice, the mechanism remains unclear.

Aim of the study: To investigate the therapeutic effects and underlying mechanisms of XS on dextran sodium sulfate (DSS)-induced chronic UC in mice and the LPS-induced RAW264.7 cell inflammation model.

Materials and methods: The chemical components of XS were identified using UPLC-Q-Orbitrap HRMS. The chronic UC mouse model and LPS-induced RAW264.7 cell inflammation model were established and treated with XS. Body weight, disease activity index (DAI), colon length, colonic mucosal damage index (CMDI), and histopathology changes in colonic tissues were assessed. The mRNA expression of inflammatory factors (IL-6, IL-1β, and TNF-α) in mice and cells was measured by qRT-PCR, while the expression of occludin and ZO-1 proteins and mRNAs in colonic tissue was detected by Western Blot (WB) and qRT-PCR. Transmission electron microscopy was used to observe the mitochondrial ultrastructure in colonic tissue. Cell viability was determined using the CCK-8 assay. Changes in Reactive Oxygen Species (ROS) and mitochondrial membrane potential (MMP) were measured by flow cytometry. A PI3K Inhibitor (LY294002) and an AMPK Inhibitor (M2238) were employed to confirm the specificity of the cellular signaling pathways. Molecular docking was performed to validate the binding ability between the active compound and the targets.

Results: We identified 50 compounds in XS, mainly Flavonoids and organic acids. In the chronic UC mouse model, XS alleviated weight loss, reduced DAI scores, increased colon length, and improved colonic mucosal and pathological changes; inhibited the mRNA expression of inflammatory factors (IL-6, IL-1β, TNF-α); and significantly increased the mRNA and protein expression of tight-junction proteins occludin and ZO-1 to reduce intestinal inflammation and protect the integrity of the colonic epithelial barrier. In vitro experiments showed that XS dose-dependently reduced the inflammatory response in LPS-induced RAW264.7 cells by inhibiting the expression of IL-6, IL-1β, and TNF-α, reducing ROS levels, restoring MMP, and improving mitochondrial function. Mechanistic studies showed that XS activated PI3K and Akt phosphorylation, upregulated Nrf2 and HO-1 expression, and reduced oxidative stress damage. XS promoted AMPK phosphorylation in the mitochondrial biogenesis pathway, increased the protein expression of SIRT1 and PGC-1α, and upregulated the expression of NRF1 and TFAM. The involvement of both pathways was confirmed in vivo and in vitro experiments, with inhibitor studies further validating the results in vitro experiments.

Conclusions: XS mitigates chronic UC by decreasing ROS levels and inhibiting the macrophage inflammatory response by regulating the PI3K/Akt/Nrf2 and AMPK/SIRT1/PGC-1α signaling pathways.

Chronic ulcerative colitis; Mitochondrial biogenesis; Mitochondrial function; Oxidative stress; Xiangsha Liujunzi decoction.