ATF3 restoration as a potential strategy in managing ulcerative colitis: Implications from Sishen pill research

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with limited therapeutic options, particularly for moderate-to-severe cases. The present study evaluated the therapeutic potential of Sishen Pill (SSP) through Activating Transcription Factor 3 (ATF3).

Colonic biopsy samples were collected from 11 UC patients and 6 healthy controls (HCs). A murine colitis model was established using dextran sodium sulfate (DSS) and treated with SSP. The therapeutic efficacy of SSP was evaluated by measuring body weight, colonic length, and inflammatory markers in wild-type (WT) mice. Transcriptomic analysis revealed differentially expressed genes in colonic tissues following Atf3 knockout. Western blotting, immunofluorescence, immunohistochemical, and Luminex assays were conducted to assess the effect of SSP on Neutrophil Extracellular Traps (NETs) formation and ATF3 signaling.

ATF3 expression was significantly reduced in the inflamed mucosa of UC patients, correlating with disease severity. UC tissues also exhibited increased spontaneous NETs formation. In DSS-induced colitis mice, ATF3 expression was similarly reduced, whereas SSP treatment upregulated ATF3, mitigated weight loss, reduced colonic shortening, alleviated histopathological damage, and lowered inflammatory cytokine levels. Atf3 knockout mice (Atf3^-/-) displayed more severe DSS-induced colitis with enhanced immune response as compared to control littermates (Atf3^+/+). Transcriptomic analyses revealed that SSP downregulated key genes involved in NETs formation pathways, tumor necrosis factor (TNF) and cytokine-cytokine receptor signaling. Experimental validation confirmed that SSP reduced the levels of NETs-related proteins [Myeloperoxidase (MPO), Peptidylarginine Deiminase 4 (PAD4), Lymphocyte Antigen 6 Complex, G (Ly6G), Neutrophil Elastase (NE), Citrullinated Histone H3 (CitH3)] in the colorectal tissue of colitis mice. It also down-regulated TNF pathway-related proteins [Phosphorylated Extracellular Signal-Regulated Kinase (p-ERK), Matrix Metalloproteinase 9 (MMP9)]. Furthermore, SSP intervention reduced pro-inflammatory factors [interleukin (IL)-6, IL1β, Granulocyte Colony-Stimulating Factor (G-CSF) and TNF-α] and decreased CXCL1/CXCR2 axis factors, including CXCL1 protein levels and diminished CXCR2⁺MPO⁺ positive expressed cells. Importantly, these beneficial effects of SSP were ATF3-dependent, as SSP did not exert its effects in Atf3^-/- mice.

SSP ameliorates colitic mice through multiple mechanisms, including the inhibition of NETs formation, attenuation of inflammatory responses, and suppression of CXCL1/CXCR2-mediated inflammation, all via modulation of ATF3 expression. These findings support the potential of SSP as a promising adjunctive therapy for UC and underscore the therapeutic potential of targeting ATF3 in future treatment strategies.