Pulsatilla decoction attenuates colorectal cancer progression in association with gut microbiota remodeling, T-cell immune restoration, and NF-κB/JAK2/STAT3 signaling suppression.

Pulsatilla Decoction (PD) exhibits anti-inflammatory properties, but its impact on colorectal cancer (CRC) and the role of gut microbiota remain incompletely understood. This study investigated the antitumor effects of PD in a xenograft mouse model of HCT116 cells reconstituted with human peripheral blood mononuclear cells. We also analyzed corresponding changes in gut microbiota, T-cell immunity, inflammatory responses, and NF-κB/JAK2/STAT3 signaling. Tumor-bearing mice were treated for three weeks with low-, medium-, or high-dose PD, or with 5-fluorouracil. Gut microbiota composition and function were analyzed using 16S rRNA and metagenomic sequencing. Immune cell populations, cytokines, and pathway-related proteins were assessed via flow cytometry, immunofluorescence, ELISA, RT-qPCR, and western blotting. To further evaluate the involvement of microbiota, we performed antibiotic pretreatment followed by fecal microbiota transplantation (FMT). PD effectively reduced tumor volume and weight without causing significant body weight loss. High-dose PD partially restored microbial diversity and community structure, increased Akkermansia muciniphila, and modulated inflammation- and metabolism-related microbial pathways. Furthermore, PD enhanced peripheral and intratumoral CD4+ and CD8+ T-cell responses, improved the splenic Treg/Th17 balance, decreased TNF-α, IL-6, and IL-17A, increased IL-10, and suppressed NF-κB, JAK2, and STAT3 activation in tumor tissues. FMT from high-dose PD-treated donors partially transferred tumor inhibition, increased tumor CD8A and granzyme B, reduced Foxp3, and lowered NF-κB, JAK2, and STAT3 levels in recipient mice not directly treated with PD. These findings suggest that PD inhibits CRC progression by coordinately modulating gut microbiota, T-cell immunity, inflammation, and NF-κB/JAK2/STAT3 signaling, with the gut microbiota partially contributing to its antitumor effects.