Fufangxiaopi formula alleviates DSS-induced colitis in mice by inhibiting inflammatory reaction, protecting intestinal barrier and regulating intestinal microecology

Fufangxiaopi Formula (FF) is a modified form of Sishen Wan, traditionally used for treating diarrhea. The application of FF for treating ulcerative colitis (UC) has achieved desirable outcomes in clinical settings. However, the underlying mechanism of the effect of FF on UC is yet to be determined. This study aimed to evaluate the protective effect and underlying mechanism of FF on mice with dextran sodium sulfate (DSS)-induced colitis. In vivo, the efficacy of FF on the symptoms associated with DSS-induced colitis in mice was clarified by observing the body weight change, colon length, DAI score, and H&E staining. The release of inflammatory mediators in mouse colon tissues was detected by ELISA and MPO, and the contents of TLR4/NF-κB signaling pathway and MAPK signaling pathway-related proteins, as well as intestinal barrier-related proteins, were detected in mouse colon tissues by western blot method. Changes in the content of barrier proteins in mouse colon tissues were detected by immunofluorescence. 16S rRNA sequencing and FMT were performed to clarify the effects of FF on intestinal flora. In vitro, the effect of FF-containing serum on LPS-induced inflammatory mediator release from RAW264.7 cells were detected by qRT-PCR. The contents of TLR4/NF The effects of FF-containing serum on B signaling pathway and MAPK signaling pathway related proteins in RAW264.7 cells and intestinal barrier related proteins in Caco-2 cells were detected by western blot. The effects of FF-containing serum on LPS-induced nuclear translocation of p65 protein in RAW264.7 cells and barrier-associated protein in Caco-2 cells were detected by immunofluorescence. In vivo studies showed that FF could significantly alleviate the symptoms of UC, including reducing colon length, weight loss, clinical score, and colon tissue injury in mice. FF could significantly reduce the secretion of proinflammatory cytokines by suppressing the activation of the TLR4/NF-κB and MAPK signaling pathways. Moreover, FF could protect the integrity of intestinal barriers by significantly increasing claudin-3, occludin, and ZO-1 expression levels. 16S rRNA sequencing and FMT elucidate that FF can alleviate symptoms associated with colitis in mice by interfering with intestinal flora. In vitro studies showed that FF drug-containing serum could significantly inhibit proinflammatory responses and attenuate the secretion of iNOS, IL-1β, TNF-α, IL-6, and COX-2 by suppressing the activation of TLR4/NF-κB and MAPK signaling pathways in RAW264.7 cells. Furthermore, FF could protect the Caco-2 cell epithelial barrier. FF could alleviate DSS-induced colitis in mice by maintaining the intestinal barrier, inhibiting the activation of TLR4/NF-κB and MAPK signaling pathways, reducing the release of proinflammatory factors, and regulating intestinal microecology.