Astragalus polysaccharides ameliorate irinotecan-induced gut toxicity by regulating gut microbiota and suppressing the MAPK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE Astragalus membranaceus var. mongholicus (Bunge) P.K.Hsiao (AM), a classic Qi-tonifying herb, has been widely used for treating a range of inflammatory diseases. Nevertheless, the protective effects of AM against irinotecan-induced gut toxicity (IGT) remains insufficiently characterized, while the active pharmacological fractions and the underlying anti-IGT mechanisms have not been fully elucidated.

Aims

This study aimed to investigate the ameliorative effects of the water extract of AM and its fractions on IGT in mice, as well as to identify the most potent anti-IGT fraction and to reveal the underlying anti-IGT mechanisms.

Methods

The water extract of AM (WEA) was administered to an IGT murine model. Therapeutic efficacy was assessed using the Disease Activity Index (DAI), while histopathology was evaluated via H&E and PAS staining. Intestinal barrier integrity was examined by measuring ZO-1, Occludin, and Muc2 expression using RT-qPCR, immunofluorescence, and immunohistochemistry. Colonic pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were quantified by ELISA. Following confirmation of efficacy, four fractions were isolated and compared. The most effective fraction, Astragalus membranaceus polysaccharides (APS), was further investigated using 16S rRNA sequencing, microbial metabolomics, transcriptomic, and Western blot.

Results

APS significantly improved body weight loss and reduced DAI in IGT mice. H&E staining showed that APS ameliorated structural damage and inflammatory infiltration in colonic tissues. PAS staining revealed a notable increase in goblet cell numbers following APS treatment. RT-PCR and immunohistochemical staining confirmed that APS enhanced the expression of intestinal barrier markers (ZO-1, Occludin, Muc2) and promoted crypt proliferation (Ki67) in colonic tissue. Consequently, APS markedly reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in colonic tissues from IGT mice. 16S rRNA sequencing showed that APS regulated gut microbiota composition, thereby increasing the beneficial metabolites (e.g. butyrate acid) in colonic lumen. It was also found that APS significantly reduced the abundance of gmβ-GUS-producing bacteria, which in turn, decreasing gmβ-GUS enzymatic activity and the intestinal exposure levels of the toxic metabolite SN-38. Finally, transcriptomic analysis of colonic tissues revealed that APS suppressed the MAPK signaling pathway (MEK, ERK, P38, JNK) and down-regulated apoptosis-related genes (Bax, Bcl-2) in IGT mice.

Conclusion

Our findings suggest that APS, the key active fraction of AM against IGT, mitigate IGT by modulating microbiota-metabolite-MAPK axis, offering novel mechanistic insight for the ethnopharmacological use of AM to mitigate chemotherapy-induced intestinal toxicity.