Chinese herbal Jianpi Jiedu formula suppressed colorectal cancer growth in vitro and in vivo via modulating hypoxia-inducible factor 1 alpha-mediated fibroblasts activation

Ethnopharmacological relevance: Jianpi Jiedu Formula (JPJDF) is a traditional Chinese medicinal decoction clinically used for its anti-cancer properties, particularly in colorectal cancer (CRC). Aim of the study: This study aims to investigate the therapeutic effects of JPJDF on CRC and elucidate its potential molecular mechanisms, with a focus on its impact on hypoxia-inducible factor 1 alpha (HIF1 alpha) and cancerassociated fibroblasts (CAFs) both in vitro and in vivo. Materials and methods: UPLC-Q-TOF-MS was used to identify the constituents of JPJDF. A chemical-induced colorectal cancer model was established and treated with JPJDF to evaluate its effects. Tumor size was measured, and histopathological analyses were performed to examine JPJDF's regulatory potential on CRC. The functional mechanism of JPJDF was predicted through network pharmacology, molecular docking, and transcriptomics. Co-culture techniques involving CRC cells and CCD-18Co fibroblasts were used to assess JPJDF's impact on fibroblast activation. The effects of HIF1 alpha on CAFs were evaluated using CCK-8 proliferation, clonal formation, and apoptotic assays, with differential marker expression quantified via qPCR and Western blotting. Results: Pharmacodynamic assessment demonstrated that JPJDF reduced tumor size without affecting body weight, indicating its safety in the chemical-induced murine CRC model. Network pharmacology analysis, combined with molecular docking and transcriptomics, revealed that JPJDF regulates HIF-1 signaling pathways and identified HIF1 alpha as a potential target for JPJDF's anti-CRC effect. JPJDF effectively suppressed CRC growth in vivo by attenuating fibroblast activation, reducing alpha-SMA expression and POSTN secretion through HIF1 alpha inhibition. HIF1 alpha knockdown in CRC cells inhibited fibroblast proliferation and clonal formation, while overexpression promoted these processes. Additionally, downregulating HIF1 alpha suppressed alpha-SMA and POSTN expression in fibroblasts, whereas overexpression enhanced fibroblast activation. Conclusion: JPJDF emerges as a promising therapeutic candidate for inhibiting CAFs activation by targeting HIF1 alpha, offering potential avenues for modulating fibroblast activation towards CAFs in CRC therapy.