Objective

2

To explore the possible mechanism of

Xianlian Jiedu Formula

（仙连解毒方， XJF） in treating dampness-heat stasis toxin syndrome of colorectal cancer.

Methods

2

Thirty C57BL/6N mice were randomly divided into normal group， model group and XJF group with 10 rats in each group. The mice in model group and XJF group were induced by high temperature and high humidity environment， high fat diet and azoxymethane/ dextran sodium sulfate to establish the model of dampness-heat stasis toxin syndrome of colorectal cancer. After successful modeling， XJF group was given 12.9 g/（kg·d） of XJF by gavage， while the normal group and the model group were given 0.2 ml of normal saline by gavage， both for 6 times per week， 16 weeks. Disease activity index （DAI） score was calculated weekly during administration， and serum diamine oxidase （DAO） and colorectal tissue myeloperoxidase （MPO） contents were detected after last administration. The colonic tissue was taken for single-cell transcriptome sequencing （scRNA-seq）. According to the cell surface marker gene， SingleR-v1.4.1 was used to identify cell subtypes of the immune cells in the tumor microenvironment （TME）. The biological function and signaling pathway of the differentially expressed genes by immune cells in each group were analyzed.

Results

2

Compared to that in the model group， DAI score in XJF group was significantly lower from week 7 to week 16 during administration （

P

＜0.01）. Compared to those in the normal group， the survival rate of mice in the model group significantly decreased， while the serum DAO and colorectal tissue MPO contents increased （

P

＜0.05 or

P

＜0.01）； compared to the model group， the XJF group had higher survival rate and lower serum DAO and colorectal tissue MPO contents （

P

＜0.05 or

P

＜0.01）. The scRNA-seq analysis showed that mouse intestinal T cells could be divided into four subtypes， including CD4

+

T cells， CD8

+

T cells， natural killer （NK） T cells and γδ T cells. Compared to those in the model group， the number and proportion of intestinal epithelial cells， fibroblasts and erythroid cells in the intestinal tissues in XJF group increased， while the number and proportion of B cells， T cells and myeloid cells decreased； among T cells， the number and proportion of active CD4

+

T cells， effector CD4

+

T cells， memory CD8

+

T cells， CD4

+

T cells， γδ T cells and NK T cells decreased， while those of CD8+ T cells， cytotoxic CD8

+

T cells and depletion CD8

+

T cells increased； the number of initial CD4

+

T cells， memory CD4

+

T cells， regulatory T cells （Tregs） and initial CD8

+

T cells decreased， while the proportion increased； the number of all kinds of myeloid cells decreased， while the proportion of macrophages and neutrophils decreased， and that of mast cells and dendritic cells increased. Differential gene enrichment analysis showed that the regulation function of XJF on CD8

+

T cells was mainly concentrated in the positive regulation of RNA polymerase Ⅱ on transcription and transcription factor activator protein-1 （AP-1） complex， and the signaling pathway was mainly concentrated in osteoclast differentiation， interleukin-17 （IL-17） signaling pathway， nuclear factor κB （NF-kB） signaling pathway， tumor necrosis factor （TNF） signaling pathway. The regulatory functions of CD4

+

T cells was mainly concentrated in chaperone cofactory-dependent protein refolding， p38 mitogen-activated protein kinase （p38MAPK） cascade negative regulation， and the signaling pathways were mainly concentrated in inflammatory bowel disease （IBD）， osteoclast differentiation， cell apoptosis， and TNF signaling pathway. The regulatory functions of myeloid cells were mainly concentrated in inflammatory response， neutrophil chemotaxis， and immune response， and the signaling pathways were mainly concentrated in IL-17 signaling pathway， TNF signaling pathway， NF-kappa B signaling pathway， and antigen presentation and processing.

Conclusion

2

XJF can be used to treat colorectal cancer with dampness-heat stasis toxin syndrome， and its mechanism may be related to regulating the number and proportion of CD8

+

T cells， CD4

+

T cells， γδ T cells， macrophages， neutrophils and other immune cells in TME， and interfering with Th1/Th2 cell differentiation， Th17 cell differentiation， IL-17， TNF， NF-κB and other signaling pathways， so as to improve the immune and inflammatory microenvironment of colorectal cancer.