Objective

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To explore the mechanism of

Xianlian Jiedu Formula

（仙连解毒方， XJF） in treating colorectal cancer mice with damp-heat stasis toxin pattern by using single-cell transcriptome sequencing.

Methods

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Thirty male C57BL/6N mice were randomly divided into control group， model group and XJF group， with 10 mice in each group. Mice in model group and XJF group were induced by high fat diet and azomethane oxide （AOM）/ dextran sulfate sodium （DSS） to establish the model of colorectal cancer with damp-heat stasis toxin pattern， while those in the control group were given normal diet. After three cycles of DSS drinking water， XJF group was given 12.9 g/kg by gavage， while the control group and model group were given equal volume of normal saline， once daily for six consecutive days a week， with one day off， for consecutive 16 weeks. Four hours after the last administration， the colorectal tissues of mice in each group were collected， and the histopathological morphology of the mice was observed by hematoxylin-eosin staining （HE）. A 2 cm-long distal colon segment located 6 cm away from the anus was taken， and tissue samples were dissociated； single-cell suspension was isolated， and RNA was extracted for qPCR amplification. Single-cell transcriptome sequencing was performed. CellRanger was used for data quality control. Seurat-v3.1.1 software package was used to perform dimensionality reduction cluster analysis， cell type identification， and gene ontology （GO） and kyoto encyclopedia of genes and genomes （KEGG） enrichment analysis of differential genes.

Results

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HE staining showed that the colon glands of the mice in the control group were arranged regularly， and the nuclei were located at the base， which were flat or cubic. Epithelial cells in the model group lost their polarity， and were nest-like and cribriform-like， with necrosis in the center， having increased nucleocytoplasmic ratio， deep nuclear staining， obvious nucleoli， and significant mitotic phenomenon； the above conditions were improved in the XJF group. Totally， 45 480 intestinal cells were captured in the three groups through single-cell transcriptome sequencing， which can be classified into 20 cell groups by dimensionality reduction clustering. The marker genes of each cell group were identified， and 9 main intestinal cell types were obtained， which were intestinal epithelial cells， B cells， natural killer T （NKT） cells， natural lymphocytes， stem cells， macrophages， neutrophils， mast cells， and fibroblasts. Compared to the model group， the XJF group had significant higher proportion of intestinal epithelial cells as well as lower proportion of B cells， NKT cells， macrophages， and neutrophils in the intestinal tissue. GO and KEGG enrichment analysis showed that XJF's effect on colorectal cancer mice with damp-heat stasis toxin pattern was mainly enriched in immune system process， antigen processing and presentation， adaptive immune response and rotation mechanism of ATP transport synthase activity， and the signaling pathways were mainly involved with immune system signals， cancer-related pathways， cell proliferation and death signals， and energy metabolism signals.

Conclusion

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XJF may play its role in treating colorectal cancer mice with damp-heat stasis toxin pattern by regulating the proportion of immune cells in intestinal tissue， and preventing the transformation of intestinal epithelial cells into colorectal cancer cells.