Molecular mechanisms of the Xiao-chai-hu-tang on chronic stress-induced colorectal cancer growth based on an integrated network pharmacology and RNA sequencing approach with experimental validation.

Background: Chronic stress is a risk factor for the development of colorectal cancer (CRC). Xiao-chai-hu-tang (XCHT) is a traditional Chinese medicine prescription and has been widely used to treat chronic stress-related diseases and cancer. However, its role in chronic stress-induced CRC remains unclear.

Methods: Our study aimed to investigate the roles of XCHT in CRC development under chronic stress. A xenografted CRC mouse model subjected to chronic restraint stress (CRS) was utilized to determine the effects of XCHT on CRC growth in vitro and in vivo. XCHT was administered via oral gavage once daily at dosages of 10.27 g/kg and 20.54 g/kg. RNA-sequencing was combined with network pharmacology to investigate potential target and pathway in this study. ELISA, RT-qPCR and immunofluorescence were performed to detect the expression of inflammation related genes. Glycolysis related genes and phenotype were evaluated by western blot, RT-qPCR and seahorse.

Results: XCHT significantly alleviated depression-like behaviors in CRS mice (p < 0.05) and effectively reduced tumor size and weight in a dose-dependent manner (p < 0.01). Mechanistic studies revealed that XCHT inhibited the CRS-induced upregulation of IL-6, attenuated the IL-6/JAK2/STAT3 signaling pathway (p < 0.05), and suppressed glycolysis by downregulating glycolytic enzymes (p < 0.01). Additionally, XCHT treatment reversed the CRS-induced decrease in immune cell infiltration, including CD4⁺ and CD8⁺ T cells, and reduced F4/80⁺ macrophage levels.

Conclusions: XCHT could reverse the tumor energy metabolism reprogramming and improve the inflammatory microenvironment in CRC under chronic stress through the IL-6/JAK2/STAT3 pathway. Therefore, XCHT might represent a promising therapeutic strategy for suppressing psychologically associated CRC progression.