Objective To investigate of the regulatory effects of Budi Shenlian Recipe on gut microbiota and the phenotypic transition of tumor-associated macrophages (TAMs) in colorectal cancer (CRC) mice.

Methods Forty male BALB/c mice were divided into blank control group, model group, positive control group, high-dose Budi Shenlian Recipe group, and low-dose Budi Shenlian Recipe group (n=8). CRC-bearing models were established by subcutaneous injection of CT26 cells. Additionally, 10 male BALB/c mice were divided into the Budi Shenlian Recipe fecal microbiota transplantation (BFMT) group and model fecal microbiota transplantation (MFMT) group (n=5). The gut microbiota of these mice was cleared using a mixed solution of quadruple antibiotics, followed by subcutaneous injection of CT26 cells to construct pseudo-germ-free CRC-bearing mice. Fecal samples from the model group and high-dose Budi Shenlian Recipe group were collected to prepare fecal microbiota solutions. The BFMT group received gavage with fecal microbiota solution from the high-dose Budi Shenlian Recipe group, while the MFMT group received gavage with fecal microbiota solution from the model group. Tumor volume changes were observed and recorded. HE staining was used to assess pathological changes in tumor tissues. 16S sequencing was performed to analyze changes in gut microbiota. Flow cytometry and immunofluorescence staining were used to evaluate the proportion of M1/M2 type TAMs in tumor tissues. ELISA was used to detect differences in TNF-α and IL-10 levels in tumor tissues.

RESULTS Compared to the model group, the tumor volume of mice in the positive control group, high-dose Budi Shenlian Recipe group, and low-dose Budi Shenlian Recipe group grew more slowly (P < 0.01). HE staining showed necrotic areas in tumor tissues and reduced mitotic figures in the positive control and Budi Shenlian Recipe groups compared to the model group. 16S rRNA sequencing showed no significant differences in Chao1 and ACE indices between the high-dose Budi Shenlian Recipe group and the model group. PCoA analysis indicated a distinct microbial community structure between the blank group and model group, with the microbial structure of CRC mice in the Budi Shenlian Recipe group closer to that of the blank group. Compared to the blank group, the model group showed a significant decrease in the proportion of Muribaculaceae, Muribaculum, Alloprevotella, and Prevotellaceae_UCG-001, and a significant increase in Lachnospiraceae_NK4A136_group, Bacteroides, and Helicobacter. After administering Budi Shenlian Recipe to CRC mice, the community structure of some mice partially reverted to the level of the blank group. Transcriptome sequencing revealed that the most significant biological process (BP) among upregulated genes was the negative regulation of macrophage migration, suggesting that Budi Shenlian Recipe can reduce macrophage migration. Moreover, compared to the model group, the proportion of TAMs cells in the tumor tissues of the Budi Shenlian Recipe group significantly decreased (P < 0.001). Simultaneously, compared to the model group, the proportion of M1 type TAMs in the Budi Shenlian Recipe group significantly increased (P < 0.000 1), while the proportion of M2 type TAMs significantly decreased (P < 0.05). Immunofluorescence analysis showed the same trend as flow cytometry. The content of TNF-α in tumor tissues of the Budi Shenlian Recipe group significantly increased (P < 0.001), and IL-10 content significantly decreased (P < 0.001). Additionally, compared to the MFMT group, the tumor volume in the BFMT group grew more slowly (P < 0.0001). HE staining showed increased necrotic areas, sparser cell arrangement, and reduced pathological mitosis in the BFMT group. Furthermore, compared to the MFMT group, the proportion of TAMs cells in the tumor tissues of the BFMT group significantly decreased (P < 0.01). Compared to the MFMT group, the proportion of M1 type TAMs cells in the BFMT group increased (P < 0.000 1), while the proportion of M2 type TAMs cells decreased (P < 0.01). Immunofluorescence analysis further confirmed that Budi Shenlian Recipe fecal microbiota transplantation can reduce the proportion of TAMs in the tumor tissues of CRC mice and promote the conversion from M2 to M1 type, thereby reducing immune suppression in the tumor microenvironment.

CONCLUSION Budi Shenlian Recipe can improve gut microbiota dysbiosis in CRC mice and exert anti-CRC effects by reducing tumor infiltration of TAMs and modulating the phenotypic transition of TAMs. There may be a certain correlation between these two effects.