Beyond Conventional Immunotherapy: Zhenqi Fuzheng Granule Synergizes with PD-1 Blockade by Remodeling Gut-Immune Metabolism in Colorectal Cancer

Introduction

The clinical landscape of colorectal cancer (CRC) treatment has been transformed by the advent of immune checkpoint inhibitors (ICIs), specifically antibodies targeting the programmed cell death protein 1 (PD-1). Despite this progress, a significant proportion of patients exhibit primary or acquired resistance, often driven by a hostile, immunosuppressive tumor microenvironment (TME). Emerging evidence suggests that the gut microbiota and its metabolic byproducts play a pivotal role in modulating systemic immunity and therapeutic responses. A recent study published in Phytomedicine by Guo et al. (2025) provides compelling evidence that Zhenqi Fuzheng Granule (ZQFZ), a traditional Chinese medicine (TCM) formulation, acts as a potent adjuvant to PD-1 therapy by targeting the SCFAs-GPR109A axis and remodeling immunometabolic pathways.

Highlights of the Research

The study demonstrates that ZQFZ significantly enhances the antitumor activity of PD-1 antibodies in murine models of CRC. The mechanism involves a multi-layered approach: first, the modulation of the gut microbiota to increase the production of short-chain fatty acids (SCFAs), particularly butyrate; second, the activation of the GPR109A receptor; and third, the subsequent inhibition of the AKT/mTOR/HIF-1α signaling pathway. This cascade leads to a reduction in tumor glycolysis and a shift in the immune landscape from suppressive to active, characterized by increased CD8+ T cell infiltration and M1 macrophage polarization.

Background and Clinical Context

Colorectal cancer remains a leading cause of cancer-related mortality worldwide. While PD-1/PD-L1 inhibitors have shown efficacy in patients with microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) tumors, the majority of CRC patients possess microsatellite stable (MSS) tumors that are largely non-responsive to immunotherapy. The immunosuppressive TME, characterized by high levels of myeloid-derived suppressor cells (MDSCs) and metabolic competition for glucose, often thwarts the efforts of infiltrating lymphocytes. ZQFZ, a clinically approved herbal prescription composed primarily of Radix Astragali and Radix Codonopsis, has been used for decades to support immune function in cancer patients. However, the molecular and microbial basis for its synergy with modern immunotherapy has remained largely undefined until now.

Study Design and Methodology

To elucidate the mechanisms of ZQFZ, researchers employed a comprehensive multi-omics and experimental approach. The phytochemical profile of ZQFZ was first established using LC-MS/MS and UPLC-MS/MS. In vivo experiments utilized the azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model, which mimics the inflammation-to-carcinoma progression seen in human CRC. Mice were treated with ZQFZ, PD-1 antibody, or a combination of both. The researchers analyzed gut microbial diversity via 16S rRNA sequencing and quantified SCFAs using gas chromatography. To confirm the role of the microbiota, fecal microbiota transplantation (FMT) was performed on antibiotic-pretreated mice. Mechanistic validation involved molecular docking, molecular dynamics simulations, and microscale thermophoresis (MST) assays to examine protein-protein and ligand-protein interactions. In vitro studies under hypoxic conditions further explored the impact of sodium butyrate (NaB) on glycolytic flux and signaling pathways in CRC cell lines.

Key Findings and Mechanistic Insights

Phytochemical Characterization

The analysis identified seven major bioactive compounds within ZQFZ. Quantitative results showed the presence of Adenosine (0.87 mg/g), Salidroside (0.11 mg/g), Astragaloside IV (0.07 mg/g), Calycosin (0.03 mg/g), Formononetin (6.7 μg/g), Chlorogenic acid (1.4 μg/g), and Apigenin (0.5 μg/g). These compounds are known for their immunomodulatory and anti-inflammatory properties.

Synergistic Antitumor Efficacy

In the AOM/DSS mouse model, while both ZQFZ and PD-1 antibody alone reduced tumor burden, the combination therapy was significantly more effective. This synergy was reflected in reduced tumor numbers, smaller tumor volumes, and improved survival rates. Crucially, the combination treatment led to a significant remodeling of the TME.

Microbiota and SCFA Modulation

ZQFZ treatment significantly altered the gut microbial composition, increasing the abundance of beneficial, SCFA-producing bacteria. This shift resulted in elevated levels of colonic SCFAs, with butyrate showing the most prominent increase. The importance of the microbiota was confirmed when FMT from ZQFZ-treated mice successfully replicated the antitumor effects and immune remodeling in recipient mice.

The GPR109A/AKT/mTOR/HIF-1α Axis

The study identified GPR109A, a receptor for butyrate, as a central mediator. ZQFZ and its metabolite butyrate activated GPR109A, which directly interacts with AKT1. MST assays confirmed a high-affinity binding between GPR109A and AKT1 (Kd = 74.5 ± 20.8 nM). This interaction effectively inhibited the AKT/mTOR/HIF-1α signaling axis. Downregulation of this pathway suppressed the expression of key glycolytic enzymes, including HK2, GLUT1, and LDHA, thereby reducing lactate production in the tumor.

Immune Microenvironment Remodeling

The metabolic shift away from glycolysis in the tumor cells relieved the metabolic stress on immune cells. Flow cytometry revealed a significant decrease in MDSC infiltration and a shift in macrophage polarization toward the pro-inflammatory M1 phenotype. Furthermore, the ratio of CD4+/CD8+ T cells was restored, and serum levels of effector cytokines such as IL-2, IL-12, and IFN-γ were upregulated, while immunosuppressive cytokines like IL-4 and IL-10 were downregulated.

Expert Commentary and Clinical Implications

The findings by Guo et al. provide a robust scientific rationale for the integration of TCM with modern immunotherapy. The identification of the GPR109A-AKT1 interaction is particularly noteworthy, as it offers a direct molecular link between microbial metabolites and intracellular oncogenic signaling. By suppressing tumor glycolysis, ZQFZ effectively ‘starves’ the tumor while ‘feeding’ the immune system, addressing one of the primary hurdles in ICI therapy. This study also highlights the potential of butyrate as a key metabolite in cancer prevention and treatment. From a clinical perspective, ZQFZ could serve as a low-toxicity adjuvant to improve outcomes for CRC patients, particularly those who are less responsive to PD-1 monotherapy. However, further clinical trials are necessary to confirm these findings in human cohorts and to determine optimal dosing and timing alongside immunotherapy.

Conclusion

This research uncovers a novel integrated mechanism whereby Zhenqi Fuzheng Granule enhances the efficacy of PD-1 antibodies through the gut microbiota-SCFAs-GPR109A axis. By inhibiting the AKT/mTOR/HIF-1α pathway and remodeling the immunometabolic landscape, ZQFZ transforms a ‘cold’ tumor environment into a ‘hot’ one, more susceptible to immune attack. These insights not only validate the traditional use of ZQFZ but also pave the way for more sophisticated, microbiota-targeted combination strategies in oncology.

References

Guo L, Yi J, Zhang A, Zheng X, Wang M, Yang F, Kong X, Meng J. Zhenqi Fuzheng Granule targets the SCFAs-GPR109A axis to enhance PD-1 antibody efficacy via immunometabolic remodeling in colorectal cancer. Phytomedicine. 2025 Nov 25;148:157312. doi: 10.1016/j.phymed.2025.157312. Epub 2025 Sep 26. PMID: 41038145.