Dissecting the Anti-Glioma Effects of Shenqi Fuzheng Injection via SRC/PI3K/AKT Pathway: Network Pharmacology Insights

Study Background and Research Question

Gliomas are among the most aggressive and therapeutically challenging primary brain tumors, accounting for 30–50% of all such malignancies. Despite advances in surgery, radiotherapy, and chemotherapy, the median survival for glioma patients remains disappointingly low, typically less than 17 months (reference study). There is a critical need for novel therapeutic strategies, particularly those that target the molecular mechanisms underlying glioma proliferation, migration, and angiogenesis. Traditional Chinese medicine formulations, such as Shenqi Fuzheng injection (SFI), are used clinically as adjuvant therapies in cancer treatment, but their molecular mechanisms of action in glioma remain incompletely understood. This study seeks to clarify the anti-glioma mechanism of SFI, focusing on its regulation of the SRC/PI3K/AKT signaling pathway.

Key Innovation from the Reference Study

The central innovation of this research lies in its integrative approach: combining network pharmacology with rigorous experimental validation to map the molecular interactions between SFI’s bioactive compounds and glioma signaling networks. The study identifies the SRC/PI3K/AKT axis as a pivotal pathway through which SFI suppresses glioma cell proliferation and migration. This mechanistic clarity represents a significant advance, providing a scientific rationale for the anti-angiogenic and anti-proliferative effects of SFI previously observed in clinical practice and animal models.

Methods and Experimental Design Insights

The investigation began with network pharmacology, an approach designed to predict and analyze the complex interactions between multi-component therapeutics and disease-associated molecular targets. The authors first identified 26 major active components in SFI, including prominent ingredients from Codonopsis pilosula and Astragalus membranaceus. These compounds were computationally mapped to 110 potential therapeutic targets. Cross-referencing with 3,343 glioma-related targets from large-scale bioinformatics databases yielded 79 shared targets, suggesting possible points of SFI action in glioma biology (reference study). To validate these predictions, a multi-tiered experimental workflow was employed:

• In vitro assays (CCK-8, EdU incorporation, plate cloning, scratch/wound healing, Transwell migration) measured proliferation and migration of human glioma cell lines (U87, T98G).
• Flow cytometry and immunofluorescence characterized cell cycle distribution and EMT marker expression.
• Western blotting quantified key proteins in the SRC/PI3K/AKT pathway.
• In vivo efficacy was assessed using a subcutaneous GL261 glioma model in C57BL/6 mice, with HE staining and immunohistochemistry documenting tumor suppression and pathway modulation.

Core Findings and Why They Matter

The study presents several mechanistically important findings:

• SFI suppresses glioma proliferation and migration: SFI exposure led to significant inhibition of U87 and T98G cell proliferation, as well as reduced migratory capacity, as demonstrated by CCK-8, EdU, and migration assays.
• Cell cycle arrest in S-phase: Flow cytometry revealed that SFI induced S-phase cell cycle block, restricting glioma cell growth.
• Downregulation of EMT markers: SFI reduced expression of epithelial-mesenchymal transition (EMT) markers, a process closely linked to tumor invasiveness and angiogenesis.
• Pathway-specific modulation: Western blot and immunohistochemistry confirmed SFI’s suppression of SRC, PI3K, and AKT phosphorylation, substantiating the predicted pathway-level interaction.
• In vivo tumor inhibition: SFI treatment dramatically reduced tumor volume in the GL261 mouse model, aligning with in vitro results and reinforcing translational potential.

Collectively, these findings clarify that SFI’s anti-glioma effects are mediated at least in part through inhibition of the SRC/PI3K/AKT signaling cascade—a pathway known to regulate cell proliferation, migration, and angiogenesis in malignant tumors. This mechanistic insight is crucial for the rational design of combination therapies and the advancement of anti-angiogenic strategies in glioma.

Comparison with Existing Internal Articles

Prior coverage, such as the overview in "Network Pharmacology Reveals SFI Blocks Glioma via SRC/PI3K/AKT", emphasized SFI’s broad anti-proliferative and anti-angiogenic actions. The present study extends these insights by providing direct experimental validation of the SRC/PI3K/AKT pathway as a mechanistic link, rather than relying solely on network predictions. Similarly, the article "Network Pharmacology Reveals SFI's Anti-Glioma Mechanism via SRC/PI3K/AKT" summarizes the same research focus, but this new publication offers a more detailed mapping of compound-target relationships and comprehensive functional assays. In the context of broader tumor angiogenesis research, literature on small molecule angiogenesis inhibitors—including precision VEGF receptor inhibitors such as AAL-993—complements the findings here by demonstrating that both herbal formulations (like SFI) and selective chemical agents can converge on common pro-angiogenic and proliferative signaling pathways. For instance, as detailed in "AAL-993: Precision VEGF Receptor Inhibitor in Tumor Angiogenesis", targeting VEGFR-2/3 effectively models anti-angiogenic responses in tumor systems, paralleling the anti-angiogenic dimensions of SFI’s action.

Limitations and Transferability

Despite its integrative design, the study’s translational reach is tempered by several factors. First, the SFI formulation is complex, containing dozens of bioactive compounds, complicating efforts to ascribe effects to specific molecules. Second, while the SRC/PI3K/AKT axis is convincingly implicated, other signaling networks likely contribute to SFI’s anti-tumor profile. Third, in vivo efficacy was demonstrated in a murine subcutaneous transplant model, which may not fully recapitulate human glioma microenvironmental complexities or blood-brain barrier constraints. Finally, the absence of clinical trial data for SFI in glioma patients limits immediate clinical translation. Nevertheless, the study’s robust combination of network pharmacology, molecular biology, and animal modeling provides a strong foundation for future investigations into targeted anti-angiogenic and anti-proliferative therapies.

Protocol Parameters

• SFI concentration range: In vitro, SFI was tested across a range of concentrations, typically 0.5–2 mg/mL, with significant inhibitory effects observed in this window for U87 and T98G glioma cell lines (reference study).
• Cell cycle analysis: Flow cytometry was performed 24–48 hours after SFI treatment to determine S-phase arrest.
• Animal model workflow: C57BL/6 mice were inoculated subcutaneously with GL261 cells, followed by daily intraperitoneal SFI administration, with tumor volume monitored over 2–3 weeks.

Practical workflows for anti-angiogenic compound testing in glioma models may also incorporate selective VEGF receptor inhibitors (e.g., AAL-993) to dissect pathway-specific effects.

Research Support Resources

For researchers aiming to model anti-angiogenic and anti-proliferative mechanisms in glioma or related tumor systems, selective VEGF receptor inhibitors offer a complementary approach to complex herbal formulations. AAL-993 (SKU C3730) is a potent and selective VEGFR-1/2/3 inhibitor that enables precise interrogation of angiogenic signaling. Its robust in vitro and in vivo activity, protocol-friendly solubility, and well-characterized selectivity profile make it a valuable tool for tumor angiogenesis research, as highlighted by APExBIO. When designing protocols to parallel or extend findings from SFI studies, the use of AAL-993 can facilitate targeted investigation of VEGF-dependent pathways in both cell-based and animal models. For further technical details, refer to the product information and relevant internal resources.