Network Pharmacology Dissects SFI’s Anti-Glioma Actions via SRC/PI3K/AKT

Study Background and Research Question

Gliomas, which account for 30–50% of all primary brain tumors, remain among the most therapeutically challenging malignancies due to their rapid proliferation, invasive growth, and poor prognosis despite surgery, radiotherapy, and chemotherapy. The median survival for patients with high-grade gliomas is less than 17 months, underscoring the urgent need to identify more effective therapeutic strategies. Shenqi Fuzheng injection (SFI), a clinical formulation derived from Codonopsis pilosula and Astragalus membranaceus, has traditionally been used in China as an adjunct to chemotherapy. However, the specific molecular mechanisms underlying SFI's anti-glioma effects have remained largely undefined. The reference study (Li et al., 2024) addresses this gap by integrating network pharmacology and experimental validation to elucidate SFI’s mode of action in glioma proliferation and migration.

Key Innovation from the Reference Study

The central innovation of the reference paper lies in its combined use of network pharmacology—a computational approach to predict multi-component drug targets—and experimental validation to map the anti-tumor mechanisms of SFI. The study systematically identified overlapping gene targets between SFI’s active components and glioma pathophysiology, leading to the discovery that the SRC/PI3K/AKT signaling pathway is a critical axis modulated by SFI. This approach not only bridges traditional Chinese medicine with molecular oncology but also reveals a concrete mechanistic link between SFI treatment and suppression of glioma cell proliferation and migration.

Methods and Experimental Design Insights

The researchers utilized a multi-step methodology:

• Network Pharmacology Target Screening: 26 major SFI components were mapped to 110 potential molecular targets. These were compared to 3,343 glioma-associated targets, revealing 79 shared targets.
• In Vitro Assays: Human glioma cell lines U87 and T98G were subjected to CCK-8 proliferation assays, EdU incorporation, plate colony formation, scratch and Transwell migration assays, as well as immunofluorescence, flow cytometry, and Western blot analyses to examine cell cycle and EMT marker expression.
• In Vivo Validation: A subcutaneous GL261 glioma model in C57BL/6 mice was used, with tumor progression monitored via HE staining and immunohistochemistry.
• Bioinformatics and Pathway Analysis: Enrichment and network analyses pinpointed the SRC/PI3K/AKT pathway as a central node affected by SFI’s active ingredients.

This workflow not only highlights the power of network pharmacology for drug mechanism deconvolution but also demonstrates rigorous experimental follow-up for computational predictions.

Core Findings and Why They Matter

The study’s principal findings are as follows:

• SFI significantly inhibited proliferation and induced S-phase cell cycle arrest in U87 and T98G glioma cells, as shown by CCK-8 and EdU assays.
• SFI suppressed cell migration and invasion, correlated with downregulation of EMT markers.
• In vivo, SFI robustly reduced tumor growth in the GL261 mouse model.
• Network analysis and Western blot validation demonstrated that SFI downregulated the SRC/PI3K/AKT signaling pathway—a pathway known to drive glioma cell survival and motility.

These results underscore the importance of the SRC/PI3K/AKT axis in glioma biology and provide mechanistic support for using anti-angiogenic compounds or pathway inhibitors in malignant glioma research. By linking SFI’s effects to suppression of EMT and tumor vascular proliferation, the study also aligns with broader anti-angiogenesis strategies in oncology.

Comparison with Existing Internal Articles

Several internal resources, including "Network Pharmacology Reveals SFI Blocks Glioma via SRC/PI3K/AKT Pathway" and "SFI Inhibits Glioma via SRC/PI3K/AKT: Network Pharmacology Insights", previously highlighted the value of network pharmacology in dissecting SFI’s anti-glioma mechanisms. Both internal articles emphasize the role of the SRC/PI3K/AKT pathway in mediating anti-tumor effects and suggest that targeting this axis could inform future research on angiogenesis inhibitors and tumor suppression. The present reference study builds on these insights by providing comprehensive experimental validation and directly connecting SFI-induced pathway modulation with functional outcomes in cell proliferation, migration, and in vivo tumor growth. This strengthens the translational relevance of SRC/PI3K/AKT as a therapeutic target in glioma and encourages further research into anti-angiogenic compounds and VEGF receptor inhibitors.

Limitations and Transferability

While the study provides compelling mechanistic data, several limitations should be noted. First, although the SRC/PI3K/AKT pathway is strongly implicated, the complexity of glioma signaling networks means that additional, uncharacterized pathways may also contribute to SFI’s effects. Second, the in vivo data are limited to a single immunocompetent mouse model, and further research will be required to assess the reproducibility of findings across glioma subtypes and in the context of the tumor microenvironment. Third, SFI is a multi-component herbal preparation, and individual active ingredients’ contributions remain only partially resolved. These factors may limit direct translation to clinical application, but the demonstration of pathway-specific inhibition provides a rational basis for future anti-angiogenic compound screening and mechanistic studies.

Protocol Parameters

• Network pharmacology screening: Identify overlapping targets between SFI components and glioma-associated genes using public databases prior to experimental validation.
• Cell culture models: Use U87 and T98G glioma cell lines for proliferation and migration assays; GL261 for in vivo studies in C57BL/6 mice.
• Assay selection: Employ CCK-8 for cell viability, EdU for DNA synthesis, scratch and Transwell assays for migration, and Western blot for signaling pathway analysis.
• SFI dosing (as in the reference study): Use concentrations validated in the paper for in vitro and in vivo assays; exact values should be optimized per cell line and animal model.
• Pathway validation: Confirm modulation of SRC/PI3K/AKT by Western blot or immunofluorescence in treated samples.

Research Support Resources

For researchers seeking to explore anti-angiogenic mechanisms or test VEGF pathway inhibition in tumor models, highly selective VEGF receptor inhibitors can be valuable tools. AAL-993 (SKU C3730) from APExBIO is a well-characterized VEGF receptor inhibitor with potent activity against VEGFR-1, VEGFR-2, and VEGFR-3, demonstrated in both in vitro kinase assays and in vivo tumor studies. Use of such compounds can complement network pharmacology and pathway analysis workflows for dissecting angiogenesis in glioma or other solid tumors. Full protocol guidelines and compound specifications are available via the manufacturer for research applications only.