Injectable Herbal Hydrogel Strikes Choline Kinase Alpha to Treat Malignant Ascites

A team of researchers in China has transformed a centuries-old herbal pairing once considered dangerously incompatible into a modern, injectable cancer therapy. In a study published in the Journal of Advanced Research, the scientists describe a carrier-free, temperature-responsive hydrogel built entirely from the active fraction of Euphorbia pekinensis Rupr. and glycyrrhizic acid, the principal component of licorice root. When injected into the abdominal cavity of mice with liver cancer, the hydrogel dramatically slowed tumor growth, curbed the buildup of malignant ascites, and left vital organs essentially unharmed. The work points to a strikingly different way of treating one of the most dreaded complications of advanced hepatocellular carcinoma, the predominant form of liver cancer and the third leading cause of cancer-related death worldwide.

Malignant ascites, the pathological accumulation of fluid in the peritoneal cavity, is a hallmark of advanced disease and signals rapid progression and metastatic spread. Standard interventions, including cytotoxic agents such as cisplatin and anti-angiogenic drugs like bevacizumab, typically provide only transient relief, and fluid reaccumulation remains common. The researchers sought a strategy that could simultaneously attack the tumor and suppress fluid production, while sidestepping the toxicity that limits conventional regimens. Their attention turned to the PI3K/AKT/mTOR signaling pathway, a master circuit governing tumor proliferation, survival, angiogenesis, and metastasis that is frequently hyperactivated in hepatocellular carcinoma through gene mutations, epigenetic changes, and upstream receptor dysregulation. Because the pathway is also implicated in treatment resistance, it represents an attractive therapeutic bullseye.

The team’s mechanistic insight centered on choline metabolism, a well-documented hallmark of cancer. Rapidly dividing malignant cells ramp up phosphocholine biosynthesis to meet relentless demands for new membrane material. Choline kinase alpha, the rate-limiting enzyme in this pipeline, is markedly overexpressed in hepatocellular carcinoma and is functionally entwined with PI3K/AKT signaling, where it mediates interactions between the epidermal growth factor receptor and mTORC2 that promote drug resistance and tumor progression. Disrupting the choline-metabolic axis, the researchers reasoned, could starve tumors of structural building blocks while severing their survival signaling. What they needed was a delivery platform that would keep the drug where it was needed, inside the peritoneal cavity, for as long as possible.

The answer emerged from an unlikely corner of pharmacological history. Euphorbia pekinensis Rupr. and Radix Glycyrrhizae have been prescribed together for centuries in traditional Chinese medicine for edema, fluid accumulation, and ascites, appearing in classical formulations such as Da Wu Yin Wan. Yet the pairing belongs to the classic eighteen incompatible medicaments, a traditional prohibition rooted in severe organ toxicity when the herbs are prepared conventionally. Modern investigations had hinted that the combination could mitigate ascites associated with hepatocellular carcinoma, but how to harness its synergy while avoiding organ damage remained unresolved. The new study resolved this tension through supramolecular engineering rather than simple co-administration.

To build the hydrogel, the researchers extracted an ethyl acetate fraction from Euphorbia pekinensis using reflux extraction with 95 percent ethanol, then combined it with glycyrrhizic acid in a one-to-one ratio and co-decocted the mixture. Heating to 65 degrees Celsius produced a sol, and on cooling to room temperature the solution gelled within minutes. Electron microscopy revealed why the combination behaves so differently from either component alone: whereas the Euphorbia fraction forms heterogeneous, irregular particles, the co-decocted material self-assembles into a homogeneous gel of uniformly distributed spherical nanoparticles roughly 200 nanometers in diameter. Rheological testing confirmed a stable gel state at low shear strain, with shear-thinning behavior that allows the material to be extruded cleanly through a syringe, a property essential for clinical injectability.

Spectroscopic and computational analyses illuminated the assembly mechanism at the molecular level. High-performance liquid chromatography showed that glycyrrhizic acid boosted the solubility of euphol, a marker compound of the herb, from a chromatographic peak area of 145.9 in isolation to 2530.3 in the combined preparation. Ultraviolet-visible and infrared spectroscopy located the binding interaction at hydroxyl groups, whose stretching vibrations shifted to lower wavenumbers in the assembly, and two-dimensional nuclear magnetic resonance experiments revealed close spatial contact between the hydroxyl group of euphol and the hydrophilic glucuronic acid end of glycyrrhizic acid. Molecular dynamics simulations depicted randomly dispersed molecules gradually forming binary aggregates through hydrogen bonding, which then intercalate and intertwine into an ordered supramolecular network. In short, the weak, noncovalent bonds between the two herbal molecules create a genuinely new state of matter, not a mere mixture.

The practical payoff of this architecture became apparent in live animals. Using fluorescent imaging in mice bearing ascites tumors, the team showed that the hydrogel remained concentrated in the abdominal cavity for at least 24 hours after intraperitoneal injection, whereas free fluorescent dye cleared rapidly. Enhanced retention was also observed in the liver and kidneys, with negligible accumulation in the heart, lungs, or spleen. This localized biodistribution maximizes drug exposure at the disease site while minimizing off-target toxicity, and it reduces the frequency of administration required, a meaningful advantage for patients undergoing repeated peritoneal procedures.

Therapeutic performance matched the elegant delivery design. In a subcutaneous xenograft model of H22 liver cancer, the hydrogel at 18.75 milligrams per kilogram per day achieved a tumor growth inhibition rate of 68.63 percent, approaching the 74.98 percent produced by cyclophosphamide at 20 milligrams per kilogram. Critically, the toxicology diverged sharply: cyclophosphamide significantly shrank the spleen, evidence of immune organ damage, while the hydrogel left liver, kidney, and spleen indices unchanged and produced no pathological abnormalities on histological staining. In a malignant ascites model established by intraperitoneal injection of H22 cells, hydrogel-treated mice showed markedly reduced abdominal circumference and slower weight gain driven by ascites accumulation, and organ indices that had fallen in untreated diseased animals were largely restored. The assembly appears to tame the herb’s inherent toxicity through an aggregation and assembly retention effect that concentrates the supramolecular complex at the tumor while sparing healthy tissue.

Transcriptomic and metabolomic profiling converged on a unified mechanism. RNA sequencing of treated tumors identified thousands of differentially expressed genes, with pathway enrichment pointing decisively to the PI3K-AKT and mTOR signaling cascades alongside apoptosis, hypoxia-inducible factor, and immune checkpoint pathways. Serum metabolomics revealed 119 key metabolites altered by treatment, with glycerophospholipid metabolism and choline metabolism in cancer emerging as the most significant enriched pathways, suggesting the hydrogel impairs choline utilization and phospholipid recycling and forces tumor cells toward mitochondrial fatty acid oxidation to sustain membrane homeostasis. A combined analysis of gene and metabolite shifts, applied for the first time in this ascites context, pointed to choline kinase alpha inhibition as the pivotal event.

Laboratory validation sealed the case. The hydrogel inhibited H22 cell growth in a concentration-dependent manner, elevated intracellular reactive oxygen species, collapsed mitochondrial membrane potential, and triggered apoptosis, the programmed cell death cascade that ultimately dismantles tumor cells. Choline kinase alpha activity fell dose-dependently with hydrogel treatment, and the known choline kinase inhibitor MN58b cooperated with the hydrogel at higher concentrations to amplify apoptosis. Western blotting and immunohistochemistry confirmed that treatment reduced the expression of choline kinase alpha and the phosphorylated, active forms of PI3K, AKT, and mTOR in both cells and tumor tissue. Together, the results establish the hydrogel as a choline kinase alpha inhibitor that suppresses the PI3K/AKT/mTOR axis to combat malignant ascites. Because the formulation is carrier-free, self-assembled from inexpensive natural molecules, and produced by simple co-decoction, the researchers argue it offers a practical blueprint for nature-derived, ascites-targeted precision therapeutics, though human trials will be needed to confirm whether the remarkable preclinical safety and efficacy translate to patients.

Subject of Research: A self-assembled herbal hydrogel that inhibits choline kinase alpha and the PI3K/AKT/mTOR pathway to treat hepatocellular carcinoma and malignant ascites.

Article Title: Injectable multi-component hydrogel as an inhibitor of choline kinase α achieved the treatment of malignant ascites by inhibiting PI3K/AKT/mTOR signaling pathway

Article References: Yang, Y., Qi, J., Zhu, Z., Wu, M., Zhao, Y., Wang, M., Lang, Y., Gu, Y., Liu, Y., & Cai, M. (2026). Injectable multi-component hydrogel as an inhibitor of choline kinase α achieved the treatment of malignant ascites by inhibiting PI3K/AKT/mTOR signaling pathway. Journal of Advanced Research, 87, 1105-1120. https://doi.org/10.1016/j.jare.2026.01.001

Image Credits: AI Generated

DOI: 10.1016/j.jare.2026.01.001

Keywords: hepatocellular carcinoma, malignant ascites, hydrogel, choline kinase alpha, PI3K/AKT/mTOR pathway, drug delivery, self-assembly, natural products, glycyrrhizic acid, Euphorbia pekinensis, metabolomics, nanomedicine