FBP1 catalyzes the irreversible hydrolysis of fructose-1,6-bisphosphate into fructose-6-phosphate, making it the rate-limiting enzyme of gluconeogenesis. In recent years it has attracted attention in oncology, where it suppresses glycolysis by restraining the HIF-1α pathway and acts as a protein phosphatase that dephosphorylates targets such as histone H3 and telomerase reverse transcriptase. But its role in diabetic wound healing had never been systematically characterized. To close that gap, the team built an in vitro model of the diabetic wound microenvironment using HaCaT keratinocytes, the workhorse cells of epidermal renewal, exposed to methylglyoxal. Methylglyoxal is a highly reactive α-dicarbonyl metabolite that accumulates in hyperglycemic tissue and drives the formation of irreversible advanced glycation end-products, fueling collagen cross-linking and microvascular damage. Dose-response experiments identified 400 micromolar methylglyoxal as a concentration that significantly suppressed keratinocyte proliferation, impaired colony formation, and crippled cell migration in scratch and transwell assays, faithfully mimicking the cellular defects seen in chronic diabetic wounds.

With the model established, the researchers turned to transcriptome-wide RNA sequencing to find the molecular drivers of dysfunction. The analysis revealed 1,375 upregulated and 724 downregulated genes in methylglyoxal-treated cells, with gene ontology annotations pointing to defects in cytoplasmic and plasma membrane components and KEGG enrichment implicating type 1 diabetes, diabetic complications, and growth regulation. Among the differentially expressed genes, one stood out: FBP1 was significantly upregulated by methylglyoxal exposure. Functional validation quickly established the enzyme as a negative regulator of repair. Keratinocytes engineered to overexpress FBP1 using lentiviral vectors showed markedly reduced proliferation and migration, while cells treated with FBP1-specific small interfering RNA proliferated and migrated more vigorously. MTT and colony formation assays quantified the effect, confirming at the statistical level of P less than 0.01 that FBP1 is a significant inhibitor of the two cellular behaviors on which wound closure most depends.

Having identified the target, the team went hunting for an inhibitor. Using the crystal structure of human FBP1 as a template, they virtually screened a library of 2,100 natural products, first filtering candidates through Lipinski’s Rule of Five and then docking them into the enzyme’s fructose-6-phosphate binding pocket with the LibDock and CDOCKER algorithms in Discovery Studio. From the top-ranked hits, in vitro enzymatic testing singled out asiatic acid, a pentacyclic triterpenoid from Centella asiatica long used in traditional wound care. Molecular docking showed asiatic acid nesting snugly in the catalytic pocket, forming hydrogen bonds with ARG25, MET30, and LYS112 and hydrophobic contacts with ALA24, ARG140, and MET177, at a docking energy of −7.15 kcal/mol. Its measured inhibitory potency, an IC50 of 2.50 micromolar, actually exceeded that of adenosine monophosphate, the enzyme’s endogenous inhibitor. A cellular thermal shift assay, in which treated cells are heated across a temperature gradient, confirmed that asiatic acid binds FBP1 directly inside living cells, stabilizing the protein between 48 and 62 degrees Celsius.

Mechanistically, asiatic acid’s benefits flowed through a well-defined signaling cascade. Western blotting showed that methylglyoxal suppressed phosphorylation of AKT and mTOR along with downstream HIF-1α and uPAR, proteins that together drive keratinocyte proliferation and migration. Treatment with 20 micromolar asiatic acid restored the p-AKT/AKT and p-mTOR/mTOR ratios and lifted HIF-1α levels 1.7-fold and uPAR levels 2.3-fold. Importantly, the compound left total FBP1 expression unchanged, demonstrating that it works through direct enzymatic inhibition rather than transcriptional repression. Phalloidin staining added a striking visual dimension: methylglyoxal and FBP1 overexpression fragmented the actin cytoskeleton, and asiatic acid partially restored filamentous actin organization, linking FBP1 inhibition to the cytoskeletal mechanics of cell migration.

But asiatic acid has a practical flaw: its high polarity limits skin permeability. The chemists responded with rational design. They acetylated hydroxyl groups on the molecule’s A-ring to balance solubility and membrane permeability, then conjugated the scaffold to three different hydrogen sulfide donors. Hydrogen sulfide, the most recently recognized gasotransmitter alongside nitric oxide and carbon monoxide, has well-documented pro-healing credentials, from AMPK-mediated angiogenesis to preservation of mitochondrial membrane potential in stressed keratinocytes. Among the resulting conjugates, one derivative, AA4, outperformed the rest by a wide margin, enhancing proliferation of methylglyoxal-injured keratinocytes more than tenfold compared with the parent compound at equivalent concentrations.

AA4 proved to be more than a simple inhibitor. In vitro enzymatic assays confirmed that it retained asiatic acid’s FBP1-blocking power, with an IC50 of 2.38 micromolar. Yet western blots revealed that AA4 also lowered FBP1 protein levels, and the mechanism proved to be chemistry of an unusual kind. When the team scavenged hydrogen sulfide using oxidized glutathione, FBP1 expression rebounded, and the reducing agent dithiothreitol reversed AA4’s effect, lifting FBP1 levels nearly fivefold. Together these experiments indicate that AA4’s hydrogen sulfide release promotes FBP1 protein degradation through persulfidation, a sulfur-based post-translational modification that marks the enzyme for breakdown. The result is a dual attack: asiatic acid directly jams the enzyme’s catalytic activity while the released gas erases the protein itself. Rescue experiments underscored the significance, with AA4 restoring 82 percent of methylglyoxal-impaired migration and 3.3-fold higher proliferation in FBP1-overexpressing cells.

The derivative’s most important gift to wounded tissue, however, may be keeping keratinocytes alive. RNA sequencing of AA4-treated cells showed 748 upregulated and 1,422 downregulated genes relative to injured controls, with strong enrichment of apoptotic pathways and a signature of increased Bcl-2 and decreased FBP1. Flow cytometry, Hoechst nuclear staining, and TUNEL assays confirmed that AA4 dramatically reduced apoptosis in both methylglyoxal-challenged and FBP1-overexpressing cells. At the protein level, injury models showed a 5.8-fold increase in the pro-apoptotic Bax/Bcl-2 ratio along with elevated cleaved Caspase-3 and PARP; AA4 treatment raised Bcl-2 3.5-fold, cut Bax by 69.3 percent, and reduced cleaved Caspase-3 and PARP by 64.4 percent. When hydrogen sulfide was scavenged, this protection vanished, and in cells where FBP1 had been knocked down, AA4 offered no further benefit, proving its anti-apoptotic effect depends entirely on the presence of its target.

The decisive test came in living animals. The team induced diabetes in mice with streptozotocin, created full-thickness dorsal wounds, and applied AA4 in a Pluronic F-127 hydrogel that released more than 80 percent of its payload within 12 hours. The results were striking. By day seven, wounds treated with 1 milligram per milliliter AA4 had closed by 90.7 percent, compared with just 50 percent in untreated diabetic controls, and the 3 milligram per milliliter formulation achieved 95.5 percent closure, outperforming both native asiatic acid and adenosine monophosphate. Histology revealed thicker stratum corneum, enhanced epidermal regeneration, and denser collagen deposition, while immunohistochemistry confirmed suppression of FBP1 in the healing tissue. The treatment also calmed the inflammatory storm that stalls diabetic wounds: mRNA levels of IL-1β, IL-6, IL-8, and TNF-α, all elevated in diabetic wounds, fell after AA4 application, and macrophage markers shifted from the pro-inflammatory M1 phenotype, marked by CD86, toward the pro-healing M2 phenotype, marked by CD206. Using the methylene blue method, the researchers verified that AA4 released hydrogen sulfide sustainably, restoring wound tissue gas levels to 45.9 micromolar and reversing the hydrogen sulfide deficiency characteristic of diabetes.

The study, whose authors say the work was conducted with ethical approval and without competing interests, is notable both for the target it unveils and for the strategy it embodies. By pairing unbiased transcriptomics with structure-guided drug design, the researchers moved from a differentially expressed gene to a chemically optimized, dual-mechanism therapeutic in a single program. FBP1, long studied in the contexts of gluconeogenesis and tumor metabolism, now emerges as a druggable node in regenerative medicine, while AA4 demonstrates how conjugating a natural product with a gasotransmitter donor can overcome pharmacological limitations. The specific persulfidation sites on FBP1 and the degradation pathways involved remain to be mapped, and clinical translation will require further development. But for patients whose wounds linger for months or years, the prospect that a derivative of a traditional medicinal plant, supercharged with a signaling gas, could more than double healing rates within a week offers a genuinely new direction, one in which precision target discovery and rational molecular engineering finally converge on one of diabetes’ most stubborn complications.