Thymosin Alpha-1 Peptide Blocks Ferroptosis to Treat Tooth Pulp Inflammation

Tooth pulpitis is one of the most common and painful oral conditions, typically treated by root canal therapy or extraction. Despite its prevalence, the molecular mechanisms driving pulp cell death during inflammation remain incompletely understood. This study is the first to systematically characterize ferroptosis — a form of iron-dependent, lipid peroxide-driven cell death — as a central mechanism in pulpitis, and to identify thymosin α1 as a potential therapeutic agent to counteract it.

The researchers performed single-cell RNA sequencing (scRNA-seq) on tissue from 3 pulpitis patients and 3 healthy controls, capturing 40,231 cells (17,814 from pulpitis; 22,417 from healthy pulp). UMAP clustering identified 12 distinct cell types including fibroblasts, macrophages, neutrophils, T cells, B cells, plasma cells, endothelial cells, mesenchymal stem cells, and Schwann cells. Fibroblasts dominated healthy pulp but were markedly reduced in pulpitis, while immune cell populations expanded substantially. Differentially expressed ferroptosis-related genes (DE-FRGs), cross-referenced against the FerrDB database of 484 ferroptosis genes, were found in nearly every cell cluster, with the ferroptosis pathway significantly enriched in fibroblasts, macrophages, MSCs, neutrophils, non-myelinating Schwann cells, and dental pulp cells (DPCs). Oxidative phosphorylation was the most significantly enriched co-pathway, implicating mitochondrial dysfunction in ferroptosis activation.

To validate ferroptosis at the tissue level, the team measured ROS and Fe2+ in human pulp specimens using DCFH-DA and FerroOrange fluorescent probes. Both markers were significantly elevated in pulpitis versus healthy pulp (p<0.05). Immunohistochemistry confirmed low GPX4 (the master ferroptosis suppressor) and high PTGS2 (a ferroptosis marker) expression in pulpitis tissue, consistent with active ferroptosis. In LPS-stimulated DPCs in vitro, RNA sequencing identified 68 ferroptosis-related differentially expressed genes, with GPX4 showing notably low expression by heatmap analysis.

Thymosin α1 (Tα1) at 1 μg/mL was selected based on CCK8 proliferation assays showing optimal DPC growth promotion at 24 hours. In LPS-stimulated DPCs, Tα1 treatment significantly increased GPX4 and ferritin light chain (FTL) protein expression and decreased PTGS2 expression (p<0.05). Intracellular Fe2+ levels, measured by FerroOrange fluorescence, were significantly reduced by Tα1, comparable to the ferroptosis inhibitor ferrostatin-1. JC-10 flow cytometry showed that the red/green fluorescence ratio — a measure of mitochondrial membrane potential — was significantly restored by Tα1 in LPS-stimulated DPCs (p<0.05). Silencing the PTMA gene (which encodes the Tα1 precursor) reversed these protective effects, confirming the specificity of the pathway. Tα1 also significantly reduced TNF-α, IL-1β, and IL-6 expression in LPS-stimulated DPCs (p<0.05).

In a rat pulpitis model, PTMA was delivered either via gelatin sponge placed at the pulp exposure site or by direct pulp injection. Both delivery methods significantly reduced inflammatory cell infiltration and PTGS2 expression, and increased GPX4 expression compared to LPS-only controls (p<0.05). Fe2+ levels in rat pulp were significantly higher in the LPS group than controls, and both PTMA delivery routes significantly reduced Fe2+ accumulation. RNA sequencing of Tα1-treated versus LPS-only DPCs confirmed broad reversal of DE-FRGs expression patterns. These converging in vitro, in vivo, and transcriptomic findings establish a mechanistic link between thymosin α1, ferroptosis suppression, and pulpitis resolution, opening a potential avenue for peptide-based pulp-preserving therapies.