6-Gingerol alleviates acute graft-versus-host disease by regulating Th17/Treg differentiation via the P2Y13/Ca²⁺ signaling pathway

Acute graft-versus-host disease (aGVHD) is a common immunological complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). This study aims to investigate the intervention effects of 6-gingerol on aGVHD and explore its potential mechanism in regulating T cell function and differentiation.

A murine allo-HSCT aGVHD model was established. SPF-grade male mice (8 to 12 weeks old, weighing 22 to 24 g) were randomly divided into a syngeneic control group (n=5), a vehicle control group (n=8,13), and 6-gingerol experimental groups at different concentrations (n=8,13). The survival status and body weight changes of recipient mice were observed, clinical scores and target organ histopathological scores were performed, and flow cytometry was used to detect changes in T cell subsets and surface markers in the spleen and liver. A murine B cell lymphoma cell line A20 graft-versus-leukemia (GVL) model was established, and tumor growth was evaluated through in vivo imaging and survival analysis. Splenic T cells were isolated and cultured in vitro, and divided into unstimulated, stimulated, and different concentrations of 6-gingerol treatment groups to assess proliferation, apoptosis, and cytokine changes. Mouse splenic CD4⁺ T cells were isolated and cultured under induction conditions for T helper 1 (Th1) cells, T helper 17 (Th17) cells, and T regulatory (Treg) cells, and were divided into a stimulated group and experimental groups with different concentrations of 6-gingerol. Changes in the differentiation direction of CD4⁺ T cells were analyzed via flow cytometry. Bulk RNA sequencing (bulk RNA-seq) was used to analyze gene expression profiles and differential pathway changes, and the transcriptomic results were validated using RT-qPCR. Receptor agonist intervention was further performed, and then the experiment was divided into: a stimulation group, a 6-gingerol monotherapy group, a 2-MeS-ADP monotherapy group, and a 6-gingerol + 2-MeS-ADP dual-drug group. The related mechanisms were validated through T-cell proliferation assays, in vitro induced differentiation assays, detection of cytoplasmic and mitochondrial Ca²⁺, and transmission electron microscopy (TEM) and quantitative analysis of mitochondrial cristae structure.

Following 6-gingerol intervention, the survival status, body weight, and clinical scores of aGVHD mice were significantly improved in the aGVHD mice (P<0. 05). HE stained pathological section scores revealed alleviated liver and intestinal injury (P<0. 05). Survival analysis results and in vivo bioluminescence imaging in the GVL model showed that 6-gingerol had no significant effect on the GVL effect. In vitro experiments demonstrated that 6-gingerol suppressed T cell proliferation (P<0. 0001) and promoted apoptosis (P<0. 01) in partially activated T cells. Under specific polarization conditions, 6-gingerol regulated CD4⁺ T cell subset differentiation, manifested as inhibition of Th1 (P<0. 05) and Th17 (P<0. 05) differentiation and promotion of Treg cell differentiation (P<0. 05). Transcriptomic analysis suggested that 6-gingerol treatment affected purinergic receptor and Ca²⁺-related pathways. RT-qPCR results showed that P2ry13 expression was significantly downregulated (P<0. 0001). Further mechanistic studies revealed that following 6-gingerol treatment, cytoplasmic Ca²⁺ levels in CD4⁺ T cells decreased (P=0. 0001), while mitochondrial Ca²⁺ accumulation in CD4⁺ T cells increased (P=0. 0002). Compared with the 6-gingerol monotherapy group, the two-drug treatment group, with the addition of the P2Y13 agonist 2-MeS-ADP showed partial recovery in T cell proliferation (P<0. 0001), Th1 and Th17/Treg differentiation (P<0. 05), and cytoplasmic and mitochondrial Ca²⁺ concentrations (P<0. 05). TEM revealed that mitochondrial ultrastructure in the two-drug group was improved compared to that of the 6-gingerol group, and quantitative analysis of mitochondrial cristae also supported this change (P<0. 0001), suggesting that 6-gingerol regulates T cell subset differentiation through the P2Y13-related Ca²⁺ signaling pathway.

6-Gingerol mitigates aGVHD by inhibiting Th17 cells and expanding Treg cells through the purinergic receptor P2Y13-related Ca²⁺ signaling pathway, providing a novel strategy for the prevention and treatment of aGVHD.