Abstract: Objective To investigate the regulatory mechanism underlying the effect of acid-sensing ion channel 3 (ASIC3) on the balance between regulatory T (Treg) cells and T helper type 17 (Th17) cells via the Ca²⁺-calcineurin (CaN)-nuclear factor of activated T cells (NFAT) signaling axis. Methods Donor platelets and recipient peripheral blood mononuclear cells (PBMCs) were co-cultured in vitro at a ratio of 250∶1 to simulate the state of platelet transfusion in vitro. By constructing ASIC3 overexpression lentivirus and co-culturing with in vitro cell model, they were divided into three groups: (1) blank control group; (2) negative control lentivirus group; (3) ASIC3 overexpression lentivirus group. The proportion of Treg (Foxp3⁺) and Th17 (IL-17A⁺) cells and intracellular Ca²⁺ level were detected by flow cytometry. The expression of ASIC3, phospholipase CB (PLCB), CaN, NFATC1 and its phosphorylated protein (p-NFATC1) were detected by Western blot. Results Compared with the blank control group, the proportion of Th17 cells in the ASIC3 overexpression lentivirus group was significantly increased (P<0.01), and the proportion of Treg cells was significantly decreased (P<0.05). Intracellular Ca²⁺ concentration increased 1.8 times (P<0.001). Protein detection showed that the expression of PLCB, CaNand p-NFATC1 in ASIC3 overexpression group were significantly up-regulated (all P<0.05). Conclusion ASIC3 overexpression up-regulates the expression of PLCB/CaN/p-NFATC1 by activating the Ca²⁺-CaN-NFAT signaling axis, promotes the continuous activation of NFAT, and then drives IL-17 transcription, eventually leading to Treg/Th17 immune imbalance.

Key words: ASIC3, Cell experiment, Treg/Th17 balance, Calcium ion, PBMCs