PAR2 Participates in the Development of Cough Hypersensitivity in Guinea Pigs by Regulating TRPA1 Through PKC

Objective: This study was conducted to validate the involvement of the PAR2-PKC-TRPA1 pathway in cough hypersensitivity (CHS) development. Methods: Guinea pigs were divided into a blank control, a citric acid-induced enhanced cough model, and drug intervention groups. The effects of the drugs on capsaicin-induced cough responsiveness in a cough model were observed. The effects of individual and combined treatments (including PAR2 agonists, TRPA1 agonists, PAR2 antagonists, TRPA1 antagonists, PKC agonists, and PKC antagonists) on PAR2, phospho-PKC (pPKC), and TRPA1 expression in bronchial tissues and the vagus ganglion (jugular and nodose) in the cough model and control groups were assessed. Additionally, whole-cell patch-clamp recordings were conducted to evaluate the effects of the drugs on vagus ganglion neuron electrophysiological activity. Results: ① Both PAR2 antagonists and TRPA1 antagonists significantly reduced cough frequency in guinea pigs with a cough, and the PAR2 antagonist inhibited coughing induced by the TRPA1 agonist. ② Western blotting and multiplex immunohistochemistry (mIHC) indicated that PAR2, pPKCα, PKCα, and TRPA1 expression in bronchial and vagus ganglion tissues was elevated in the cough model compared with the control, with TRPA1 expression being regulated by PAR2 and PKC being involved in this regulatory process. ③ Whole-cell patch-clamp recordings demonstrated that TRPA1 agonists induced an inward current in nodose ganglion neurons, which was further amplified by PAR2 agonists; this amplification effect was blocked by PKC antagonist. Additionally, PAR2 antagonists inhibited the inward current induced by TRPA1 agonists. ④ At various concentrations, including the optimal antitussive concentration, PAR2 antagonists did not significantly affect pulse amplitude, arterial oxygen saturation, heart rate, body temperature, or respiratory rate in guinea pigs. Conclusion: PAR2 regulates TRPA1 through PKC in cough syndrome (CHS) pathogenesis, making targeting PAR2 a safe and effective therapeutic strategy for CHS.