The extract from Quzhou Aurantii Fructus attenuates cough variant asthma through inhibiting the TRPV1/Ca2+/NFAT/TSLP pathway and ferroptosis via TRPV1 mediation in ovalbumin-induced mice

Ethnopharmacological relevance: Cough variant asthma (CVA), a prevalent chronic inflammatory disease, is the most common cause of chronic cough. Over the years, the aqueous extract of Quzhou Aurantii Fructus (QAFA) has been widely used to treat respiratory diseases, particularly cough. Aim of the study: This study aimed to elucidate the therapeutic effect of QAFA on allergen-induced CVA, providing deep insights into the underlying mechanisms. Materials and methods: Ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was employed to characterize the compositions, while UPLC was used to quantify the contents of its major components in QAFA. CVA model was established via sensitization and atomization with ovalbumin (OVA), and received 600 and 1200 mg/kg of QAFA via intragastric gavage. Cough response was assessed by stimulation with capsaicin (CAP). Then, airway hyperresponsiveness (AHR), ELISA, western blotting, RT-qPCR, and histological analyses, were applied to assess pulmonary function, pathological changes, and investigate mechanisms in CVA mice following QAFA treatment through the TRPV1/Ca2+-dependent NFATinduced expression of TSLP and ferroptosis. Additionally, the effects and mechanisms of QAFA were validated using IL-4, CAP for stimulation, capsazepine (CPZ) for inhibition, and TRPV1 siRNA transfection in cells. Results: Chemical analysis revealed that QAFA primarily contained sixteen compounds, with four main components including narirutin, naringin, hesperidin, and neohesperidin. In vivo, QAFA treatment alleviated cough and AHR, while concurrently reducing airway inflammation and mucus secretion in CVA mice. These effects were achieved by suppressing the TRPV1/NFAT/TSLP pathway and modulating the expression of ferroptosis-related proteins. In vitro, siTRPV1-transfected BEAS-2B cells demonstrated the involvement of the TRPV1 channel in IL-4-mediated Ca2+ influxes, ferroptosis, and regulation of TSLP production. QAFA and CPZ suppressed IL-4induced TSLP production via the TRPV1/NFAT pathway and regulated the levels of ferroptosis-related proteins, while CAP counteracted the effect of QAFA on TSLP production in BEAS-2B cells. Furthermore, QAFA reduced IL-4 or CAP induced Ca2+ influx and IL-4 induced ferroptosis through TRPV1 mediation.