Mechanistic of Mulberry Apricot Coughing Granules in Regulating Dysregulated Fluid Metabolism and Inhibiting Ferroptosis in Cough Variant Asthma

Background Cough variant asthma (CVA) is a distinct type of asthma manifested as dysregulation of fluid metabolism and modulation of ferroptosis. Objectives The aim of this study was to demonstrate the regulatory effects of Mulberry Apricot Coughing Granules on fluid metabolism dysregulation in CVA patients and elucidate its mechanism in inhibiting ferroptosis. Materials and Methods In this study, Mulberry Apricot Coughing Granules were prepared and analyzed for their major active components. Subsequently, in vitro experiments were conducted using human bronchial epithelial cells (BEAS-2B), which were divided into the following groups: control group (CG, basal medium), RSL3 group (treated with Ras-selective lethal substance 3), 0.2 mg/mL group, 0.1 mg/mL group, and 0.05 mg/mL group (treated with Mulberry Apricot Coughing Granules extract at concentrations of 0.2, 0.1, and 0.05 mg/mL, respectively), as well as the RSL3 + 0.2 mg/mL group (treated with RSL3 + 0.2 mg/mL Mulberry Apricot Coughing Granules extract). The levels of inflammatory factors, including interleukin (IL)-4, IL-6, and tumor necrosis factor (TNF)-α, were measured in each group, and reactive oxygen species (ROS) and malondialdehyde (MDA) levels were determined using assay kits. In addition, the expression levels of glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4) were assessed. Subsequently, a CVA mouse model was established using a sensitizing solution, with groups including the normal group (NG, physiological saline), CVA group (sensitizing solution), and treatment group (TG, sensitizing solution + Mulberry Apricot Coughing Granules treatment). Urine output and water intake were observed, and plasma anti-diuretic hormone (ADH) levels and urinary aquaporin-2 (AQP-2) content were measured. The expression levels of aquaporin-5 (AQP5), mucin 5AC (MUC5AC), and cystic fibrosis transmembrane conductance regulator (CFTR) were evaluated using quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting techniques. Results The chromatographic peaks of the water extract from Mulberry Apricot Coughing Granules and the amygdalin standard exhibited identical positions with clear separation. The distribution of active ingredients in Mulberry Apricot Coughing Granules was uniform. In the 0.2, 0.1, and 0.05 mg/mL groups, concentrations of IL-4, IL-6, and TNF-α were markedly inferior to CG ( p < .05), with the 0.2 mg/mL group showing the lowest levels ( p < .05). The levels of ROS and MDA, as well as the mRNA and protein expression of ACSL4, in the RSL3 group were significantly higher than those in the CG ( p < .05), while the mRNA and protein expression of GPX4 were decreased ( p < .05). In contrast, the levels of ROS and MDA and the mRNA and protein expression of ACSL4 were reduced in the 0.2 mg/mL group and the RSL3 + 0.2 mg/mL group, while the mRNA and protein expression of GPX4 were increased compared to the RSL3 group, with the 0.2 mg/mL group showing a better effect ( p < .05). In the CVA model, relative to NG, urine volume, water intake, AQP5, MUC5AC mRNA, and protein expression level were decreased, while plasma ADH concentration, urine AQP-2 content, CFTR mRNA, and protein expression level were increased in the CVA group ( p < .05). Mice in TG showed increased urine volume, water intake, AQP5, MUC5AC mRNA, and protein expression level, as well as decreased plasma ADH concentration, urine AQP-2 content, CFTR mRNA, and protein expression level versus CVA group ( p < .05). Conclusion Mulberry Apricot Coughing Granules regulate oxidative stress and lipid peroxidation by upregulating the antioxidant protein GPX4 and reducing ACSL4 expression level, thereby inhibiting ferroptosis and improving inflammatory responses. Moreover, Mulberry Apricot Coughing Granules may improve fluid metabolism abnormalities in CVA by enhancing the expression levels of AQP5 and MUC5AC while reducing CFTR expression levels. These findings suggest that Mulberry Apricot Coughing Granules may exert therapeutic effects on CVA through multiple mechanisms.