Pakistan Journal of Pharmaceutical Sciences

Abstract: Background: Asthma is a chronic airway inflammatory disease with limited curative options. Ephedrine (EP), a natural alkaloid, has shown anti asthmatic effects, but its molecular mechanism remains incompletely understood. Ferroptosis, an iron dependent lipid peroxidation cell death pathway, aggravates airway epithelial injury in asthma. Pyruvate kinase M2 (PKM2) generates lactate, which can drive protein lactylation; however, whether EP acts by modulating PKM2-dependent lactylation of acyl-CoA synthetase long-chain family member 4 (ACSL4) to inhibit ferroptosis is unknown. Objectives: To investigated whether EP attenuates ferroptosis-driven asthmatic progression by suppressing PKM2-dependent lactylation of ACSL4. Methods: LPS-stimulated BEAS-2B cells and ovalbumin (OVA)-sensitized rats were used as in-vitro and in-vivo asthma models, respectively. Cell proliferation (CCK-8), apoptosis (TUNEL), inflammatory cytokines (ELISA) and oxidative/lipid peroxidation markers (ROS, Fe²?, MDA, LPO, SOD) were measured. PKM2 and ACSL4 expression were determined by qPCR and Western blot; ACSL4 lactylation was assessed by co-immunoprecipitation. In-vivo, asthma behavioral scores, airway resistance (RL), dynamic compliance (Cdyn), lung histology (HE) and bronchoalveolar lavage fluid cytokines were evaluated. Results: In-vitro, EP restored cell viability, reduced apoptosis and cytokine release and inhibited ferroptosis (P < 0.05). LPS upregulated PKM2; EP reversed this, whereas PKM2 overexpression abolished EP’s protection (P < 0.05). PKM2 overexpression increased ACSL4 transcription and lactylation, effects attenuated by EP. Silencing ACSL4 reversed PKM2 overexpression induced injury and ferroptosis (P < 0.05). In-vivo, EP alleviated OVA induced asthmatic symptoms and pulmonary pathological changes. Conclusion: EP mitigates asthma by down-regulating PKM2, thereby blocking ACSL4 lactylation and ferroptosis-mediated airway epithelial injury. The PKM2 ACSL4 lactylation axis represents a novel therapeutic target.