3,4‐Dimethoxychalcone alleviates limb ischemia/reperfusion injury by TFEB‐mediated autophagy enhancement and antioxidative response

Abstract Caloric restriction mimetics (CRMs) replicate the positive effects of caloric restriction (CR) and have demonstrated therapeutic benefits in neuroinflammatory and cardiovascular diseases. However, it remains uncertain whether CRMs enhance functional recovery following ischemia/reperfusion (I/R) injury, as well as the specific mechanisms involved in this process. This study examines the therapeutic potential of the CRM 3,4‐dimethoxychalcone (3,4‐DC) in limb I/R injury. Histology, tissue swelling analysis, and laser doppler imaging (LDI) were used to assess the viability of the limbs. Western blotting and immunofluorescence were utilized to examine apoptosis levels, oxidative stress (OS), autophagy, transcription factor EB (TFEB) activity, and mucolipin 1 (MCOLN1)‐calcineurin signaling pathway. The administration of 3,4‐DC notably alleviated hypoperfusion, tissue swelling, skeletal muscle fiber damage, and cellular injury in the limb caused by I/R. The pharmacological blockade of autophagy negated the antioxidant and antiapoptotic effects of 3,4‐DC. Moreover, RNA interference‐mediated TFEB silencing eliminated the 3,4‐DC‐induced restoration of autophagy, antioxidant response, and antiapoptotic effects. Additionally, our findings revealed that 3,4‐DC modulates TFEB activity via the MCOLN1‐calcineurin signaling pathway. 3,4‐DC facilitates functional recovery by enhancing TFEB‐driven autophagy, while simultaneously suppressing oxidative stress and apoptosis following I/R injury, suggesting its potential value in clinical applications.