Yidong Cui,1 Qingming Liu,2 Qiqiang Zhang,3 Xuemei Di,3 Hai Zhang3 

1Department of Orthopedic Surgery, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Jinan, 250014, People’s Republic of China; 2Department of Neurology, Shandong Second Provincial General Hospital, Jinan, 250012, People’s Republic of China; 3Department of Pharmacy, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai, 200092, People’s Republic of China

Correspondence: Hai Zhang, Department of Pharmacy, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, No. 2699 West Gaoke Road, Shanghai, 200092, People’s Republic of China, Tel/Fax +8621-20261401, Email zhxdks2005@126.com

Background: Aconitum carmichaelii (Fuzi) has been conventionally used to cure a variety of ailments, such as pain, cold sensations, and numbness of limb muscles (Bi Zheng) in China. Our prior investigations identified Benzoylaconine (BAC) as a bioactive alkaloid derived from Aconitum carmichaelii, with other studies also demonstrating its significant pharmacological potential.
Purpose: This study aimed to explore the potential of BAC as a protective agent against skeletal muscle ischemia-reperfusion (I/R) injury and to elucidate the underlying mechanisms.
Methods: In vivo models involved subjecting Sprague-Dawley rats to I/R through femoral artery ligation followed by reperfusion, while in vitro models utilized C2C12 cells subjected to hypoxia/reoxygenation (H/R). CCK-8 assay was used to assess cell viability. TUNEL staining and flow cytometric analysis were used to measure cell apoptosis. Biochemical assay was used to assess skeletal muscle injury and oxidative stress. Immunofluorescence and Western blot were performed to determine protein levels.
Results: BAC effectively protected muscle tissue from I/R injury, enhancing cell viability (p< 0.01), elevating SOD levels (p< 0.05), and reducing CK (p< 0.01), LDH (p< 0.01), ROS (p< 0.01), MDA (p< 0.01), and apoptosis-related molecules in vivo and in vitro (p< 0.05, p< 0.01). Mechanistically, BAC increased the expression of IF1, phosphorylated AMPK, facilitated the translocation of nuclear Nrf2, and induced the expression of HO-1 (p< 0.01). Notably, AMPK inhibitor Compound C significantly hindered the ability of BAC to ameliorate H/R-induced cell injury (p< 0.05), oxidative stress(p< 0.01), and apoptosis (p< 0.05), as well as promote Nrf2 nuclear translocation (p< 0.01). Moreover, silencing of IF1 with siRNA abolished BAC-induced activation of AMPK/Nrf2 axis (p< 0.01).
Conclusion: Our study provides novel evidence supporting the potential of BAC as a myocyte-protective agent against I/R injury, and we establish a previously unknown mechanism involving the activation of the IF1-dependent AMPK/Nrf2 axis in mediating the protective effects of BAC.

Keywords: Benzoylaconine, ischemia-reperfusion injury, oxidative stress, AMPK, IF1