Sheng Mai San ameliorated heat stress-induced liver injury via regulating energy metabolism and AMPK/Drp1-dependent autophagy process

• Sheng Mai San (SMS) can effectively ameliorate liver dysfunction, suppress inflammatory response and oxidative stress, and repair histological destruction after heat exposure. • Targeted energy metabolomics confirmed that SMS can regulate glycolysis and tricarboxylic acid (TCA) cycle to improve energy metabolism disorder in heat stress- induced rat liver. • An AMPK-mediated Drp1-dependent mitophagy and mitochondria rebuilding process plays an important role in SMS protection in heat stress-induced liver injury. Liver damage is one of the most common complications in humans and animals after heat stress (HS). Sheng Mai San (SMS), a traditional Chinese medicine prescription that originated in the Jin Dynasty, exert a therapeutic effect on HS. However, how SMS prevents liver injury after heat exposure remains unknown. This study aimed to investigate the pharmacological effect and molecular mechanisms of SMS on HS-induced liver injury. A comprehensive strategy via incorporating pharmacodynamics, targeted metabolomics, and molecular biology technology was adopted to investigate energy metabolism changes and the therapeutic mechanisms of SMS in HS-induced rat liver injury. First, Sprague-Dawley rats were subjected to HS (38 °C/ 75% RH/ 2 h/ day) for 7 consecutive days to establish the HS model, and SMS was given orally for treatment 2 h before heat exposure. Thereafter, liver function and pathological changes in liver tissue were evaluated. Finally, the underlying mechanisms of SMS were determined using targeted energy metabolomics to comprehensively analyze the metabolic pathways and were further verified through Western-blot and qRT-PCR assays. Our results showed that SMS alleviated HS-induced liver dysfunction by reducing the alanine aminotransferase (ALT), aspartate aminotransferase (AST), and AST/ALT ratios in serum and improving hepatic pathological damage. Meanwhile, SMS suppressed inflammatory response, oxidative injury, and overexpression of heat shock proteins in liver tissue after heat exposure. With the help of targeted energy metabolomics, we found that SMS could effectively regulate glycolysis and tricarboxylic acid (TCA) cycle to relieve energy metabolism disorder. Furthermore, we confirmed that SMS can facilitate the phosphorylation of AMP-activated protein kinase (AMPK) to maintain mitochondrial homeostasis through a dynamin protein 1 (Drp1)-dependent mitophagy process. On the basis of energy metabolomics, the present study for the first time systematically illustrated the protective effect of SMS on HS-induced liver injury, and preliminarily confirmed that an AMPK-mediated Drp1-dependent mitophagy and mitochondria rebuilding process plays an important role in SMS intervention on HS-induced rat liver. Together, our study lends further support to the use of SMS in treating HS condition.