Angong Niuhuang Pill ameliorates cerebral ischemic injury in mice by selectively activating mitophagy

Angong Niuhuang Pill (ANP) is a famous Chinese materia medica preparation prescribed for treating ischemic stroke. However, the protective mechanisms of ANP in acute ischemic stroke remain unclear and need to be explored. Our study aims to investigate the therapeutic effects of ANP on focal cerebral ischemia and explore the molecular mechanisms underlying its heat-clearing and detoxifying effects. The permanent middle cerebral artery occlusion (pMCAO) model was established in C57BL/6 male mice, which were then treated with ANP (25, 100, or 400 mg/kg) or 80 mg/kg butylphthalide. To further verify the involvement of mitophagy in ANP function, mice were also treated with the autophagy inhibitor wortmannin or the mitophagy inhibitor mdivi-1 at ischemic onset. Acute administration with 100, or 400 mg/kg ANP significantly improved neurological behavior and decreased cerebral infarct volume, suggesting that ANP conferred neuroprotective effects dose-dependently. Further, western blot analysis showed that ANP treatment significantly decreased expression of cytoplasmic cytochrome C and Bax, but elevated levels of mitochondrial cytochrome C and Bcl-2. TUNEL staining also indicated that ANP ameliorated cell apoptosis. ANP did not promote ischemia-induced lipidation of LC3, but significantly reduced the protein level of SQSTM1. Moreover, ANP further reduced mitochondrial contents and enhanced translocation of Parkin and SQSTM1 to the mitochondria. Importantly, the neuroprotective effects of ANP could be abolished by wortmannin and mdivi-1, respectively. Our findings showed that ANP exerted protective effects in response to ischemic injury and enhanced selective mitophagy without increasing autophagic flux.