Dan Wu,1,* Xiaohui Yan,1,* Siqi Mei,1 Hao Hua,1 Xiaoyu Fei,1 Xinyu Xu,1 Yan Liu,1 Minghua Wu,1 Jian Zhu,2 Wenlei Li1 

1Department of Neurology, Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China; 2Department of Endocrinology, Affiliated Hospital of Jiangnan University, Jiangnan University, Wuxi, Jiangsu, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Wenlei Li, Department of Neurology, Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China, Email yfy120@njucm.edu.cn Jian Zhu, Department of Endocrinology, Affiliated Hospital of Jiangnan University, Jiangnan University, Wuxi, Jiangsu, People’s Republic of China, Email drzhujian@hotmail.com

Background: Ischemic stroke (IS) remains a leading cause of long-term disability. Neurovascular regeneration and remodeling of the corticospinal tracts are essential for neurological functional recovery. Zuogui pill (ZGP) has good efficacy in treating cerebral ischemia, but the mechanism remains unclear.
Purpose: To investigate the effects of ZGP on angiogenesis, neurogenesis, corticospinal tract (CST) remodeling, and further evaluate its mechanisms of action in mice with ischemic stroke.
Methods: Network pharmacology was used to analyze the active components, related targets, and mechanisms of ZGP’s action in promoting neurovascular regeneration after ischemic stroke. Using a photothrombotic (PT) stroke mouse model, ZGP’s effects on neurological recovery were assessed using behavioral tests. Angiogenesis and neurogenesis were evaluated by immunofluorescence of glucose transporter type 1(Glut-1) +/5-bromo-20-deoxyuridine (BrdU) + vessels and doublecortin (DCX)+/BrdU+ cells. CST remodeling was evaluated through diffusion tensor imaging (DTI). The levels of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and mammalian target of rapamycin (mTOR) expression were tested by Western blot.
Results: Network pharmacology identified 94 active ingredients and 83 overlapping targets related to IS and neurovascular regeneration. mTOR was identified as one of the core targets. Behavioral tests demonstrated ZGP significantly reduced error rates in irregular ladder walking (ZGP-H vs Stroke: p=0.003) and shortened sticker removal time (ZGP-H vs Stroke: p=0.003). Immunofluorescence revealed ZGP enhanced angiogenesis (Glut-1+/BrdU+ vessels: ZGP-H vs Stroke, p=0.018), neural progenitor cell proliferation and migration (BrdU+/DCX+ cells: ZGP-H vs Stroke: p=0.014). DTI showed increased fractional anisotropy (FA) in ipsilateral CST regions (ZGP-H vs Stroke: 0.001Conclusion: ZGP promotes neurovascular regeneration, CST remodeling, and neurological function recovery after ischemic stroke. The positive impacts of ZGP are linked to heightened VEGF and BDNF expression and the activation of the mTOR pathway.

Keywords: zuogui pill, ischemic stroke, angiogenesis, neurogenesis, corticospinal tract remodeling, mTOR