Abstract

   Background: Clinical research found that Hedysarum Multijugum
   Maxim.-Chuanxiong Rhizoma Compound (HCC) has definite curative effect
   on cerebral ischemic diseases, such as ischemic stroke and cerebral
   ischemia-reperfusion injury (CIR). However, its mechanism for treating
   cerebral ischemia is still not fully explained.

   Methods: The traditional Chinese medicine related database were
   utilized to obtain the components of HCC. The Pharmmapper were used to
   predict HCC’s potential targets. The CIR genes were obtained from
   Genecards and OMIM and the protein-protein interaction (PPI) data of
   HCC’s targets and IS genes were obtained from String database. After
   that, the DAVID platform was applied for Gene Ontology (GO) enrichment
   analysis and pathway enrichment analysis. Finally, a series of animal
   experiments were carried out to further explore the mechanism of HCC
   intervention in CIR.

   Results: The prediction results of systematic pharmacology showed that
   HCC can regulate CIR-related targets (such as AKT1, MAPK1, CASP3,
   EGFR), biological processes (such as angiogenesis, neuronal axonal
   injury, blood coagulation, calcium homeostasis) and signaling pathways
   (such as HIF-1, VEGF, Ras, FoxO signaling). The experiments showed that
   HCC can improve the neurological deficit score, decrease the volume of
   cerebral infarction and up-regulate the expression of HIF-1α/VEGF and
   VEGFR protein and mRNA (p < 0.05).

   Conclusion: HCC may play a therapeutic role by regulating CIR-related
   targets, biological processes and signaling pathways found on this
   study.

   Keywords: hedysarum multijugum maxim-chuanxiong rhizoma compound,
   cerebral ischemia-reperfusion injury, Ischemic stroke, cerebral
   ischemia, HIF-VEGF pathway, systematic pharmacology, chinese medicine

Introduction

   Cerebrovascular disease is a common disease in the clinic, which
   seriously endangers human health and life. As the population of aging
   increases, the morbidity, mortality and disability rate of
   cerebrovascular disease were increasing year by year ([38]Thomas, 1996;
   [39]Sacco and Rundek, 2012; [40]Liu et al., 2015). Among them, ischemic
   cerebrovascular disease accounts for a large proportion. The key to
   cerebral ischemia treatment is to quickly restore cerebral blood
   perfusion and maintain smooth blood flow ([41]Frizzell, 2005;
   [42]Behravan et al., 2014). However, the brain injury may be further
   aggravated after the restoration of blood flow perfusion, that is
   cerebral ischemia-reperfusion injury (CIR). Ischemia-reperfusion injury
   (IRI) refers to the pathological phenomenon that the degree of tissue
   damage is increased after the blood supply to the ischemic tissue is
   restored for a certain period of time ([43]Wu et al., 2018). The harm
   of CIR is huge. It involves many complicated links and factors, which
   has been the focus of scientists’ research for many years. With the
   deepening of the research, while looking for neuroprotective drugs, a
   variety of comprehensive intervention strategies for CIR such as mild
   hypothermia, atmospheric hyperbaric therapy and ischemic
   preconditioning and ischemic postconditioning were also proposed
   ([44]Lapi and Colantuoni, 2015; [45]Patel and McMullen, 2017; [46]Leech
   et al., 2019); the drugs include: N-methyl-D-aspartic acid (NMDA)
   receptor antagonist, Ca[2] + channel blocker, ICAM-1 antibody,
   CDP-choline, and so on ([47]Lapi and Colantuoni, 2015; [48]Patel and
   McMullen, 2017). At present, natural plant ingredients have been found
   to improve microcirculation barriers after CIR, and Chinese medicine
   formulations have gradually become a new direction for new drug
   development ([49]Wang et al., 2019).

   Hedysarum Multijugum Maxim.-Chuanxiong Rhizoma Compound (HCC) was first
   used by the First Affiliated Hospital of Hunan University of Chinese
   Medicine. Clinical research showed that HCC has definite curative
   effect on cerebral ischemic diseases (such as ischemic stroke and CIR),
   and its clinical effective rate is over 90% ([50]Ge, 2014). This
   Chinese medicine formula is composed of Hedysarum Multijugum Maxim.
   [Leguminosae; Astragali Radix (Huang Qi)], Ligusticum striatum DC.
   [Apiaceae; Chuanxiong Rhizoma (Chuan Xiong)], Pheretima Aspergillum
   (E.Perrier) [Megascolecidae; Pheretima (Di Long)], Bombyx Batryticatus
   [A desiccated body formed by the 4–5 instar larvae of Bombyx mori
   Linnaeus (family: Bombycidae) infected with white Beauveria bassiana
   (Bals.) Vuillant; (Jiang Can)], and can reduce serum tumor necrosis
   factor (TNF) -α and plasma thromboxane B2 (TXB2) levels and increase
   plasma 6-Keto-PGF1α ([51]He et al., 2002). Our previous research also
   found that HCC can protect neurons in the hippocampal CA[2] region by
   regulating Fpn expression to balance iron levels after cerebral
   ischemia. This suggests that imbalance of intracellular iron balance
   may be a new mechanism of cerebral ischemia ([52]Liao et al., 2015).
   However, the mechanism of HCC in treating cerebral ischemia is still
   not fully explained. Systematic pharmacology is an emerging discipline
   based on the intersection and integration of multidisciplinary
   technologies such as classic pharmacology, computer technology,
   bioinformatics, and network pharmacology, which systematically studies
   the interactions between drugs and the human body at multiple levels,
   including molecules, cells, organs, and networks ([53]Zeng et al.,
   2017; [54]Zeng and Yang, 2017; [55]Bao et al., 2019; [56]Yang et al.,
   2019a; [57]Yang et al., 2019b). Our previous research used systematic
   pharmacological strategies to reveal the mechanism of Chinese medicine
   formula in the treatment of complex diseases in the fields of oncology
   and cardiovascular ([58]Zeng et al., 2017; [59]Zeng and Yang, 2017;
   [60]Bao et al., 2019; [61]Yang et al., 2019a; [62]Yang et al., 2019b).
   Therefore, this study hopes to reveal the complex mechanism of HCC
   through a systematic pharmacology strategy (integrating network
   pharmacology experimental pharmacology).

Materials and Methods

HCC’s Compounds Collection

   The components of HCC were collected from the traditional Chinese
   Medicine (TCM) Database at Taiwan ([63]http://tcm.cmu.edu.tw/zh-tw/)
   ([64]Chen et al., 2014) and the Traditional Chinese Medicine Systems
   Pharmacology Database (TcmSPTM, [65]http://tcmspw.com/tcmsp.php)
   ([66]Ru et al., 2014). The components with oral bioavailability (OB) ≥
   30%, Caco-2 permeability > −0.4 and drug-likeness (DL) ≥ 0.18 were
   considered as the potential bioactive compounds of HCC ([67]Walters and
   Murcko, 2002; [68]Ano et al., 2004; [69]Hu et al., 2009; [70]Xu et al.,
   2012; [71]Ru et al., 2014). Meanwhile, since the application of
   biological models to predict HCC compounds has limitations
   ([72]Metodiewa et al., 1997), a large number of references were