An Integrated Analysis of Network Pharmacology and Experimental Validation to Reveal the Mechanism of Chinese Medicine Formula Naotaifang in Treating Cerebral Ischemia-Reperfusion Injury

Abstract

Background

Cerebral ischemia-reperfusion injury (CIRI) is a crucial factor leading to a poor prognosis for ischemic stroke patients. As a novel Chinese medicine formula, Naotaifang (NTF) was proven to exhibit a neuroprotective effect against ischemic stroke, clinically, and to alleviate CIRI in animals. However, the mechanisms underlying the beneficial effect have not been fully elucidated.

Methods

In this study, we combined a network pharmacology approach and an in vivo experiment to explore the specific effects and underlying mechanisms of NTF in the treatment of ischemia-reperfusion injury. A research strategy based on network pharmacology, combining target prediction, network construction, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and molecular docking was used to predict the targets of NTF in treating the ischemic stroke and CIRI. On the other hand, we used HPLC and HRMS to identify biologically active components of NTF. Middle cerebral artery occlusion models in rats were utilized to evaluate the effect and the underlying mechanisms of NTF against CIRI after ischemic stroke.

Results

Network pharmacology analysis revealed 43 potential targets and 14 signaling pathways for the treatment of NTF against CIRI after ischemic stroke. Functional enrichment analysis showed that a STAT3/PI3K/AKT signaling pathway serves as the target for in vivo experimental study validation. The results of animal experiments showed that NTF significantly alleviated CIRI by decreasing neurological score, infarct volume, numbers of apoptotic neuronal cells, increasing density of dendritic spines and survival of neurons. Furthermore, NTF could increase the expression of p-STAT3, PI3K, p-AKT. In addition, the detection of apoptosis-related factors showed that the NTF could raise the expression of Bcl-2 and reduce the expression of Bax.

Conclusion

This network pharmacological and experimental study indicated that NTF, as a therapeutic candidate for the management of CIRI following ischemic stroke, may exert a protective effect through the STAT3/PI3K/AKT signaling pathway.

Keywords:

• cerebral ischemia-reperfusion injury
• stroke
• network pharmacology
• molecular docking
• STAT3/PI3K/AKT signaling pathway

Acknowledgments

Special thanks to Yanan Luo for English language editing.

Abbreviations

CIRI, cerebral ischemia-reperfusion injury; NTF, Naotaifang; OB, oral bioavailability; DL, drug-likeness; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes; rtPA, recombinant tissue plasminogen activator; FDA, American Food and Drug Administration; TCM, traditional Chinese medicine; BYHWD, Buyang Huanwu decoction; HQ, Radix Astragali; CX, Rhizoma Ligustici; DL, Pheretima; JC, Bombyx Batryticatus; TCMSP, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform; OMIM, Online Mendelian Inheritance in Man; DC, degree centrality; BC, betweenness centrality; CC, closeness centrality; HPLC, high performance liquid chromatography; HRMS, high-resolution mass spectrometer; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; SD, standard deviation.

Data Sharing Statement

The data supporting the findings of this study are available from the corresponding author of this manuscript upon reasonable request and with permission of all the authors.

Ethics Statement

The animal study was reviewed and approved by the Experimental Animal Health Ethics Committee of Hunan University of Chinese Medicine.

Author Contributions

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that there are no conflicts of interest for this work.

Additional information

Funding

This work was supported by the fund of the National Key Research and Development Program of the Ministry of Science and Technology (2018YFC1704904), and the fund for Youth Top Talent Project of Hubei Provincial Health and Family Planning Commission (EWT-2019-48), and the Project Supported by Scientific Research Fund of Hunan Provincial Education Department (20A366).