ARTICLE doi: 10.12032/ATR20200603 Asian Toxicology Research A network pharmacology approach combined with animal experiment to investigate the blood enriching effect of Gei herba Wen-Bi Mu1, 2#, Can-Can Duan1, 2#, Zhi-Ping Zhong1, 2, Kuan Chen1, 2, Jian-Yong Zhang1, 2* 1School of Pharmacy, Zunyi Medical University, Zunyi 563000, China; 2Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi 563000, China. #Authors contributed equally to this article. *Corresponding to: Jian-Yong Zhang. Zunyi Medical University, No. 6 Xuefu West Road, Xinpu District, Zunyi 563000, China. Email: [email protected]. Highlights (1) A network pharmacology approach and animal experiments were established to explore the nourishing blood effect of Lanbuzheng (Gei herba). (2) The main active components, targets and pathways of Lanbuzheng (Gei herba) of blood deficiency were predicted by network pharmacology. (3) It’s verified that Lanbuzheng (Gei herba) can treat blood deficiency by improving the peripheral blood routine index and organ index in animal experiment. Submit a manuscript: https://www.tmrjournals.com/atr ATR | August 2020 | vol. 2 | no. 3 | 109 doi: 10.12032/ATR20200603 ARTICLE Abstract Background: To explore active components of Lanbuzheng (Gei herba) and its underlying complex mechanism in treating blood deficiency induced by chemotherapy drug based on network pharmacology and mice experimental validation. Methods: Active components of Lanbuzheng (Gei herba) were screened by Lipinski’s rule of five. Targets acted with active components were predicted by PharmMapper database, and targets whose function associated with blood deficiency were screened by Therapeutic Target Database and UniProt. The networks of component-target and target-pathway were constructed by Cytoscape. The levels of peripheral blood and organ indexes were detected in the animal experiments. Results: One hundred and seventy-three components of Lanbuzheng (Gei herba) were collected, and 60 active components were screened according to the rule of five. According to the degree value of compounds, the top 5 compounds were docosyl trans ferulate, C32 decursin, agrimonolide 6-O-β-D-glucoside, degree = 11, 173-ethoxyphaeophorbide, and eugenol. Finally, 59 targets associated with blood deficiency were obtained and the top 5 targets were MAPK14, TTR, CDK2, AKR1B1 and AR. Based on the interaction network of component- target and target-pathway, it’s found that 60 active components could act with 59 targets and 44 pathways for treating blood deficiency. And then, the mice experiments showed that Lanbuzheng (Gei herba) could enrich blood by increasing the levels of red blood cell, white blood cell, hemoglobin, red blood cell specific volume and platelet, and the indexes of liver, thymus and spleen, which validated the treating effect of Lanbuzheng (Gei herba). Conclusion: In this study, a network pharmacology approach and animal experiments were established to explore the nourishing blood effect of Lanbuzheng (Gei herba). The results demonstrated that Lanbuzheng (Gei herba) could improve blood deficiency and provide a theoretical basis for the further research on the in-depth mechanism of Lanbuzheng (Gei herba). Key words: Gei herba, Network pharmacology, Blood deficiency, Blood enriching, Mice experiments Author contributions: Wen-Bi Mu, Can-Can Duan and Jian-Yong Zhang contributed to the design and conception of the experiment; Kuan Chen contributed to the collection of Lanbuzheng (Gei herba) compounds and the screening of active compounds; Wen-Bi Mu and Can-Can Duan contributed to not only the drawn of compound-target and protein- protein interactions network, but also the analysis of gene ontology biological process and kyoto encyclopedia of genes and gnomes pathway; Wen-Bi Mu, Can-Can Duan and Zhi-Ping Zhong contributed to the feeding, administration and blood deficiency related indicators of animal experimen; Wen-Bi Mu contributed to the analysis of data and the drawing of figures; Wen-Bi Mu contributed the writing of paper and Jian-Yong Zhang contributed revised the modification of paper. All authors reviewed and approved the submitted version of the paper. Competing interests: The authors declare that they have no conflict of interest. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81760746 and 81803838), Education Department of Guizhou Province of China (GNYL [2017] 006), Provincial Department of Education youth talent support program (qiankehe KY [2017] 078), Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302), Science and Technology Department of Zunyi City of Zuniyi City of Guizhou Province of China ([2016] 35, [2016] 33) and Innovation talent team of Guizhou science and Technology Department (qiankehe platform talents [2020] 5007). Abbreviations: GH, Lanbuzheng (Gei herba); TCM, traditional Chinese medicine; BD, blood deficiency; G-CSF, granulocyte- colony stimulating factor; TTD, Therapeutic Target Database; PPI, protein-protein interactions; GO, gene ontology; KEGG, kyoto encyclopedia of genes and gnomes; APH, acetyl phenylhydrazine; HGB, levels of hemoglobin; RBC, red blood count; WBC, white blood count; HCT, hematocrit; PLT, total platelets; LI, liver index; TI, thymus index; SI, spleen index; PPI, protein-protein interaction; MGH, middle-dose solution of GH treatment group; HGH, high-dose solution of GH treatment group; SWT, the Classic ancient prescription Siwu Tang treatment group; LGH, low-dose solution of GH treatment group; D, degree. Citation: Wen-Bi Mu, Can-Can Duan, Zhi-Ping Zhong et al. A network pharmacology approach combined with animal experiment to investigate the blood enriching effect of Gei herba. Asian Toxicology Research 2020, 2 (3): 109– 121. Executive Editor: Yu-Ping Shi. Submitted: 08 May 2020, Accepted: 01 July 2020, Online: 09 July 2020 110 | no. 3 | vol. 2 | August 2020 | ATR Submit a manuscript: https://www.tmrjournals.com/atr ARTICLE doi: 10.12032/ATR20200603 pharmacology was extremely fit with the TCM with Background multiple compounds and multiple targets due to targeting multiple nodes in interconnected molecular Blood deficiency (BD) syndrome, as a common clinical systems [19]. Now, network pharmacology has been symptom of traditional Chinese medicine (TCM) [1], applied to study of the mechanism of many blood- was a pathological state induced by insufficient blood supplemented TCMs, such as the Chinese patent production or decreased blood support function. And the medicine Xinshenghua granule which consisted of pathological features are similar to modern Danggui (Angelicae sinensis radix), Yimucao (Leonuri hematopoietic impairment diseases [2], such as herba), Chuanxiong (Chuanxiong rhizoma), Taoren malnutrition anemia, anemia induced by tumor (Persicae semen), Ganjiangtan (Zingiberis rhizome chemotherapy, low immunity and so on. Modern carbonisata), Zhigancao (Glycyrrhizae radix et rhizome pharmacology showed that iron, recombinant praeparata cum melle) and Honghua (Carthami flos) at granulocyte-colony stimulating factor and a proportion of 80: 90: 30: 8: 5: 5: 5 [17], the classic erythropoietin were presently common medicines for ancient prescription Danggui buxue decoction which the treatment of hematopoietic impairment diseases in consisted of Danggui (Angelicae sinensis radix) and clinic [3]. However, these medicine treatments showed Huangqi (Astragali radix) at a ratio of 1:5 [20–21] and certain side effect, high cost and individuality [4–5]. Danggui (Angelicae sinensis radix)-Honghua Nowadays, TCM has attracted more and more attention (Carthami flos) herb pair [22]. Therefore, network from researchers for the treatment of BD due to fewer pharmacology is a new preferred approach to predict the toxic side effects, lower price and more safety. Based on mechanism of TCM. TCM theory, enriching blood and Qi (it is the most basic In this study, network pharmacology was employed element that constitutes human body and maintains life to predict the blood enriching effect of GH, then the activities in TCM) are the main methods to treat BD, related mechanism was explored, finally, animal and TCM treatment of BD has achieved good results experiments were performed to validation. both in clinical and animal experiments. For example, Danggui (Angelicae sinensis radix) and the classic Material and methods ancient prescription Danggui Buxue decoction which consisted of Danggui (Angelicae sinensis radix) and Active compounds of GH screening Huangqi (Astragali radix) at a ratio of 1:5 have been Chinese Pharmacopoeia 2015 version, Wanfang shown a good replenishing effect [6–7]. database (http://www.wanfangdata.com.cn/), Chinese Lanbuzheng (Gei herba, GH) is the dried whole grass National Knowledge Infrastructure database of Geum aleppicum Jacq. and Geum japonicum Thunb. (http://www.cnki.net/), Traditional Chinese Medicine var. chinense Bolle in 2015 Chinese Pharmacopoeia Systems Pharmacology Database and Analysis [8], which is used as a common blood-replenishing Platform database (http://tcmspnw.com), Pubmed herbal by ethnic minorities in Guizhou, China. (http://www.ncbi.nlm.nih.gov/pubmed/), were used to According to TCM theory, GH shows multi-activities search the components of GH. Then, active compounds including strengthening the spleen, replenishing Qi and were screened