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 according to the Lipinski’s rule of five by blood, promoting blood circulation and detoxification. PaDEL-Desceiptor software [23]. Generally, the Modern pharmacological study has found that GH could molecular weight less than 500 daltons, hydrogen bond cure ischemic diseases, inflammatory process disease donors and an octanol-water partition coefficient logP and tumor [8]. And then, GH has been reported for less than 5, and hydrogen bond acceptors and rotatable various pharmacological activities including blood- bonds less than 10 were thought to be the candidate supplying, anti-inflammatory, promoting blood and components by Lipinski’s rule of five. oxygen supply, and anti-vertigo [9–12]. Additionally, GH could down-regulate the expression of Target prediction and compound-target inflammatory factors in senile dementia mice [13], construction improve the learning and memory ability of vascular The structures of the active compounds were dementia mice by inhibiting the expression of NF-kB transformed into Spatial Data File and canonical smiles and IL-6 in hippocampus [14]. These anti-inflammatory structure format by PubChem Compound activities of GH may be beneficial for treating diseases (https://www.ncbi.nlm.nih.gov/pccompound/). And related with BD. However, folk clinical trials of blood then, PharmMapper database enriching effect of GH are mostly scattered reports, and (http://59.78.96.61/pharmmapper) was used to predict animal experiments are only proved by the hemorrhagic targets acted with these compounds, all obtained targets BD model. The curative effect of GH on blood were retrieved from Therapeutic Target Database (TTD, supplementation has still not been recognized. http://bidd.nus.edu.sg/group/cjttd/) [24] and UniProt Network pharmacology is a new strategy to explore (https://www.uniprot.org/) to explore the function. the biological process based on network construction Based on the characteristics of BD, targets will be and topology analysis [15–18]. And network screened if its function associated to immune diseases, Submit a manuscript: https://www.tmrjournals.com/atr ATR | August 2020 | vol. 2 | no. 3 | 111
doi: 10.12032/ATR20200603 ARTICLE anemia, tumors and blood system diseases. diet and water. The animal experiments were approved Furthermore, these screened targets were imported by the Ethical Committee of Zunyi Medical University into Cytoscape3.7.1 (http://www.cytoscape.org/) to (approval No.: ZUMER 2015-2-026). Seventy-two construct compound-target network. healthy female mice were randomly divided into 6 groups (12 mice for each group): control group (con), Protein-protein interactions analysis model group (mod), the Classic ancient prescription These screened targets were imported into STRING Siwu Tang treatment group (SWT), high-dose solution (version 10.5, https://string-db.org/) to study the of GH treatment group (HGH), middle-dose solution of interaction among them, and the protein-protein GH treatment group (MGH), low-dose solution of GH interactions (PPI) network was constructed by treatment group (LGH). According to the method Cytoscape 3.7.1, the line thickness of two targets reported in the literature [25–26], the BD model mice indicated the strength of supported data, the thicker the was induced by acetyl phenylhydrazine (APH) (lot No. lines, the stronger the interactions. RS1014S4011Z, Shanghai, China) and cyclophosphamide (lot No. 13122425, Jiangsu, China). Gene ontology and pathway enrichment analysis First of all, mice in con，SWT and GH groups were DAVID platform (functional annotation result summary, hypodermically injected with 20 mg/kg APH on day 1. https://david.ncifcrf.gov/summary.jsp) was hired to Then, 2 h after the hypodermic injection with 10 mg/kg perform gene ontology (GO) and Kyoto encyclopedia of APH on day 4, mice were intraperitoneally injected with genes and gnomes (KEGG) pathway enrichment cyclophosphamide saline solution on day 4, 5, 6 and 7 analysis. The threshold P-value < 0.05, and the main at a dose of 20 mg/kg. The mice in con group were also signal pathways which targets acted were obtained. hypodermically injected with equivalent normal saline. Furthermore, the Omicshare online analysis platform Mice in HGH, MGH and LGH groups were was used to visualize the results of enrichment, analysis intragastrically given GH at a dose of 20, 10, 5 g/kg/d, the main signal pathway of GH treating BD and reveal respectively. Mice in SWT group were intragastrically the mechanism. Finally, target-pathway network was given the Classic ancient prescription Siwu Tang at a constructed by Cytoscape 3.7.1. dose of 10 g/(kg·d). Simultaneously, mice in con and mod groups were intragastrically given with equivalent Animal experiment for verifing the efficacy of GH distilled water. The administration course is 7 days in The solution of GH: 250 g of GH dry herbs was weighed total, 24 h after the last intragastric administration, the and placed in a medicine pot, 2000 mL of distilled water blood of mice was collected into heparinized was added and soaked for 30 min, then boil and keep anticoagulant tubes by posterior orbital venous plexus slightly boiled for 30 min. The drug residue was approach for blood routine detection. The levels of decoctioned again in the same method and filtered while hemoglobin (HGB), red blood count (RBC), white still hot. The two filtrates were combined and blood count (WBC), hematocrit (HCT) and total concentrated by rotation to 1 g/mL of the original drug, platelets (PLT) were analyzed by fully automatic all liquid medicines were divided into aliquots and hematology analyzer (lot No. XS-1000i, Kobe, Japan). stored at a low temperature of −20 °C. The mice were killed by spinal dislocation, and the liver, The solution of the Classic ancient prescription Siwu thymus and spleen of mice were separated aseptically. Tang: Shudihuang (Rehmanniae radix praeparata), Then, the liver index (LI), thymus index (TI) and spleen Danggui (Angelicae sinensis radix), Baishao (Paeoniae index (SI) were calculated by the formula: LI/TI/SI = radix alba), Chuanxiong (Chuanxiong rhizoma) (45 g: (tissue weight (mg)/mouse weight (g)) × 10. 30 g: 30 g: 18 g) were weighed according to the Additionally, a part of liver was used to detect ATPase prescription of the Classic ancient prescription Siwu according to the instructions. Tang (the ancient book Taiping Huimin Heyaofang The mice were killed by cervical dislocation, the liver (Welfare Pharmacy, written by Liu Jingyuan in 1151 tissue (0.2 g–0.1 g) was washed with pre-cooled C.E.)), and 1000 mL water was added and soaked for 30 physiological saline and weighed, the total volume of min, then boil and keep slightly boiled for 30 min. The pre-cooled homogenate medium (pH 7.4, 0.01 mol/L medicine residue was fried again in the same method Tris-HCl, 0.0001 mol/L ethylenediaminetetraacetic acid and filtered while still hot. The two filtrates were disodium, 0.01 mol/L sucrose, 0.8% sodium chloride combined and concentrated by rotation to 0.5 g/mL of solution) was 9 times the tissue mass, repeatedly the original drug, all liquid medicines were divided into ultrasonically crushed it, and then centrifuged at 1,000 aliquots and stored at a low temperature of −20 °C. r/min for 5 minutes using a low-temperature and low- BD syndrome is more common in women in daily speed centrifuge. The supernatant was treated according life, so female mice (17–22g) with 4 weeks old were to the instructions of the ATPase kit (lot No. 20140825, supplied by the Third Military Medical University Nanjing, China), and the activity of Na+-K+-ATPase in Animal Experiment Center (Chongqing, China). The mouse liver tissue homogenate was determined by the mice were raised under standard temperature, humidity phosphorus determination method. According to the and light conditions, and had access to a standard rodent measurement results, the content of Na+-K+-ATPase in 112 | no. 3 | vol. 2 | August 2020 | ATR Submit a manuscript: https://www.tmrjournals.com/atr
ARTICLE doi: 10.12032/ATR20200603 the liver tissues of mice was calculated to evaluate the represents the association between the targets. effect of GH on energy metabolism in mice. Additionally, the top 10 targets were shown in Figure 3 according to the proportion of targets, including SRC (D Statistical analysis = 16), MAPK14 (D = 11), MAPK8 (D = 11), AR (D = The experimental data was expressed as mean ± 9), NOS3 (D = 7), MAP2K1 (D = 7), ESR1 (D = 7), standard deviation, the IBM SPSS Statistics 19.0 PGR (D = 6), FGG (D =6) and F2 (D = 5), these software was used to perform one-way statistical targets may be key targets for GH prevention and analysis of variance on the experimental data. The treatment of BD. homogeneity of variance was analyzed using the lysergic acid diethylamide method, and the uneven Functional enrichment analysis variance was determined by Dunnett’s T3. P < 0.05 Fifty-nine targets acted with GH were imported into the indicated that the difference was statistically significant. DAVID for GO and pathway enrichment analysis. Then The graph was drawn by Graphad and Cytoscape 3.7.1, the hierarchical relationships of biological processes and the table was drawn by Excel 97-2003. were obtained. There were 132 biological processes, whose P value no more than 0.05, and the top 10 were Results displayed in Table 3 and Figure 4, respectively. These targets mainly involved steroid hormone mediated Active compounds of GH signaling pathway, transcription initiation from RNA Firstly, 173 ingredients of GH were collected totally. polymerase II promoter, negative regulation of Based on Lipinski’s rule of five, only 60 active apoptotic process, positive regulation of MAPK compounds were screened and showed in Table 1. cascade, et al. It’s suggested that the BD syndrome may According to the degree (D) value of compounds, the be prevented by GH on the above biological processes. top 5 compounds (D ≥ 10) were C33 (docosyl trans The KEGG pathway enrichment analysis was ferulate, D = 13), C32 (decursin, D = 11), C2 performed on 59 targets of GH by the STRING online (agrimonolide 6-O-β-D-glucoside, D = 11), C15 (173- analysis platform, the D of enrichment was measured. ethoxyphaeophorbide, D = 10) and C20 (eugenol, D = As shown in Figure 5, the first 20 terms with the most 10). significant P value were picked. The results showed that these targets were mainly enriched in the three signaling Target prediction and compound-target network pathways of PI3K-Akt signaling pathway, prolactin construction signaling pathway and Rap1 signaling pathway. There were 595 related targets of active compounds in Additionally, the target-pathway network was GH, then the functions of each target were obtained constructed, which was composed of 112 nodes and 279 from TTD, UniProt and published literatures, and edges and shown in Figure 6. The targets were showed repeated targets were deleted. Finally, a total of 59 in the smaller circle with pink colors, and the pathways targets related BD were reserved and shown in Table 2. were shown in blue circle. There are multiple targets on Afterwords, compound-target network was constructed one pathway, and the same target may act on multiple and shown in Figure 1. In the network, the D of a node pathways (Figure 6). represents the number of routes connected to the node. The larger the D value, the more important the node Peripheral blood results of mice plays in the network. The compound-target network The effect of GH on peripheral blood in mice was shown consisted of 119 nodes and 438 edges, 60 green nodes in Figure 7. The levels of RBC, WBC, HGB and HCT represent compounds of GH, 59 yellow nodes represent in mod group were significantly decreased compared target of compounds. According to the D value of with control group (P < 0.05 or P < 0.01), which targets, the top 5 targets were MAPK14 (D = 29), TTR indicated that the BD model was constructed (D = 28), CDK2 (D = 21), AKR1B1 (D = 20) and AR successfully. After SWT treatment, the levels of WBC, (D = 19). HGB, HCT and PLT were increased in MGH and HGH groups (P < 0.05 or P < 0.01), the level of WBC was PPI analysis increased in MGH group (P < 0.05), and the levels of A total of 59 BD-related targets were imported into the HCT and HGB were increased in LGH, MGH and HGH STRING database to obtain PPI network among these groups (P < 0.05 or P < 0.01). targets, then score with greater than or equal to 0.7 were eventually retained, and PPI network was re-arranged Effect of GH on organ index of BD mice by D and exhibited in Figure 2. Each node represents a The results of organ index were shown in Figure 8. target, the color change and the node size correspond to Compared with con group, the levels of TI, SI and LI its D value. The brighter the color, the greater the D were decreased in mod group (P < 0.05 or P < 0.01), value, the red triangle has the largest D value, and the which demonstrated that BD model was constructed gray triangle has the smallest D value. And the larger successfully. These index were increased after SWT and the node size, the greater the D value. And the edge GH intervened, SI and LI were increased in SWT group Submit a manuscript: https://www.tmrjournals.com/atr ATR | August 2020 | vol. 2 | no. 3 | 113
doi: 10.12032/ATR20200603 ARTICLE (P < 0.05 or P < 0.01). The levels of SI, and LI were better potential ability of supplementing blood by increased in LGH, MGH and HGH groups (P < 0.05 or network pharmacology. Furthermore, the efficacy of P < 0.01), the levels of TI were increased in LGH and GH was verified by animal experiments, which laid the HGH groups (P < 0.05). foundation for further development. Network pharmacology is aimed at targeting multiple Activity of Na+-K+-ATPase in liver of mice nodes in interconnected molecular systems, rather than The ATP activity in liver tissue was calculated and the individual molecules, to achieve better efficacy and results were shown in Figure 9. The Na+-K+-ATPase fewer adverse effects, which was extremely fit with the activity of mod group was decreased compared with con theory of multiple compounds and multiple targets in group (P < 0.01). Compared with mod group, the TCM [27]. In this study, GH was employed to study activities of Na+-K+-ATPase in HGH, MGH and LGH blood enriching effect by network pharmacology groups were all increased (P < 0.05). approach. Different networks were constructed, such as component-target network, target-pathway network. Discussion According to the D, component-target network analysis showed that the main active ingredients (D ≥ 10) of GH In this study, network pharmacology and experimental were docosyl trans ferulate (D = 13), decursin (D = 11), pharmacology were used to explore the effect of the agrimonolide 6-O-β-D-glucoside (D = 11), 173- national medicine GH on the BD caused by ethoxyphaeophorbide (D = 10), Eugenol (D = 10), chemotherapy. First of all, it was found that GH has the caffeic acid (D = 10) and benzoic acid (D = 10),
Table 1 Active compounds of GH Number Name D Number Name D (7R, 8S)-3-methoxy-3’, 7-epoxy-8, 4’-oxyneo- C1 9 C31 Daucosterol 5 ligna-4, 9, 9’-triol-9’-O-α-L-rhamnopyranoside C2 Agrimonolide 6-O-β-D-glucoside 11 C32 Decursin 11 （9Z, 11E)-13-hydroxy-9, 11-octadecadienoic C3 5 C33 Docosyl trans ferulate 13 acid ethylester C4 1, 2-phthalic acid-bis-2-methylheptyl ester 9 C34 Ellagic acid 7 1 beta, 2 alpha 19 alpha, trihydroxy-3-oxo-12- C5 9 C35 Ethyl gallate 9 ursen-28-oic acid C6 1-O-methyl-6-O-caffeoyl-β-D-glucopyranose 3 C36 N-eicosyl trans ferulate 6 2 alpha, 3 alpha, 19 alpha, 23-tetrahydroxy-12- C7 7 C37 N-teracosyl docosyl trans ferulate 10 ursen-28-oic acid C8 2α, 3α-2, 3-dihydroxy-urs-12-en-28-olc acid 8 C38 N-tritriacontanol 7 C9 3, 4, 5-trihydroxy benzaldehyde 9 C39 Palmitic acid 7 C10 3, 4-dihydroxybenzoic acid 9 C40 P-hydroxybenzoic acid 8 C11 4-methyl-5, 6-dihydro-2-pyranone 6 C41 P-hydroxycinnamic acid 10 C12 5-hydroxymethylfurfural 8 C42 Ursolic acid 7 C13 Methyl 11, 14, 17-icosatrienoate 6 C43 Vanillin 7 C14 20β, 28-epoxy-28-hydroxytaraxasteran-3β-ol 8 C44 Euscaphic acid 8 C15 173-ethoxyphaeophorbide 10 C45 Glycerol-1-monopalmitate 10 C16 A-amirin 7 C46 Isololiolide 5 C17 Amburoside A 5 C47 Kaempferol 4 C18 Apocynin 6 C48 Linoleic acid 4 C19 α-tocoquinone 7 C49 Loliolide 5 C20 Eugenol 10 C50 Luteolin 7 C21 Hydroxybenzoic acid 8 C51 Megastigman-7-ene-3, 5, 6, 9-tetrol 5 C22 Trans-phytol 5 C52 3-O-trans-p-coumaroyltormentic acid 8 C23 Caffeic acid 10 C53 Epifridelinol 5 C24 Chlorogenic acid 4 C54 Galliic acid 7 C25 Caryophyllene oxide 6 C55 Tormetic acid 7 C26 Salicylic acid 9 C56 Benzoic acid 10 C27 Vanillin 8 C57 Quercetin 7 C28 Beta-sitosterol 8 C58 4’, 5, 7-trihydroxyflavone 4 C29 Blumenol A 7 C59 3’-O-methyl-3, 4-methylenedioxo ellagic acid 6 C30 Cupressoside A 6 C60 3, 3’-Di-O-methyl ellagic acid 6 GH, Lanbuzheng (Gei herba); D, degree.
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ARTICLE doi: 10.12032/ATR20200603 which indicated that these ingredients may be the main erythropoiesis, and the anemia of inflammation is pharmacodynamic basis for GH to treat BD. Eugenol regarded as the most frequent anemia in hospitalized could protect the liver against ischemia/reperfusion and chronically ill patients [30–31]. So, eugenol and injury by down-regulating inflammatory mediator level caffeic acid may improve BD by inhibiting [28]. Study has found that chronic aplastic anemia could inflammation. However, the effects of other active be effectively improved by caffeic acid tablets in ingredients on BD are not clear. clinical [29]. Research had confirmed that This study found that the core targets of GH action inflammation-inducible cytokines could including MAPK14 (D = 29), TTR (D = 28), CDK2 (D block intestinal iron absorption and cause iron retention = 21), AKR1B1 (D = 20) and AR (D = 19). Abnormal in reticuloendothelial cells resulting in iron-restricted expression of Mapk14 mRNA was inhibited in a BD
Table 2 Targets of GH Number Target Gene name Number Target Gene name Histo-blood group ABO system T-1 ABO T-31 Glutathione S-transferase A1 GSTA1 transferase T-2 Angiotensin-converting enzyme ACE T-32 Insulin receptor INSR Vascular endothelial growth factor T-3 Aldose reductase AKR1B1 T-33 KDR receptor 2 Aldo-keto reductase family 1 T-4 AKR1C3 T-34 Kinesin-like protein KIF11 KIF11 member C3 Proto-oncogene tyrosine-protein kinase T-5 Annexin A5 ANXA5 T-35 LCK LCK T-6 Androgen receptor AR T-36 Leukotriene A-4 hydrolase LTA4H Dual specificity mitogen-activated T-7 Arginase-1 ARG1 T-37 MAP2K1 protein kinase kinase 1 Branched-chain-amino-acid T-8 BCAT2 T-38 Mitogen-activated protein kinase 14 MAPK14 aminotransferase, mitochondrial T-9 Bone morphogenetic protein 2 BMP2 T-39 Mitogen-activated protein kinase 8 MAPK8 B-Raf proto-oncogene T-10 BRAF T-40 Hepatocyte growth factor receptor MET serine/threonine-protein kinase T-11 Cystathionine beta-synthase CBS T-41 Macrophage migration inhibitory factor MIF T-12 Cell division protein kinase 2 CDK2 T-42 Stromelysin-1 MMP3 T-13 Cell division protein kinase 6 CDK6 T-43 Neutrophil collagenase MMP8 T-14 Cytochrome P450 2C8 CYP2C8 T-44 Nitric oxide synthase, inducible NOS2 T-15 Dihydrofolate reductase DHFR T-45 Nitric oxide synthase, endothelial NOS3 Eukaryotic translation initiation T-16 EIF4E T-46 NAD(P)H dehydrogenase [quinone] 1 NQO1 factor 4E Nuclear receptor subfamily 1 group I T-17 Leukocyte elastase ELANE T-47 NR1I2 member 2 T-18 Ephrin type-A receptor 2 EPHA2 T-48 Mineralocorticoid receptor NR3C2 T-19 Ephrin type-B receptor 4 EPHB4 T-49 Progesterone receptor PGR T-20 Estrogen receptor ESR1 T-50 Pyruvate kinase isozymes R/L PKLR T-21 Estrogen receptor beta ESR2 T-51 Urokinase-type plasminogen activator PLAU Peroxisome proliferator-activated T-22 Prothrombin F2 T-52 PPARA receptor alpha T-23 Coagulation factor VII F7 T-53 Retinoic acid receptor gamma RARG Basic fibroblast growth factor T-24 FGFR1 T-54 Renin REN receptor 1 T-25 Fibrinogen gamma chain FGG T-55 Retinoic acid receptor RXR-beta RXRB Glucose-6-phosphate 1- Proto-oncogene tyrosine-protein kinase T-26 G6PD T-56 SRC dehydrogenase Src T-27 Lactoylglutathione lyase GLO1 T-57 Estrogen sulfotransferase SULT1E1 T-28 Glucose-6-phosphate isomerase GPI T-58 Thyroid hormone receptor beta THRB T-29 Glycogen synthase kinase-3 beta GSK3B T-59 Transthyretin TTR Glutathione reductase, T-30 GSR mitochondrial GH, Lanbuzheng (Gei herba).
Submit a manuscript: https://www.tmrjournals.com/atr ATR | August 2020 | vol. 2 | no. 3 | 115
doi: 10.12032/ATR20200603 ARTICLE
Figure 1 The compound-target network of GH. The yellow circle nodes targets, the green chevron nodes represent active components. GH, Lanbuzheng (Gei herba).
Figure 2 The PPIs network. Node size is in proportion to its degree. PPI, protein-protein interaction.
Table 3 Biological process analysis results of GH treatment of BD targets (top10) GO term Count % P Steroid hormone mediated signaling pathway 8 13.56 9.33 E −10 Transcription initiation from RNA polymerase II promoter 10 16.95 2.35 E −09 Peptidyl-tyrosine phosphorylation 8 13.56 9.25 E −07 Negative regulation of apoptotic process 10 16.95 2.38 E −05 Positive regulation of ERK1 and ERK2 cascade 7 11.86 3.04 E −05 Oxidation-reduction process 11 18.64 3.12 E −05 Positive regulation of gene expression 8 13.56 3.17 E −05 Positive regulation of transcription, DNA-templated 10 16.95 6.25 E −05 Negative regulation of gene expression 6 10.17 1.09 E −04 Positive regulation of MAPK cascade 5 8.47 1.75 E −04 GH, Lanbuzheng (Gei herba); BD, blood deficiency; GO, gene ontology.
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ARTICLE doi: 10.12032/ATR20200603
Figure 3 The top 10 potential effective targets in GH. Different colors represent different targets, the names and proportions of top10 targets were shown in the figure. GH, Lanbuzheng (Gei herba).
Figure 4 The top 10 biological processes of GH. The bar graph represents the P value, and the line graph represents the number of targets. GH, Lanbuzheng (Gei herba).
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doi: 10.12032/ATR20200603 ARTICLE
Figure 5 The top 20 of pathway enrichment. The x-axis shows the rich factor, the y-axis shows the KEGG pathway terms, the circle size shows the gene counts, and the color related with -log10 (P-value). KEGG, Kyoto encyclopedia of genes and gnomes.
Figure 6 The Target-pathway network of GH. The pink rectangle represents the target, the blue triangle represents the pathway. GH, Lanbuzheng (Gei herba). 118 | no. 3 | vol. 2 | August 2020 | ATR Submit a manuscript: https://www.tmrjournals.com/atr
ARTICLE doi: 10.12032/ATR20200603
Figure 7 Effects of GH on BD mice blood peripheral. **, compared with control group, P < 0.01; #, compared with the model group, P < 0.05; ##, compared with the model group, P < 0.01. A–E: the levels of red blood count (RBC), white blood count (WBC), hemoglobin (HGB), total platelets (PLT) and hematocrit (HCT) were analyzed by fully automatic hematology analyzer. Con, control group; mod, model group; SWT, the Classic ancient prescription Siwu Tang treatment group; HGH, high-dose solution of GH treatment group; MGH, middle-dose solution of GH treatment group; LGH, low-dose solution of GH treatment group; BD, blood deficiency; GH, Lanbuzheng (Gei herba).
Figure 8 Influence of the organ index of GH on BD mice. **, compared with control, P < 0.01; #, compared with the model group, P < 0.05; ##, compared with the model group, P < 0.01. A–C: the levels of thymus index (TI), spleen index (SI) and liver index (LI) in mice. Con, control group; mod, model group; SWT, the Classic ancient prescription Siwu Tang treatment group; HGH, high-dose solution of GH treatment group; MGH, middle-dose solution of GH treatment group; LGH, low-dose solution of GH treatment group. BD, blood deficiency; GH, Lanbuzheng (Gei herba).
model by the Classic ancient prescription Sijunzi [35]. And AKR1B1 could interact with Akt protein decoction which consisted of Renshen (Ginseng radix contributing inflammatory factors overproduction, et rhizoma), Baizhu (Atractylodis macrocephalae promoting the neuro inflammation response [36]. rhizoma), Fuling (Poria), Gancao (Glycyrrhizae radix Indicating AKR1B1 may improve BD by inflammatory et rhizoma) at a proportion of 9: 9: 9: 6 [32]. CDK2 reactions. The function of other target proteins in BD could treat liver ischemia and reperfusion injury by are not yet clear and remains to be further studied. inhibiting local immune response and preventing DAVID was used to perform KEGG pathway hepatocyte death [33]. Study showed that immune- enrichment analysis, annotation analysis based on mediated hematopoietic stem cell destruction could KEGG pathway and screened with P value. It was found cause aplastic anemia [34], so CDK2 may joint into that the core targets in the network were mainly hematopoietion by moducating immune. AKR1B1 enriched in two pathways, PI3K-Akt signaling pathway caused inflammation of THP-1 cells by activation of and Rap1 signaling pathway. It has found that PI3K- NF-kB and the expression of pro-inflammatory genes Akt signaling pathway could inhibit the inflammation Submit a manuscript: https://www.tmrjournals.com/atr ATR | August 2020 | vol. 2 | no. 3 | 119
doi: 10.12032/ATR20200603 ARTICLE mechanism of GH treatment of BD.
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