Experimental design and setting.
To compare the effects of Buxue Yimu Pills (BYP), ferrous sulfate, and a combination of both treatments on GA, a prospective, randomized controlled trial was conducted between 2017 and 2019 at Gynecology departments of three public hospitals, including Peking Union Medical College Hospital (PUMCH), West China Hospital of Sichuan University, and Hangzhou Maternity and Child Health Care Hospital. This study was approved by the Ethics Committee of PUMCH (No. ZS-1254) at January 2017, and registered at the United States National Institutes of Health (registration number NCT03232554, www.clinicaltrials.gov).
Adult females aged 18–50 years with hemoglobin of 70-110 g/L associated with gynecological disorders were enrolled following the informed consent. Exclusion criteria were: (1) patients with uncontrolled gastrointestinal inflammation or bleeding or urological bleeding; (2) patients presented with other diseases leading to iron deficiency; (3) pregnant women; (4) patients complicated with mental abnormalities or other severe diseases; (5) patients with a history of malignancy, chemotherapy or radiotherapy; (6) patients treated with iron supplements, blood transfusion or participating in any other clinical trial within the past month. Eligible participants were randomized into three groups: BYP group received Buxue Yimu Pills (24 g/day); oral iron group received ferrous sulfate tablets (0.9 g/day), and BYP & oral iron group received both drugs above simultaneously. All drugs were provided by ZhuZhou QianJin Pharmaceutical Co., Ltd. The total treatment periods were four weeks.
Assessments were scheduled pre-and post-treatment. A basic physical examination including vital signs, weight, height, body mass index (BMI), blood pressure was performed. Venous blood samples were collected to detect complete blood count, including hemoglobin (Hb), mean cellular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cells (RBC), hematocrit (HCT), reticulocyte (RET) count. Iron indexes of serum iron (SI), serum ferritin (SF), and total iron-binding capacity (TIBC) were analyzed. Moreover, the safety assessment, including liver function, renal function, and a blood coagulation index, were also evaluated (data not shown).
Statistical analysis was carried out using SPSS version 20.0 (IBM). Data from normally distributed parameters are presented as means plus or minus standard deviation (SD); paired t-tests were used to compare the intragroup difference pre- and post-treatment, and one-way analysis of variance (ANOVA) was applied for intergroup comparisons. Variables not normally distributed are expressed as median plus 25-75 interquartile range (IQR); intragroup and intergroup comparisons were evaluated with Wilcoxon matched-pairs signed-rank tests and Kruskal-Wallis tests. A p-value of less than 0.05 (p < 0.05) was considered significant in all statistical analyses.
Network pharmacology analysis
Bioactive compounds and compound targets of Buxue Yimu Pills
Composite compounds were searched manually with the major ingredients of BYP “Angelicae Sinensis Radix”, “Hedysarum Multijugum Maxim”, “Colla Corii Asini”, “Leonuri Herba” and “Citrus Reticulata”, in Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TcmSP, http://lsp.nwu.edu.cn/tcmsp.php) 3, and Database of Traditional Chinese Medicine and chemical composition from Shanghai Institute of Organic Chemistry (http://www.organchem.csdb.cn/scdb/main/tcm_introduce.asp). Next, a combination of oral bioavailability ≥ 30% along with drug-likeness ≥0.18 was applied to explore the bioactive compounds. Potential treatment targets of those bioactive compounds were subsequently obtained from TcmSP; and converted to target genes with official symbols using Uniprot Knowledgebase (http://www.uniprot.org/).
Target genes of GA
GA-associated targets were retrieved from the Online Mendelian Inheritance in Man (OMIM) database (https://www.omim.org/) and GeneCard Database (https://www.genecards.org/), using “gynecological anemia ”as the keyword. Data were extracted and summarized without duplication.
Potential therapeutic targets are the intersections between the compound targets of BYP and GA-associated targets, which were generated with the VennDiagram R package. After importing the main ingredients, bioactive compounds, and potential therapeutic targets, the compound-target-disease network was built and visualized by Cytoscape software (version 3.6.1, http://cytoscape.org/).
Based on the obtained potential therapeutic targets, enrichment analysis was performed using the online tool Metascape (https://metascape.org) 15, including Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.
A protein-protein interaction (PPI) network was constructed via STRING (Version 11.0, https://string-db.org/) with a minimum required interaction score of 0.400. We subsequently used the Molecular Complex Detection tool (MCODE) plugin to identify significant clusters in this PPI network.