Abstract Background Zhijing Powder (ZJP) is a traditional Chinese medicine containing two kinds of Chinese medicine. Those studies analyze the molecular mechanism of ZJP in treating hypertension through network pharmacology, combined with animal experiments. Methods First, the effective ingredients and potential targets of the drug were obtained through drug databases, while the targets of disease obtained through disease target databases. The potential targets, cellular bioanalysis and signaling pathways were found in some platforms by analyzing collected targets. Further experiments were conducted to verify the effect and mechanism of drugs on cold and high salt in an induced-hypertension rat model. Results There are 17 effective components of centipedes and 10 of scorpions, with 464 drug targets obtained after screening. A total of 1263 hypertension targets were obtained after screening and integration, resulting in a protein-protein interaction network (PPI) with 145 points and 1310 edges. Gene ontology (GO) analysis shows that blood circulation regulation and activation of G protein-coupled receptors are mainly biological processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis shows that neuroactive ligand-receptor interaction, calcium signaling pathways, PI3K-AKT signaling pathways are the most abundant gene-enriched pathway. Animal experiments indicated that ZJP can reduce blood pressure (BP), affect expression of the PI3K-AKT signaling pathway, and improve oxidative stress in the body. Conclusion ZJP ameliorates oxidative stress and reduces BP in hypertensive rats caused by cold stimuli and high salt, revealing its effect on the expression of the PI3K/AKT signaling pathway in the rat aorta. Keywords: Hypertension, Oxidative stress, PI3K-AKT signaling Pathway, Traditional Chinese medicine, Zhijing powder, Network Pharmacology Graphical abstract Image 1 [35]Open in a new tab Highlights * • Traditional Chinese Medicine theory as a guide. * • ZJP has demonstrated clinical efficacy by Master Shimao Li. * • The ZJP component has pharmacological experimental basis. * • Network pharmacological results were demonstrated by animal experiments. * • Results obtained from integrative analysis of theory and prediction validation. 1. Introduction Hypertension is a cardiovascular syndrome characterized by elevated pressure of circulating arteries as its main clinical manifestation, which is the leading risk factor of cardiovascular disease. According to statistics, about 1.38 billion adults suffered from high blood pressure (BP) in 2010 globally, and prevalence rate is still rising [[36]1]. The etiology is complex, so it is likely that hypertension is caused by the interaction of genes, lifestyle, and environment [[37][2], [38][3], [39][4]]. Studies show that cold stimulation and high salt intake can activate the sympathetic and renin angiotensin-aldosterone system (RASS) [[40]5]. Moreover, cold stimulation and a high salt diet allow the body to generate active oxygen, reducing bioavailability of nitric oxide, leading to vascular endothelial dysfunction and exacerbating hypertension [[41]6,[42]7]. The mechanism of hypertension is a challenging process associated with many factors. Although there has been some progress in the study of hypertension, the exact mechanism of treatment is still unclear. Therefore, a novel method must explore the optimal treatment mechanism. Traditional Chinese medicine (TCM) has characteristics of multi-compound, multi-target, and multi-channel aspects [[43]8]. Some Chinese medicine has significant anti-inflammatory, antioxidant, and other effects [[44][9], [45][10], [46][11]]. In addition, a unique feature of TCM is that it has unexpected therapeutic effects on complex diseases: there exists an early understanding of hypertension [[47]12,[48]13]. According to clinical manifestations of hypertension, it belongs to the category of “headache” or “dizziness” in Chinese medicine. TCM finds that pathogenic factors of hypertension mainly include 5 elements, e.g., wind, phlegm, stasis, fire, and deficiency, and a key to the onset of hypertension is disorder of yin and yang, gas and blood loss, and sputum's mutual resistance. Chinese medicine is a new way to study the treatment of hypertension. Zhijing Powder (ZJP), a classic Chinese medicine, is composed of two animal medicine, and often used to regulate body functions such as alleviating blood coagulation, activating meridians, and relieving stasis [[49][14], [50][15], [51][16], [52][17]]. Scorpion (Buthus martensii Karsch Arthropoda; Arachricla; Scorpionida; Chinese pinyin: Quan Xie), have a long history of medicinal use towards relieving wind and suppressing spasm, described in “Chinese Pharmacopoeia.” Modern pharmacological studies show that the active ingredients in scorpions activate the PI3K/AKT signaling pathway [[53]18], promoting the release of nitric oxide (NO) by endothelial cells [[54]19]. NO is an important endothelial factor produced by nitric oxide enzyme (eNOS) [[55]20], which promotes vascular diastolic changes, regulates vascular tension, permeability, and so on [[56]21]. Centipedes (Scolopendra subspinipes mutilans L. Koch; phylum arthropods; Chilopoda; scolopendridae; Chinese pinyin: Wu Gong) function to dispel wind, relieve spasm, promote blood circulation and remove blood stasis, thus detoxifying and decreasing stagnation, recorded in Shen Nong Ben Cao Jing (Shennong's Herbal Classic) [[57]22]. Furthermore, it was demonstrated that the medical composition of centipedes can reduce malondialdehyde (MDA) levels and increase superoxide dismutase (SOD) levels to achieve anti-inflammatory and antioxidant stress effects [[58]23]. Although some knowledge has been gained on the medicinal components of centipedes and scorpions, the core mechanism of ZJP in treating hypertension is not clear. Network pharmacology is a novel medicine design including system biology, network analysis, connectivity, redundancy, along with multi-effects [[59]24]; it provides a basis for the study of molecular mechanisms for drug treatment of diseases by extracting target combinations, medicine, or compound combinations that identify key targets, bioactive compounds, and metabolic pathways [[60]25]. It also provides a potential perspective to assess drug discovery, improvement in clinical outcomes, and comprehension of side effects and toxicity [[61]26]. This is in line with a holistic view of Chinese medicine theory. Therefore, the purpose of this study is to use network pharmacology to identify bioactive ingredients and targets of ZJP. We conducted animal experiments to analyze its potential mechanisms, which will facilitate an in-depth understanding of the treatment for hypertension. 2. Materials and methods 2.1. Identification of active compounds of ZJP The active compounds of ZJP are from the batman-TCM ([62]http://bionet.ncpsb.org.cn/batman-tcm/) [[63]27] database by searching for “WUGONG” and “QUANXIE” as keywords, the medical name by Pinyin, and combining relevant literature on “centipedes” and “scorpions” to supplement a grasp of its chemical composition. Organizing and analyzing the collected chemical components, using Swiss TargetPrediction ([64]http://www.swisstargetprediction.ch/) [[65]28] to draw a composition diagram, and save it in MDL SDfile format. The collected chemicals were entered into the SwissADME [[66]29] for analysis, with GI absorption selected as high, and at least two chemicals with “YES” results in drug-likeness were used as active chemical components of ZJP. 2.2. Chemical component target collection The collected chemical components of the MDL SDfile file are entered in Swiss TargetPrediction for target prediction. Select “Homo sapiens”, download all genetic target information, filter the downloaded gene information, and select the target with a probability greater than 0 as the genetic basis of its chemical component. 2.3. Target screening for hypertension Disease targets are obtained from the Drugbank database ([67]https://go.drugbank.com/) [[68]30], the TTD database ([69]http://db.idrblab.net/ttd/) [[70]31], the DisGeNET database ([71]https://www.disgenet.org/home/) [[72]32], the GeneCard database ([73]https://www.genecards.org/) [[74]33], and the PharmGKB database ([75]https://www.pharmgkb.org/) [[76]34] by searching for “hypertension” as the key word. The above-mentioned databases collect the target, filter the information, and delete duplicate ideas. 2.4. Acquisition of drug-disease intersection targets Using the intrinsic aggregate operation in Venn Graph ([77]http://www.bioinformatics.com.cn/static/others/jvenn/example.html) in Microsystems, genetic target information of ZJP and hypertension were analyzed with common aspects of diseases and drugs: the Venn Graph was mapped. 2.5. Protein-protein interaction (PPI) network construction The drug and disease common targets are stored in the STRING database [[78]35], multiple proteins are selected, with the species limited to ‘homo sapiens,’ the unrelated free proteins in network are removed, and remaining parameters are constructed per default settings to build the PPI network map. To further screen core proteins. The TSV file obtained from the STRING database was processed by Cytoscape3.72 software [[79]36], and core targets were then screened according to the medium degree value. 2.6. GO biological function and KEGG pathway analysis The Metascape database ([80]http://metascape.org) [[81]37] was used for the Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of drug-disease intersection targets. 2.7. Potential targets – pathway networks The common targets of diseases and drugs, with predicted pathways are imported in Cytoscape 3.72 software to create a “Potential Targets - Pathways” network diagram. 2.8. Animal experiments 2.8.1. Experiment design A total of 36 adult male Sprague-Dawley rats (age, 6 weeks; weight, 160–200 g) were obtained from the Laboratory Animal Center of Hebei Medical University. Rats were raised in plastic cages (n = 5/cage or n = 4/cage) and were given normal diets for an acclimation period of a week. All animals were approved by The Ethics Committee for Animal Experiments of Hebei University of Chinese Medicine (DWLL2020084). Animals were randomly divided into four groups (n = 8/group): the control group was fed tap water and ordinary food; the model group was fed tap water and an enriched salt diet (8%); the ZJP-alone group was fed tap water and ordinary food, while the ZJP treatment group was fed tap water and an enrichment salt diet (8%). In addition, the model and ZJP groups were regularly placed in a freezer at −10 °C ± 2 °C for 2 h (from 9:00am - 11:00am). After 6 weeks, the ZJP and ZJP-alone group were treated by gavage for 2 weeks, while the model and control groups had saline by gavage. 2.8.2. Oral gavage preparation ZJP was composed of scorpions (10 g), and centipedes (10 g), and purchased from the National Medical Hall of Hebei University of Chinese Medicine (Shijiazhuang, China). The dosage of ZJP was based on the clinical efficacy dosage of Shimao Li, a master of TCM. Normal saline was purchased in Shijiazhuang NO.4 Pharmaceutical. 2.8.3. Measurement of blood pressure Rats’ BPs were measured by the rodent BP analysis system (Visitech Systems, lnc., Apex, NC, USA) in the afternoon (13:30–19:00) once a week. All rats were placed in a test room with a temperature of 25 °C for 30 min, put on the platform for monitoring and setting parameters—which was predicted five times, measured 10 times, with the data on BP taken from ten average levels. 2.8.4. Preparation of tissue and plasma After the last administration, all rats fasted for 24 h: they were anaesthetized with sodium barbiturate (2%) in the abdominal cavity with blood through the femoral artery on both sides. The blood sample was centrifuged at 3000 rpm for 10 min after keeping it at room temperature for 2 h, extracted as the supernate, and poured into a 200 μL EP tube stored in a refrigerator at −80 °C. The thoracic aorta was dissected after opening the chest, with excess connective tissue in fixation fluid. 2.8.5. Histopathological analysis The removed chest aorta was fixed, the conventional paraffin encapsulated, sliced with xylene dewax, ethanol wash, HE and Masson staining, routine dehydration, neutral resin sealing, using an optical microscope observation and photography. The National Institutes of Health ImageJ program was used for quantification. The arterial lumen and total vessel area were measured by the ImageJ program. The vessel area was calculated by subtracting the area of the arterial lumen and the total vessel area. To quantify the area of vascular fibrosis, Masson-stained images were converted to a gray scale in the ImageJ program, the stained area segmented with thresholding. The measured threshold area served as the fibrotic area. 2.8.6. Serological analysis The activity of creatine kinase (CK, Catalog: A032-1-1), nitric oxide (NO, Catalog: A012-1-2), superoxide dismutase (SOD, Catalog: A001-3-1), hydrogen peroxide enzyme (MDA, Catalog: A003-1-1), glutathione peroxidase (GSH-PX, Catalog: A006-1-1), catalase (CAT, Catalog: A007-1-1) in the serum were detected by colorimetric assay with commercial kits, purchased from Nanjing Jiancheng Bioengineering Institute, China. The specific operation was in strict accordance with instructions. 2.8.7. Western-blot analysis Take each rat's thoracic aortic tissue, PBS, and efficient liquid ice cracking protein sparing modified fast (PSMF) (including 1%), 4 °C after centrifuging to clear total protein in the BCA kit(Catalog: G2026, Servicebio technology Co., Ltd., Wuhan, China) to determine protein concentration, as per protein concentration calculation sample, with 95 °C water bath pot boil for 10 min-electrophoresis, transfer, skimmed milk powder for a closed parallel fight after the incubation, and the specific protein was anti-PI3K (Catalog: bs-2067R, Beijing Bioss Biotechnology Co., Ltd, Beijing, China, 1: 600 dilution), anti-AKT (Catalog: bs-0115R, Beijing Bioss Biotechnology Co., Ltd, Beijing, China, 1: 600 dilution), and anti-GAPDH (Catalog: bs-2188R, Bioss Biotechnology Co., Ltd, Beijing, China, 1: 2000 dilution). The polyvinylidene fluoride (PVDF) membrane was placed on the smooth surface of the glue-making glass backplane, and luminescence droplets were evenly added to the PVDF membrane with a 200 μL pipetting gun. Exposure was opened with ImageLab software, and target protein bands were detected. The gray value of each strip was measured with ImageJ software and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference. The ratio of PI3K/AKT protein to the gray value of the internal reference with GAPDH protein band was the relative expression level, compared to the normal control group for statistics. 2.8.8. Statistical analysis IBM SPSS24.0 software (Chicago, IL, USA) was used for data analysis, and all measurement data were expressed as mean ± SD (x ± s). One-way analysis of variance was used for comparison between multiple groups, while the LSD test was used for pairwise comparison of variance between groups, the Dunnett-t test was used for uneven variances, and P < 0.05 was considered statistically significant. 3. Results 3.1. Screening of active components and targets in ZJP Through batman-TCM and the literature, 23 effective chemical components of centipedes and 16 effective components of scorpions were found. Targeted screening based on GI absorption and Drug-likeness, and non-targeted screening were conducted, with 17 effective components of centipedes and 10 effective components of scorpions. There were 514 targets for centipedes and 229 targets for scorpions, anticipated by Swiss TargetPrediction, and a total of 743 targets were obtained. After deleting repeated targets, a total of 464 drug targets were obtained. A network of component-targets was constructed by Cytoscape3.72 ([82]Fig. 1) . Table 1. List of active components. Number Active components Centipedes WG1 (3S) – 1,2,3,4-tetrahydro-carbo-line-3-carboxylic acid WG2 3,8-Dihydroxyquinoline (Jineol) WG3 7,8-Dimethylpyrrolidine WG4 8-Hydroxy-1h-2-benzopyran WG5 N-Acetyl-2-phenylethylamine WG6 Alanine WG7 Hypoxanthine WG8 Uracil WG9 Proline WG10 Glycerin1-monostearate WG11 Centipede alkali b WG12 Scolosprine A WG13 Vanillic acid WG14 Valine WG15 Acetylcholine WG16 Indole-3-acetamide Scorpions QX1 (−)4-(2′-Iso-octanoicacid)-6-hydroxy-1-methyl cyclohexene QX2 (−)22E,24-3-Cholestone-4,22(23)-diene-25-alcohol QX3 2β-,22-Dihydroxy,3-acetoxyl,20-methoxy-cardenolidol QX4 3β-Acetoxyl,2,14,22-trihydroxy,19-hydroxymethyl,9α,5β,14β-card20(22) enolide QX5 Alanine QX6 Glycerol QX7 Trigonelline QX8 Leucine QX9 Uracil QX10 Proline [83]Open in a new tab Fig. 1. [84]Fig. 1 [85]Open in a new tab Relationships among the active components and targets of centipedes and scorpions. Pink represents centipede active components, purple represents the scorpion active components, orange represents components shared by centipedes and scorpions, green represents targets of centipedes and scorpions. There are 17 active components of centipede and 10 active components of scorpion, and 464 drug targets. The code numbers of drugs are presented in [86]Table 1. (For interpretation of the references to color in this figure legend, the reader is referred