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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 3 | Page : 355-368
Mechanism of wuweijiangyasan in the treatment of spontaneous hypertension based on network pharmacology
Ai-Ping Chen1, Zi-Juan Zhang1, Jing-Zhong Li2, Ling Zuo1, Ya-Xing Cheng1, Dong Deng1, Xue-Li Li1, Xiao-Yun Ma1, Da Man1, Ming-Huang Zheng3, Jian Chen1, Bo Wen1, Juan Wang1, Jian-Guo Zhou4, Hui-Hui Zhao1
1 School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
2 Center for Information and Education Technology, Beijing University of Chinese Medicine, Beijing, 100029, China
3 School of Management, Beijing University of Chinese Medicine, Beijing 100029, China
4 Department of Management, Beijing Youjian Medical Research Institute, Beijing 100025, China
Correspondence Address:
Prof. Hui-Hui Zhao
School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029
China
Dr. Jian-Guo Zhou
Beijing Youjian Medical Research Institute, Beijing 100025
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.351793
Background: Hypertension affects over 1 billion people globally and is the top risk factor of cardiovascular morbidity and mortality. Wuweijiangyasan (WWJYS), as an empirical prescription, has stable depressurization effects. This study investigated the chemical composition and pharmacodynamic effects of WWJYS in regulating the blood pressure (BP), emotion, and blood lipid of spontaneous hypertensive rats, and further explored the depressurization mechanism of WWJYS. Materials and Methods: This study used network pharmacology to identify the origins and predict targets of WWJYS, and artificial intelligence-based molecular docking is used to further predict targets and mechanisms. The chemical constituents of WWJYS were analyzed and identified by ultra high-performance liquid chromatography–mass spectrometry (MS)/MS. Results: In the WWJYS group, the systolic BP level significantly was decreased, and the HR was stable. The irritability became stable after the 5-week treatment compared with the model group (P < 0.05). Rats' rotation tolerance time increased after 2-weeks stabilization. Compared with the model group, angiotensin-converting enzyme 2 protein and mRNA of the WWJYS group increased significantly (P < 0.05). Network pharmacology collected 64 compounds and identified 22 potential targets of WWJYS for antihypertensive activity. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that WWJYS might regulate smooth muscle cells, affect inflammatory response and improve endothelial function through multiple pathways. The molecular docking study further supported that the target proteins have good combinations with the main active components of WWJYS. Conclusions: The data indicated that WWJYS had significant depressurization, analgesic, and sedative, as well as lipid-lowering effects, and the depressurization mechanism of WWJYS may function in multiple signal pathways, especially in improving blood vessel function and intervening inflammation.
Keywords: Wuweijiangyasan, hypertension, network pharmacology, mechanism
Hypertension, a common and frequently occurring disease with a gradual increasing morbidity,[1] can cause damage to the heart, brain, kidneys, and other target organs.[2],[3] Hypertension affects over 1 billion people globally and is the top risk factor of cardiovascular morbidity and mortality.[4] Although different types of antihypertensive drugs are available, many patients may develop nonadherence to drugs due to the intolerance caused by side effects.[5],[6],[7] For patients who cannot tolerate antihypertensive drugs or whose blood pressure (BP) cannot be controlled after taking antihypertensives, a new type of interventional procedure with excellent tolerability, safety, and efficacy is needed to manage hypertension.[8],[9],[10],[11]
It is well-known that traditional Chinese herbal medicine has obvious advantages in syndrome differentiation with fewer side effects and lower dependence in the treatment of hypertension,[12] which deserves in-depth exploration.[1] Wuweijiangyasan (WWJYS), as an empirical prescription, has stable depressurization effects. It was first formulated by Chen Wenbo, a distinguished national-level practitioner of traditional Chinese medicine (TCM). According to Professor Chen, by taking the decoction of 3 g of each Cortex Lycii, Radix Puerariae Lobatae, Radix et Rhizoma Salviae Miltiorrhizae, Crataegus pinnatifida Bunge, and root of Arctii Fructus, respectively, three times a day, obvious effects in lowering blood lipid, blood sugar level, and BP can be achieved.
Based on the efficacy of WWJYS reported previously, the present study was conducted. The analysis of the functions of the compound composition is presented as below: Cortex Lycii plays the role of monarch medicine, while Radix Puerariae Lobatae and Radix et Rhizoma Salviae Miltiorrhizae are the minister medicine. Fructus Crataegi is adjuvant medicine, then the guide medicine is the root of Arctii Fructus. Cortex Lycii is an antipyretic which dispels steaming heat and relieves lung heat; Radix Puerariae Lobatae is also an antipyretic that releases the muscle, invigorates yang, and promotes eruption. Radix et Rhizoma Salviae Miltiorrhizae is employed to activate blood circulation to remove stasis, cool blood, eliminate carbuncle, nourish blood and tranquilize the nerves. Fructus Crataegi is used for accretion to activate blood and disperse stasis. The effects of Arctii Fructus root are heat clearing and detoxicating. Combined with the effects of these five herbs, WWJYS can regulate both qi and blood, clear liver and reduce fire, clear heat and detoxify the body, activate blood circulation, and remove blood stasis. Qi and blood are targeted in the treatment of hypertension which is considered to be caused by yin deficiency of liver and kidney according to the theory of TCM.
This study aimed to evaluate the efficacy of WWJYS and explore the depressurization mechanism of this empirical prescription.
Materials and MethodsInstruments
The Thermo LTQ Orbitrap ultra high-performance liquid chromatography–mass spectrometry (UHPLC-MS)/MS was obtained from the America Thermo Fisher Scientific. The BT 25S Sartorius electronic balance was purchased from the Sartorius Scientific Instruments (Beijing, China). The RE-52AA rotary evaporator was from the Shanghai Yarong biochemical instrument factory (Shanghai, China). The Softron BP-2010A noninvasive tail artery piezometer was from the Softron Beijing Biotechnology (Beijing, China).
Experimental drugs
Danshensu (MUST-16030205), dihydrotanshinone I (MUST-16032705) and tanshinone A (MUST-16032502), tanshinone I (MUST-16030210), chlorogenic acid (MUST-16031610), cryptochlorogenic acid (MUST-16022403), neochlorogenic acid (MUST-16021806), 1, 3-two caffeoylquinic acid (MUST16022610), 1, 5-two caffeoylquinic acid (MUST-16040105), (+)-catechin (MUST-16030812), procyanidin B2 (MUST-16022608), biochanin A (MUST-16030604), and genistin (MUST-13080501) were purchased from Chengdu Mann Stewart Biological Technology (Chengdu, China). Cryptotanshinone (151022) and epicatechin (150312) were purchased from Chengdu Pfeiffer Biotechnology (Chengdu, China). Genistein (G106672) and daidzein (G109561) were purchased from Shanghai Pure Biochemical Polytron Technologies (Shanghai, China). Puerarin (20130310) and daidzein (20130728) were purchased from Shanghai Source Leaves Biological Technology (Shanghai, China). The quality control scores of all materials were higher than 98%.
Niuhuang Jiangya pill (1.6 g each, Z11020199) was purchased from Beijing Tongrentang Technology Development (Beijing, China). Valsartan capsule (80 mg, H20040217) was from Beijing Novartis Pharmaceutical (Beijing, China). BCA protein assay kit was from Beijing Applygen Gene Technology (Beijing, China) and angiotensin-converting enzyme (ACE) 1 (ab77990), ACE2 (ab108252), and β-actin (ab8227) were all purchased from Abcam (Cambridge, UK).
Identification of origins of Wuweijiangyasan
Radix Puerariae Lobatae (Gegen), Radix et Rhizoma Salviae Miltiorrhizae (Danshen), the root of Arctii Fructus(Niubanggen), Cortex Lycii (Digupi), Fructus Crataegi (Shanzha) were purchased from Beijing Tongrentang Pharmacy (Beijing, China). The botanical identity of each herb in WWJYS was well-defined.
Preparation of sample solution
According to the compatibility ratio of clinical medication, the water decoction method similar to clinical medication mode was used to extract the medicinal materials. Pueraria lobata, Salvia miltiorrhiza, root of Arctii Fructus, Cortex Lycii, and Fructus Crataegi, (each weighed 25 g) were mixed, decocted with 12 times of distilled water for 1 h, and then filtered under heat. The obtained decoction was decocted with 10 times of distilled water for 1 h, filtered under heat, combined with 2 times of filtrate, concentrated to 1 g/mL as sample reserve solution, and stored at 4°C. Samples of each Pueraria lobata, Salvia miltiorrhiza, root of Arctii Fructus, Cortex Lycii, and Fructus Crataegi, were prepared by the same method.
The taxonomically validated names
By searching through the website of the Kew Science and referring to Pharmacopoeia of China (2015), the validated names of the five herbal medicines were obtained. The names were listed as follows: Puerariae Lobatae Radix, Pueraria montana var. lobata (Wild) Maesen and S. M. Almeida ex Sanjappa and Predeep (Gegen); Salviae Miltiorrhizae Radix et Rhizoma, Salvia miltiorrhiza Bunge (Danshen); root of Arctii Fructus, root of Arctium lappa L.(Niubanggen); Lycii Cortex, Lycium chinense Mill (Digupi); Crataegi Fructus, Crataegus pinnatifida Bunge (Shanzha).
Animals
This study selected 14-week-old spontaneous hypertensive rat (SHR), 14-week-old WKY rats, and ICR mice (weight 18-22 g) from Beijing Vital River Laboratory Animal Technology (License No. SCXK [Beijing] 2012-0001; Beijing, China). Each experimental group included 10 animals. This study was approved by the Animal Care and Use Committee of the Development Center for Biotechnology. Animals were maintained in the standard animal room of Beijing University of Chinese Medicine Research Center, meeting the institutional guiding principles for animal research. The researcher's qualification for laboratory animal performance was 1116040500219.
Wuweijiangyasan preparation
The abovementioned five Chinese herbs (75 g each) were mixed, 4500 mL distilled water was added to decoct for 1 h and filtered. Then, 3750 mL distilled water was added into the residue and decocted for 1 h. After removing the dregs, the filtered solution was condensed into a concentration of 2 g/mL and stored at −20°C. The decoction was diluted before use.
Depressurization and behavioral experiments
One-week adaptation for vehicle administration (tap water) and BP measurement was allowed before initiation of the experimental protocol. The experiments were carried out over the course of 10 weeks. The SHRs were divided into four groups: Model group, WWJYS group (3.20 g/kg, twice the clinical dose), Niuhuang Jiangya pills (NHJY) group (0.144 g/kg), and valsartan capsule (Val) group (0.016 g/kg). WKY rats were used as the control.
Systolic BP (SBP) and heart rate (HR) measurements were conducted twice a week in awakening rats by Softron BP-2010A rat noninvasive tail artery pressure gauges. Measurements were repeated thrice.
The irritable and peevish characteristics of hypertension patients were similar to those of rats. According to a bibliographic search,[12],[13] the irritability degree could be divided into four levels: Level III, with a score of 3 points (screaming, jumping or even biting and fighting when tail lifted); level II, with a score of 2 points (screaming, even biting when caught by the neck); level I, with a score of 1 point (screaming and jumping when caught by the neck); and level 0, with a score of 0 point (none of the above occurred).
According to recent research,[14] lab rats were placed on the JD-01 gsz-type balanced rotating instrument platform at a velocity of 60 r/min with the time from the beginning of the rotation recorded. If the rats did not fall down in 2 min, the test was manually terminated, and the rotation time of the rat was recorded as 120 s. Each rat was continuously measured thrice. The experiment was performed weekly.
After the rats were sacrificed, the heart, kidneys, and liver were removed quickly. The middle line along the lateral side of the rat's leg was carefully cut with surgical scissors until the rat's tibia and fibula were exposed. The fascia and connective tissue were separated by forceps along the proximal humerus and then peeled off at the knee with forceps. The humerus was dissected from the knee joint cavity. Finally, a complete “S” type tibia was obtained by surgical shearing at the distal end of the humerus. The length of the rat tibia was measured with a Vernier caliper.
Analgesic sedative experiment
ICR mice were randomly divided into five groups: control group, positive drug group, WWJYS-2.75 g/kg group (equal to clinical dose), WWJYS-5.50 g/kg group (2 times more than clinical dose), and WWJYS-11.00 g/kg group (4 times more than clinical dose).
Each group was given drugs by intragastric administration once a day for 5 days. Inflammatory pain resulted in abdominal concave, trunk and hind limb stretch, and hip high behavior response. The number of writhing in 15 min after acetic acid injection was observed, and the inhibition rate was calculated as the comparison index. Inhibition rate (%) = (Number of writhing in the negative control group-number of writhing in the administration group)/number of writhing ×100% of the negative control group.
The mice were placed in the upper container of the tachycardia before, 0.5 h and 1.5 h, respectively, after the administration. Once the mice remained calm, the infrared light started shining on the mice's feet, and recorded the time of movement.
The mice were suspended from the tail at 0.5 h, 1 h, and 2 h, respectively, after the administration. Then, the activity time within 5 min was recorded by the image tracking system [Figure 1].
Western blot analysis
The heart and kidneys of the rats were pulverized and solubilized in a lysis buffer. Protein quantification was performed using the BCA protein assay kit to ensure equal loading (10 μg protein) in each lane. Proteins were separated on SDS-PAGE gels and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk powder and incubated overnight at 4°C with the first antibodies directed against ACE1 and ACE2. After washing blots to remove excessive primary antibody binding, blots were incubated for 1 h with secondary antibodies. Antibody binding was detected through enhanced chemiluminescence (Millipore, USA), and the membranes were scanned. Immunoblot band intensity was analyzed with Image Lab5.2.1 software. Measured intensities were corrected using the internal control (β-actin).
Real-time quantitative polymerase chain reaction
Real-time polymerase chain reaction (RT-PCR) was used to detect the differences of ACE1 and ACE2 mRNA expression in the heart and kidneys of SHR rats among the groups. The primer sequences used for RT-PCR were as follows: ACE1 (forward-GTGCCTAGATCCCAAGGTGAC, reverse-TCGTGGAACTGGAACTGGATG), ACE2 (forward-CTTACGAGCCTCCTGTCACC, reverse-ATGCCAACCACTACCGTTCC), and GAPDH (forward-TGCACCACCAACTGCTTAG, reverse-GGATGCAGGGATGATGTTC).
Chromatographic and mass spectrum conditions
First, methanol was diluted into a 50 mg/mL solution. A proper amount of the solution was taken in a centrifuge tube and centrifuged at 12000 r/min for LC/MS analysis. Thermo BDS Hypersil C18 column (2.1 mm × 150 mm, 2.4 m) was used. The mobile phase was A phase 0.1% formic acid water and B phase acetonitrile. The gradient elution conditions were 0 min 3% B, 15 min 25% B, 22 min 40% B, 30 min 90% B, and 32 min 90% B. The flow rate was 0.3 mL/min, the column temperature was 35 °C, and the sample volume was 3 μL.
The electron spray ionization (ESI) and positive and negative ions were detected. Spray pressure was 344.74 kPa and dry gas (N2) flow rate was 5 L/min. The temperature of the drying air and gasification chamber were 330°C and 350°C respectively. The capillary voltage was 4 kV, the corona discharge current was 4.0 μA (fragmentor), broken voltage was 100 V, and scanning range was 100 m/z–1000 m/z.
Network pharmacology analysis
Database preparation
The candidate compounds of WWJYS were collected from HERB database (http://herb.ac.cn/), which integrates multiple popular TCM databases. All pharmacokinetic properties were identified from TCMSP database (https://tcmsp-e. com/). Oral bioavailability (OB), drug likeness (DL), and Caco-2, as three crucial indicators of pharmacokinetic properties, were selected to identify WWJYS active ingredients and the threshold was OB ≥30%, DL D0.18, and Caco-2 ≥0.4. All the corresponding targets of active components were acquired from TCMSP database. The official gene symbols and UniProt ID of corresponding proteins were obtained from UniProt database (https://www.uniprot.org). Components without known targets or standard names were deleted.
”Spontaneous hypertension”, “essential hypertension” and “primary hypertension” were the keywords to gather spontaneous hypertension-related genes from the following five databases: Genecards (http://www.genecards.org), OMIM (http://omim.org/), DisGenet (https://www.disgenet.org/), TTD (http://db.idrblab.net/ttd/), and PharmGKB (https://www.pharmgkb.org/). Overlapping targets obtained from SH-related genes and WWJYS target genes would be considered as key targets for WWJYS against spontaneous hypertension.
OMIM (http://omim. org/)
DisGenet (https://www.disgenet.org/), TTD (http://db.idrblab.net/ttd/) and PharmGKB (https://www.pharmgkb.org/). Overlapping targets obtained from SH-related genes and WWJYS target genes would be considered as key targets for WWJYS against spontaneous hypertension.,
Network analysis
Herbs, corresponding active components, and key target genes were imported into Cytoscape 3.7.2 to build the network model of “herb-compound-key targets”. CytoNCA plug-in was applied to perform network topology analysis and screen key nodes in light of the degree centrality (DC) and between centrality. Construction of protein–protein interaction (PPI) network of key target proteins was completed in the STRING database (http://string-db.org/, ver. 11.0). The PPI network was then inputted into Cytoscape and the key genes was selected. R version 4.1.1 and R package clusterProfiler was used for Gene Ontology (GO) knowledgebase (http://geneontology. org/), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (https://www.genome.jp/kegg/) and data visualization.
Molecular docking
The binding efficiency of key target proteins and major active components in WWJYS were evaluated by molecular docking. The SDF structures of compounds were collected in PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The protein sequences were collected from UniProt database, and their crystal structures were predicted by AlphaFold2, the structures with the highest per-residue confidence score (pLDDT) were saved as PDB files for later use for docking. The receptors and ligands were prepared with PyMOL and AutoDock and then docked through Vina.
Statistical analysis
All analyses were performed using the IBM SPSS Statistics 20.0 software (IBM, Armonk, NY, USA). Data were expressed as mean ± standard deviation. As for multiple sets of measurement data, if normal distribution, one-way analysis of variance was used; if not, data were processed using the Kruskal–Wallis rank-sum test. Paired-samples t-test was used to compare the differences in each group before and after the experiment. The threshold of P < 0.05 was considered statistically significant.
ResultsWuweijiangyasan depressurization
As shown in [Figure 2]a, SBP of the model group increased continuously compared with that of the control group, but there was no significant difference (P > 0.05). In the WWJYS group, SBP level significantly decreased compared with the model group (P < 0.05). [Figure 2]b shows that the SBP was maintained a stable and low level after 4 weeks of treatment. As shown in [Figure 2]c and [Figure 2]d, the HR of the WWJYS group remained stable. The HR and SBP decreased and then remained stable after 5 weeks of treatment, indicating the trend of the two indexes were consistent with each other.
Figure 2: (a) Changes of mean systolic blood pressure at different time periods in rats. As compared with the model group, *denotes a significant difference (P < 0.05). As compared with the control group, #denotes a significant difference (P < 0.05). (b) Systolic blood pressure trend at different time periods of the five groups. (c and d) Trend of heart rate at different time points among the five groups. Data are presented as mean ± standard deviation, n = 10 per groupImproved emotions
As shown in [Figure 3]a, the irritability of each group was evaluated. The WWJYS group tended to be stable after 5 weeks of treatment, and then remained at the lowest level, compared with the other groups. However, the rats in the model group were rather irritable, and the fluctuation range was significant. [Figure 3]b reveals that the rats' rotation tolerance time in the WWJYS group increased after 2 weeks of treatment and then remained stable henceforth.
Figure 3: (a) Degrees of irritability in rats at different time periods. (b) Comparison of the rotation tolerance time in rats at different time periods. Data are presented as mean ± standard deviation, n = 10 rats per groupTest of writhing frequency in 15 min and inhibitory rate of acetic acid showed that aspirin and middle-dose WWJYS could significantly reduce writhing frequency in mice [P < 0.05, [Table 1]].
As for analgesic effect in thermo stimulation, the pain threshold of mice in the control group before and after treatment showed no statistical difference (P > 0.05). Low-dose WWJYS treatment for 1.5 h could significantly increase the pain threshold of mice, and high-dose WWJYS treatment for 0.5 h and 1.5 h received similar results [P < 0.05, [Table 2]].
The 5-min tail-hanging test showed no statistical difference in sedative effect before and 0.5 h, 1 h, and 2 h after the diazepam administration in the control group (P > 0.05). The time of tail-hanging activity had a significant difference between before and 2 h after the administration in the high-dose WWJYS group, which showed WWJYS can reduce the 5-minute tail-hanging activity time, while extending the immobility time [P < 0.05, [Table 3]].
Lipid-lowering effect
[Figure 4] reveals the weight of WKY rats had increased the most. The weight of SHR rats in the model group increased faster and was heavier than that in the WWJYS group. The levels of cholesterol, triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) decreased in the WWJYS group, while the cholesterol and TG levels in the NHJY group, the Val group, and the control group all increased [P < 0.05, [Table 4]].
Figure 4: Trend of weight change among the Wuweijiangyasan, model and control groups. Data are presented as mean ± standard deviation, n = 10 in each group
Table 4: Effects on serum cholesterol, triglyceride, low-density lipoprotein cholesterol in spontaneous hypertensive rats (mmol/L)Improvement of target organ damage
As shown in [Figure 5], the heart weight, heart weight-to-tibial length ratio, liver weight, and liver weight-to-tibial ratio in the model group were significantly higher than those in the WKY group (P < 0.05). The liver weight and liver weight-to-tibial length ratio of the WWJYS group were significantly lower than those of the model group (P < 0.05).
Figure 5: Comparison of visceral index in each group. Data are presented as mean ± standard deviation, n = 10 in each group. When compared with the model group, *denotes a significant difference (P < 0.05); #denotes a significant difference (P < 0.05) when compared with the control groupFigure 6a showed that the myocardium of rats in the WWJYS group became slightly swollen and small. The blood stasis of glomerulus in the WWJYS group was improved, and the lesion of curved duct was alleviated [Figure 6]b.
Figure 6: Comparison of cardiac (a) and renal (b) pathological diagnosis in each groupEffects of Wuweijiangyasan on angiotensin-converting enzyme 1 and angiotensin-converting enzyme 2 expression in cardiac and kidney
Compared with the model group, cardiac angiotensin-converting enzyme 2 (ACE2) protein of the WWJYS group increased significantly, which was consistent with the expression of mRNA [P < 0.05; [Figure 7]a, [Figure 7]b, [Figure 7]c]. The expression level of ACE1 mRNA in the WWJYS group was significantly lower than that of the model group, while the ACE2 mRNA was upregulated significantly (P < 0.05).
Figure 7: Effects of Wuweijiangyasan on angiotensin-converting enzyme 1 and angiotensin-converting enzyme 2 expression in heart and kidney. (a and b) Western blot analysis of cardiac angiotensin-converting enzyme 1 and angiotensin-converting enzyme 2 expression. (c) Real-time polymerase chain reaction analysis of cardiac angiotensin-converting enzyme 1 and angiotensin-converting enzyme 2 expression. (d and e) Western blot analysis of angiotensin-converting enzyme 1 and angiotensin-converting enzyme 2 expression in kidney. (f) Real-time polymerase chain reaction analysis of angiotensin-converting enzyme 1 and angiotensin-converting enzyme 2 expression in kidney. Data are presented as mean ± standard deviation, n = 3 in each group. When compared with the model group, *denotes a significant difference (P < 0.05); when compared with the control, #denotes a significant difference (P < 0.05)As shown in [Figure 7]d, [Figure 7]e, [Figure 7]f, compared with the model group, the kidney ACE1 protein expression level of the Val group and WWJYS group was obviously higher (P < 0.05). The expression level of ACE2 protein in the Val group and WWJYS group significantly decreased compared with that in the model group (P < 0.05). There was no significant difference in the expression of ACE1 mRNA in the other four groups. Compared with the model group, the expression level of ACE2 mRNA in the WWJYS group and Val group was significantly increased (P < 0.05).
Bioactive compounds and key targets identification of Wuweijiangyasan
A total of 64 bioactive compounds were identified in the HERB database, and 226 targets were identified in the TCMSP database. By comparing the gene names related to spontaneously hypertension in the TTD, PharmGKB, Uniprot, GeneCards, and DisGeNET databases [Figure 8]a, 193 overlapping targets were reserved as candidate targets [Figure 8]b. The “Drugs-bioactive components-potential targets” network containing 247 nodes and 911 edges was summarized in [Figure 8]c. The compounds with more nodes might be the main bioactive ingredient of WWJYS. The top five components connected with targets were quercetin (122), apigenin (60), tanshinone IIA (33), formononetin (29), and dihydrotanshinlactone (27). About 193 common targets were imported into the STRING database, as shown in [Figure 8]d. The targets with DC values greater than one or two times the median were extracted by Cytoscape, and 22 key targets in the number of nodes were screened out [Figure 8]e. The top five target genes with the highest node number are: serine/threonine kinase 1(AKT1), tumor protein P53 (TP53), interleukin 6 (IL6), tumor necrosis factor (TNF), and Jun proto-oncogene (JUN).
Figure 8: Network Pharmacology results of Wuweijiangyasan. (a) Venn plot of targets screening of spontaneous hypertension in five databases; (b) Overlapping targets of Wuweijiangyasan and spontaneous hypertension; (c) “Drugs-bioactive components-potential targets” network diagram of Wuweijiangyasan. The outer circle represents the bioactive components, whose colors indicate which herb they belong to. The inner circle represents potential targets; the color depth of the node represents the value of the degree. (d) protein–protein interaction network; (e) Key targets topology. The color of targets changes from blue to yellow according to the higher DC value. The left shows all potential targets, the middle shows the targets whose DC value is >1 time the median, and the right shows the targets whose DC value is >2 times the median, which are the key targets of this studyPathway enrichment of key targets
A total of 22 key targets were involved in the GO and KEGG enrichment analysis, including AKT1, TP53, IL6, TNF, JUN, PPARG, MMP9, CCL2, CTNNB1, ESR1, CXCL8, ERBB2, HSP90AA, MYC, PTGS2, CCND1, STAT3, FOS, IL1B, CASP3, EGFR, and HIF1A. To further understand the intersection genes, GO enrichment analysis was conducted. GO analysis results showed that 1534 biological processes (BP), 21 cell components (CC), and 97 molecular functions (MF) were obtained through clusterProfiler [Figure 9]a. Target proteins of the BP category were mostly associated with epithelial cell proliferation, regulation and proliferation of smooth muscle cell, regulation of NO synthase activity, etc., Target proteins in the MF category were predominantly associated with DNA-binding transcription factor binding, protein phosphatase binding, cytokine receptor activity, and binding, etc., and CC target proteins were categorized as belonging to RNA polymerase II transcription regulator complex, transcription regulator complex, euchromatin, etc. KEGG pathway enrichment was conducted to cluster the major effects that related to WWJYS. The top 30 KEGG pathways were screened out (FDR adjusted P value (q) <0.05) as shown in [Figure 9]b. In the bubble chart, the X-axis represented the number of target genes (Gene Ratio), and the Y-axis represented the KEGG pathway or GO term with significant enrichment of target genes. The size of the dots intuitively reflected the size of the gene ratio, and the color depth of the dots reflected different q value ranges. KEGG analysis indicated that these target proteins mostly participated in the regulation of lipid and atherosclerosis, fluid shear stress and atherosclerosis, IL-17 signaling pathway, and TNF signaling pathway. The results showed WWJYS might regulate smooth muscle cells, affect the inflammatory response, and improve endothelial function through the abovementioned pathways.
Figure 9: Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis results of WuweijiangyasanMolecular docking
We selected the top five active ingredients of WWJYS to dock with the top five target proteins in the PPI network as well as the ACE and ACE2 that we have confirmed can be regulated by WWJYS through the previous experiment. Protein structures predicted by AlphaFold2 are shown in [Figure 10]. The absolute values of the docking score indicate the affinity of the components with the targets and the stability of the conformation. The absolute value >5 indicates a good binding activity, >7.0 indicates a strong binding activity. The molecular docking results revealed all these five compounds exhibited good interaction with ACE, ACE2, AKT1, TP53, IL6, TNF, and JUN.
Figure 10: Effects of Wuweijiangyasan on angiotensin-converting enzyme 1 and angiotensin converting enzyme 2 expression in kidney. (A-G) Protein structures of angiotensin-converting enzyme, angiotensin-converting enzyme 2, TP53, JUN, AKT1, IL6 and TNF predicted by AlphaFold2. Different colors indicate different confidence levels of amino acid residues; (H) Docking results of target proteins and active componentsVerification of bioactive compounds
The optimal separation of the components of the WWJYS in water decoction was achieved under 2.4 terms chromatography, as shown in [Figure 11]. The UHPLC-LTQ-Orbitrap MS/MS results showed flavonoids, diterpene quinone, and amides in WWJYS were well responded to in the positive ion mode, while phenolic acid and other organic acids exhibited good response in negative ion mode. The composition of WWJYS was speculated based on the precise molecular compounds within an element information quality deviation range of 10 ppm. Then after the combination of fragment information from secondary mass spectrometry, the reported data, and retention time, 77 compounds were finally identified in five components of this antihypertensive formula. Among these compounds, 36 were from Radix Puerariae Lobatae, 24 from Radix et Rhizoma Salviae Miltiorrhizae, 7 from root of Arctii Fructus, 6 from Cortex Lycii, 6 from Fructus Crataegi, and 5-caffeoylquinic acid were from Fructus Crataegi, Cortex Lycii, and root of Arctii Fructus. Formononetin, tanshinaldehyde, tanshinone IIA, miltirone, and other bioactive components were consistent with previously predicted results.
Figure 11: ESI-MS total ion chromatogram of Wuweijiangyasan in positive (a) and negative (b) ion mode Discussion Preliminary results[15],[16] showed that WWJYS had an antihypertensive effect on SHR rats after the 2-week administration from the onset. The SBP level of WWJYS maintained steady and continuously decreased. In the experiment, the organ weight-to-tibial length ratio as a new evaluation method could effectively avoid the experimental result error caused by the difference in individual nutrition and thus is better than the traditional evaluation method.[17],[18]
The previous research showed that overweight and dyslipidemia were risk factors for SBP elevation. It was known that blood lipids were closely related to hypertension and even had a causal relationship.[19],[20],[21] This study found that WWJYS reduced blood lipids and thus exerted obvious effects on serum cholesterol, TG and LDL-C in SHR rats compared with the model group. The five herbal ingredients of WWJYS all could lower blood lipids to a certain degree. Previous research reported that Crataegus,[22],[23],[24],[25]Radix et Rhizoma Salviae Miltiorrhizae, and Radix Puerariae Lobatae[24],[26] can reduce blood lipid. In addition, the body weight of rats in the WWJYS group was maintained steady before and after administration. Radix et Rhizoma Salviae Miltiorrhizae and Radix Puerariae Lobatae could inhibit atherogenesis in high-risk hypertensive subjects without serious adverse events or abnormal biochemistry changes. The combination of Radix et Rhizoma Salviae Miltiorrhizae and Radix Puerariae Lobatae significantly improved atherogenesis in high-risk hypertensive patients, with the potential in primary atherosclerosis prevention.[27] The root of Arctii Fructus is an herbal medicine used to treat hypertension, gout, hepatitis, and other inflammatory disorders.
WWJYS can also exert analgesic and sedative effects. As an antihypertensive formula, WWJYS's depressurization effect might be related to its sedative effect. Clinically, the BP of patients with hypertension can sometimes be decreased by improving the quality of sleep. Therefore, this study speculated that sedation might help to decrease BP. Patients with hypertension in rotation tolerance test present with dizziness. Manifestations of hypertension, such as irritability, dizziness, and dysphoria, were also shown in patients in the irritability degree test.
ACE, a rate-limiting enzyme of the RAAS system, plays an important role in RAAS26.[26],[27],[28],[29] The imbalance between ACE1 and ACE2 can negatively regulate BP resistance. Angiotensin-converting enzyme is widely recognized to play an important role in the pathogenesis of cardiovascular diseases, including hypertension.[21],[30],[31],[32] In view of the importance of ACE in the pathogenesis of hypertension, the effect of WWJYS on mRNA and protein expression levels of ACE1 and ACE2 was first examined in the heart and kidney. To confirm the hypothesis, a western blot was performed and found that compared with the model group, WWJYS could upregulate the cardiac ACE2 protein. However, the ACE1 protein expression in the heart and kidney tissues in the WWJYS group is inconsistent with previous reports. Therefore, it can be speculated that the WWJYS lowered BP by up-regulating the expression of ACE2 mRNA and protein in the heart of SHR rats. From the genetic perspective, compared with the model group, WWJYS could lower the expression level of ACE1 mRNA while increasing ACE2 mRNA expression level in rats' hearts. Meanwhile, WWJYS up-regulated the expression of kidney ACE2 mRNA. These results suggest that the regulation of vascular function mainly mediated by the RAAS system may be the main mechanism of WWJYS.
Then, this study explored the mechanisms of a multicomponent, multigene-targeting of WWJYS by establishing an “herbs-bioactive components-potential targets” network. 64 compounds were predicted, and some of which were also validated in UHPLC-LTQ-Orbitrap MS/MS results. Among the chemical compounds, quercetin could induce angiogenesis, decrease myocardial oxidative stress, and alleviate endothelial dysfunction.[32],[33] Apigenin can improve endothelium-dependent relaxation in the DOCA-salt rats, reduce the production of ROS in the renal interlobar artery and increase the production of NO.[34] Formononetin enhances NO levels or endothelial cell function and also induces vasodilation by K precontractions in rat aortas (EC + 50 107.2 μM), and its vasodilatory activity was concerned with the PI3K/Akt pathway.[35] Salvianolic acids and diterpene quinone from Radix et Rhizoma Salviae Miltiorrhizae exert cardiovascular protective effects.[36],[37],[38] Tanshinone IIA could reduce inflammatory response targeting IL6[39] and exert its protective effect on endothelial cells by inhibiting ferroptosis through NRF2 pathway.[40] In addition, Fructus Crataegi also presents a lasting depressurization effect.[37] In our study, we conducted a comprehensive analysis of the chemical components of the WWJYS. These identified compounds in WWJYS, which have been reported in the literature, might be the major effective components of WWJYS. We then collected 22 key targets through network pharmacology analysis, and GO enrichment highlighted the main processes of epithelial cell proliferation, regulation and proliferation of smooth muscle cell, and regulation of NO synthase activity. KEGG analysis indicated the role of WWJYS in lipid regulation (consistent with experimental results), fluid shear stress and atherosclerosis, and inflammatory-related pathways. Interestingly, the results showed WWJYS might regulate smooth muscle cells, affect inflammatory response and improve endothelial function through the abovementioned pathways. Although the RAAS pathway was not prominent in KEGG analysis, several predicted key targets, including IL6 and TNF, were associated with the RAAS system from oxidative stress, inflammation, and endothelial function regulation.[41] It will be interesting to explore whether WWJYS can affect the occurrence of hypertension from the upstream of the RAAS pathway.
Docking simulation, a screening method to rapidly assess a test compound's binding activity, is especially helpful in early-stage pharmacology studies. However, due to the limitation of experimental structure determination, most protein structures currently only include a fragment of the sequence, and the comprehensive structure coverage of the proteome is still a prominent and great challenge. AI-based protein structure prediction represented by AlphaFold2 may become a useful research tool for rapid high-throughput screening of small molecular targets by providing reliable protein structures at a rapid and large scale. Using artificial intelligence and deep learning, the newly developed AlphaFold2 is able to accurately predict the three-dimensional structure of individual proteins based on amino acid sequence information.[42] This study is the first to use AlphaFold2 to predict the structure of full-length sequence proteins and perform molecular docking. Although this study explored the mechanism of WWJYS, there are still certain limitations. The research data came from the existing database, so the integrity and authenticity of the results depend on the quality of the data. Therefore, in the future study, detailed pharmacological mechanisms by which WWJYS affects spontaneous hypertension will be investigated.
ConclusionsThis study explored the mechanism of WWJYS, a TCM formula, in treating hypertension from the perspective of sedation and depressurization. WWJYS could exert a stable and lasting depressurization effect. Besides, WWJYS could maintain a stable HR, lower blood lipids, and reduce body weight. Meanwhile, it found that the up-regulation of the heart RAAS could lead to a decrease in BP, and the depressurization mechanism of WWJYS might function in multiple signal pathways, especially in improving blood vessel function and intervening inflammation.
Ethics statement
All animal treatment and experiments were approved by the Experimental Animal Administration Committee of Beijing University of Chinese medicine.(Permission number: SCXK (Jing)-2012-0001).
Availability of data and materials
The datasets analyzed during the current study are available from the corresponding author on reasonable request.
Financial support and sponsorship
This study was supported by the National Natural Science Foundation of China (No. 81473521, 81973697).
Conflicts of interest
There are no conflicts of interest.
References
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