- Academic Editor
Background: Polygonum hydropiper L (PH) was widely used to
treat dysentery, gastroenteritis, diarrhea and other diseases. Coptis
chinensis (CC) had the effects of clearing dampness-heat, purging fire, and
detoxifying. Study confirmed that flavonoids in PH and alkaloids in CC alleviated
inflammation to inhibit the development of intestinal inflammation. However, how
PH-CC affects UC was unclear. Therefore, the aim of this study is to analyze the
mechanism of PH-CC on ulcerative colitis (UC) through network pharmacology and
in vivo experiments. Methods: The active ingredients and
targets of PH-CC and targets of UC were screened based on related databases. The
core targets of PH-CC on UC was predicted by protein-protein interaction network
(PPI), and then the Gene Ontology-biological processes (GO-BP) function
enrichment analysis was conducted using the Database for Annotation,
Visualization and Integrated Discovery (DAVID) database. The binding activity
between pyroptosis proteins, core targets and effective ingredients were verified
based on molecular docking technology. Finally, combined with the results of
network pharmacology and literature research, the mechanism of PH-CC against UC
was verified by in vivo experiments. Results:
There were 23 active components and 191 potential targets in PH-CC, 5275
targets in UC, and 141 co-targets. GO-BP functional analysis of 141 co-targets
showed that the first 20 biological processes were closely related to
inflammation and lipopolysaccharide (LPS) stimulation. Furthermore, core targets
had good binding activity with the corresponding compounds. Animal experiment
indicated that PH-CC effectively prevented weight loss in UC mice, reduced the
disease activity index (DAI) score, maintained colon length, suppressed
myeloperoxidase (MPO) activity, inhibited pyroptosis protein expression, and
downregulated the levels of IL-18 and IL-1
Ulcerative colitis (UC) is a subtype of inflammatory bowel disease, presenting with bloody stool, fever, weight loss, abdominal pain, and diarrhea [1]. More and more people are suffering from UC continuously worldwide continuously worldwide, indicating that UC is gradually becoming a global public health concern [2]. However, the pathogenesis of UC is complex, and some researchers believe that immune dysregulation, genetics, bacterial infection, and eating habits might be the main risk factors for UC [3]. The inflammatory response damages the intestinal mucosa and is involved in the development of UC. In the absence of sufficient treatment, long-term inflammatory response can lead to colon cancer. Pyroptosis, different from cell apoptosis, cell death, and autophagy, can lead to cell expansion and rupture, exacerbating the inflammatory response [4]. Previous studies illuminated that the protein expression of NOD-like receptor protein domain 3 (NLRP3), Caspase-1, and Gasdermin D are upregulated in the colon tissue and induce pyroptosis in UC [5].
Accumulating evidence indicates that Polygonum hydropiper L (PH) is
commonly used in the treatment of dysentery, gastroenteritis, diarrhea, rheumatic
joint pain, swelling and other diseases [6, 7]. In addition, the main ingredients
of flavonoids, such as rutin, quercitrin, and quercetin, have certain
anti-inflammatory effects [8]. Coptis chinensis (CC) has
anti-inflammatory, blood sugar- and lipid-lowering properties. Alkaloids in CC
have a certain therapeutic effect on intestinal mucosal injury in colitis rats
and inhibit the p38/NF-
The flavonoids in PH and the alkaloids in CC have anti-inflammatory effects and can effectively treat inflammation. Fengliao Changweikang showed a certain therapeutic effect on gastrointestinal diseases in clinics [10], but Daphniphyllum Calycinum Benth, one of its constituents, is a toxic traditional Chinese medicine with scarce resources. The properties and flavors of CC are similar to that of Daphniphyllum Calycinum Benth. At the same time, it is safe, non-toxic, widely distributed, easily cultivated, and simply processed. Therefore, Daphniphyllum Calycinum Benth was substituted with CC and combined with PH to form a herb-pair. The systematic pharmacological research of Polygonum hydropiper L-Coptis chinensis (PH-CC) on UC with inflammation as the major symptom provides an experimental basis for developing new drugs for UC. The multi-component, multi-target, and multi-link properties of traditional Chinese medicine help the body to produce a comprehensive effect. Thus, this study used network pharmacology to predict the effective components and key targets of PH-CC against UC, and the potential mechanisms of PH-CC against UC was verified in vivo. The analysis method is shown in Fig. 1.
Analysis strategy of Polygonum hydropiper L-Coptis chinensis (PH-CC) against ulcerative colitis (UC). CNKI, China National Knowledge Infrastructure; TCMSP, traditional Chinese medicine systems pharmacology; OMIM, Online Mendelian Inheritance in Man; PPI, Protein-Protein Interaction; GO-BP, Gene Ontology-biological process; DSS, Dextran Sulfate Sodium Salt.
The chemical components of PH-CC were searched using Wed of Science
(https://www.webofscience.com/wos/), PubMed (https://pubmed.ncbi.nlm.nih.gov/),
and China National Knowledge Infrastructure (CNKI, https://www.cnki.net/)
databases. All chemical components, based on the traditional Chinese medicine
systems pharmacology (TCMSP) database (https://old.tcmsp-e.com/tcmsp.php), were
retrieved one by one to get the active components and potential targets of PH-CC
(oral bioavailability (OB)
DisGeNet database (https://www.disgenet.org/), GeneCards database (https://www.genecards.org/), and Online Mendelian Inheritance in Man (OMIM) database (https://www.omim.org/) were used to screen related targets of UC. After deleting the repeated targets of UC, the co-targets of PH-CC against UC and the Venn diagram were obtained were obtained through Venny2.1 software (https://bioinfogp.cnb.csic.es/tools/venny/). Subsequently, the co-targets were used to construct Protein-Protein Interaction (PPI) networks in the search tool for recurring instances of neighboring genes (STRING, https://string-db.org/), where free nodes were hidden. Genes with scores greater than 0.4 were input into Cytoscape 3.9.0 software for visualization, and the key targets of PH-CC against UC were screened with a degree value greater than 2 times the median.
The key targets of PH-CC regulating UC were analyzed for biological process (BP) in the Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/). Finally, the first 20 biological processes were selected to draw a bubble diagram, and the results were analyzed using the bioinformatics database (http://www.bioinformatics.com.cn/).
The abnormal activation of NLRP3 pyroptosis pathway may be an important reason for inducing UC through literature review [11]. Therefore, we selected the key proteins (NLRP3, Caspase-1, Gasdermin D) on this pathway for molecular docking verification with the active ingredients (quercetin and kaempferol) with the largest degree value. The small molecule ligands are obtained as follows: the 3D structure of the active ingredient was downloaded from TCMSP and Pubchem (https://pubchem.ncbi.nlm.nih.gov/) databases, and the above results was saved in Mol2 format through OpenBabel 3.1.1 software (https://github.com/openbabel/openbabel/releases). The macromolecular receptor proteins are obtained as follows: The 3D structure of the core target was obtained through the UniProt (https://www.uniprot.org/) and RCSB Protein Data Bank (RCSB PDB, https://www.rcsb.org/) databases and saved in PDB format. Subsequently, the above results were removed water molecules and protein residues and saved in PDBQT format by the Pymol. Finally, the ligands and receptors were docked by AutoDockTools1.5.7 software (https://autodock.scripps.edu/). The binding activity of components and targets was analyzed by calculating the binding energy.
Dextran sulfate sodium (DSS, 160110) was obtained from MP Biomedicals (molecular
weight: 36,000–50,000, Aurora, OH, USA). Fecal occult blood qualitative test kit
(ml095013) was obtained from Shanghai Enzyme Linked Biotechnology Co., Ltd.
China. Myeloperoxidase (MPO, A044-1-1) activity assay kit was purchased from
Jiancheng Bio-engineering Institute (Nanjing, China). IL-1
Polygonum hydropiper L was collected from Wuzhishan City (Hainan, China) and identified by Prof. Niankai Zeng, Hainan Medical University. The voucher specimen (No. 20191016) was deposited in the Laboratory of Traditional Chinese Medicine, School of Pharmacy, Hainan Medical University. Coptis chinensis (No: 220201) was provided by Shijiazhuang Chengxin Traditional Chinese Medicine Co., Ltd. (Hebei, China) and identified by Prof. Niankai Zeng, Hainan Medical University.
After weighing the medicinal materials of Stems and leaves of PH and Rhizome of CC (mixed at 2:1), 60% alcohol with 9 times the volume of the total mass of PH-CC was used as the extracting solution to soak the medicinal materials of PH-CC overnight. Medicinal materials of PH-CC were extracted for 1 h and filtered. Subsequently, the residue was added with 8 times the volume of 60% alcohol to extract for 1 h, and the two filtrates were combined. The filtrate was concentrated under reduced pressure and purified by AB-8 macroporous resin. The 60% alcohol eluent was retained, and the eluent was concentrated under reduced pressure to obtain thick liquid medicine, which was dried into a powder using the freeze dryer and stored.
Sixty male SPF BALB/C mice (6–8 weeks,18–22 g) were supplied by Changsha
Tianqin Biotechnology Co., Ltd., China (SCXK (xiang) 2022-0011). All mice were
kept in rooms under controlled laboratory conditions (22
After adaptive feeding for one week, sixty mice were randomly divided into 6 groups (n = 10): normal group, model group, mesalazine group (800 mg/kg), and PH-CC group (114 mg/kg, 228 mg/kg). Except for the normal group, mice in other groups were free to drink 3% DSS solution for 10 days, and 3% DSS solution was replaced every day. After drinking the 3% DSS solution for one day, the mesalazine group (800 mg/kg) and PH-CC group (114 mg/kg, 228 mg/kg) orally received treatment once a day for 9 days, and meanwhile, the other groups were given deionized water by gavage (Fig. 2). On the 11th day, mice were anesthetized with 1% pentobarbital sodium, and orbital blood and colonic tissues were collected.
The experimental design diagram.
Fecal occult blood: Using fecal occult blood qualitative test kit. Percentage of
weight loss: The score was calculated according to the percentage of weight loss,
no weight loss (0), weight loss 1%–5% (1), weight loss 5%–10% (2), weight
loss 10%–15% (3), weight loss
The colon tissue of all groups was fixed in 4% paraformaldehyde solution. After conventional dehydration, transparency, paraffin embedding, slicing, baking, dewaxing, and H&E staining, the slices were sealed. The pathological changes of colon tissue were assessed using an optical microscope.
Colon tissues were weighed and cut into small pieces. According to the weight-to-volume ratio (1:19), 5% homogenate suspensions were prepared by adding a homogenate medium. Following the manufacturer’s instructions, MPO activity was detected.
The expression of IL-18 and IL-1
Target proteins were subjected to electrophoresis, membrane transfer and
blocking (1 h). Subsequently, they were incubated with the corresponding primary
antibodies at 4 °C overnight. The primary antibodies were as follows: NLRP3
(1:1000), Caspase-1 (1:1000), Gasdermin D (1:1000), IL-1
All data were expressed as mean
37 ingredients of PH-CC were obtained. Previous studies showed that quercitrin [13], hyperoside [14], rutin [15], jatrorrhizine [16], and columbamine [17] have strong anti-inflammatory activity; therefore, they are suitable for further studies. In total, 23 active ingredients and 191 targets were finally obtained by screening and deleting repeated targets (Table 1). In addition, 5275 UC targets were achieved, and 141 co-targets were obtained by intersecting PH-CC and UC targets (Fig. 3A). The network graph of “active ingredients-intersection targets” (Fig. 3B) demonstrate that the effect of PH-CC on UC was achieved through the interaction of “multi-active ingredients and multi-targets”, in which the active ingredients were ranked by degree and the top 4 were quercetin, kaempferol, rutin and tetrahydroberberine.
The screening of active components and targets. (A) The co-targets of PH-CC and UC. (B) The network of PH-CC active component targets.
Number | Mol ID | Molecule name | Molecule weight | OB (%) | DL |
1 | MOL001454 | berberine | 336.39 | 36.86 | 0.78 |
2 | MOL002897 | epiberberine | 336.39 | 43.09 | 0.78 |
3 | MOL001458 | coptisine | 320.34 | 30.67 | 0.86 |
4 | MOL000785 | palmatine | 352.44 | 64.60 | 0.65 |
5 | MOL00789 | jatrorrhizine | 338.41 | 19.65 | 0.59 |
6 | MOL001455 | Tetrahydroberberine | 339.42 | 53.83 | 0.77 |
7 | MOL000098 | quercetin | 302.25 | 46.43 | 0.28 |
8 | MOL001554 | Scopolamine | 303.39 | 67.97 | 0.27 |
9 | MOL002907 | Corchoroside A_qt | 404.55 | 104.95 | 0.78 |
10 | MOL000622 | Magnograndiolide | 266.37 | 63.71 | 0.19 |
11 | MOL001457 | columbamine | 338.41 | 26.94 | 0.59 |
12 | MOL002668 | Worenine | 334.37 | 45.83 | 0.87 |
13 | MOL002904 | 8-Oxyberberine | 351.38 | 36.68 | 0.82 |
14 | MOL004368 | Hyperoside | 464.41 | 6.94 | 0.77 |
15 | MOL000354 | isorhamnetin | 316.28 | 49.60 | 0.31 |
16 | MOL000422 | kaempferol | 286.25 | 41.88 | 0.24 |
17 | MOL000701 | quercitrin | 448.41 | 4.04 | 0.74 |
18 | MOL000415 | rutin | 610.57 | 3.20 | 0.68 |
19 | MOL002714 | baicalein | 270.25 | 33.52 | 0.21 |
20 | MOL000392 | formononetin | 268.28 | 69.67 | 0.21 |
21 | MOL005828 | nobiletin | 402.43 | 61.67 | 0.52 |
22 | MOL001002 | ellagic acid | 302.20 | 43.06 | 0.43 |
23 | MOL012920 | sinomenine | 329.43 | 30.98 | 0.46 |
OB, oral bioavailability; DL, drug-likeness.
The PPI network information of 141 co-targets of PH-CC on UC was obtained in the STRING database, and disconnected nodes were hidden. Result showed that the network contained 140 nodes and 2734 edges. Subsequently, The above results are visualized and analyzed through the Cytoscape 3.9.0 (Fig. 4A,B). In total, 20 core targets were obtained, indicating that these targets may be the core targets of PH-CC in UC.
The analysis of PPI network results. (A) PPI network exported from search tool for recurring instances of neighboring genes (STRING, https://string-db.org/) database. (B) PPI network visualized with Cytoscape 3.9.0.
The first 20 biological processes (BP, p
GO-BP analysis of the co-targets of PH-CC and UC.
The docking results of pyroptosis proteins and active ingredients are shown in
Table 2. The absolute value of binding energy
The results of molecular docking for active components and core
targets. (A) Quercetin and NLRP3. (B) Quercetin and Caspase-1. (C) Quercetin and
Gasdermin D. (D) Quercetin and IL-1
Targets | Phytochemicals | Binding energy (kcal/mol) |
NLRP3 | quercetin | –6.0 |
kaempferol | –6.1 | |
Caspase-1 | quercetin | –6.9 |
kaempferol | –6.2 | |
Gasdermin D | quercetin | –7.1 |
kaempferol | –6.8 | |
IL-1 |
quercetin | –7.6 |
kaempferol | –7.6 |
Mice in the normal group had smooth hair, normal eating and drinking, brown-black granular feces, and stable body weight. Mice subjected to DSS-induced UC gradually showed lower food and water intake and obvious weight loss, were curled up and depressed, and had rough hair. Loose stool was noticeable since the third day, and occult blood and hematochezia appeared on the fifth day. After treatment with PH-CC, the symptoms of mice became milder than those in the model group. Colon length was significantly shorter, and the DAI score and MPO activity were significantly upregulated in DSS-induced mice (Fig. 7). Treatment with PH-CC reduced the above indexes.These results demonstrated that PH-CC might mitigate DSS-induced UC.
PH-CC alleviated the symptoms of DSS-induced UC in mice.
(A) Disease activity index. (B) Myeloperoxidase (MPO) activity. (C,D)
The length of the colon.
Histological result demonstrated that the colon of normal mice was intact, with normal mucosal epithelium and without inflammatory cell infiltration, congestion, and ulceration. The colonic mucosa and crypt structure of UC mice were severely damaged, showing numerous inflammatory cells, gland disappearance, and deranged structure. The symptoms and colon injury were obviously relieved, crypt destruction and inflammatory cell infiltration were alleviated, and glandular structure was preserved in the PH-CC and mesalazine group (Fig. 8).
Effect of PH-CC on histopathological changes of colon of UC mice
(HE
Compared with the normal group, the serum levels of IL-1
PH-CC inhibited the inflammatory response in UC mice. The serum
levels of IL-1
Compared with the normal group, the protein levels of NLRP3, Caspase-1,
Gasdermin D, IL-1
Effect of PH-CC on the expression of pyroptosis
proteins. Effect of PH-CC on pyroptosis proteins electrophoresis in UC mice colon tissue (A), The protein levels of IL-18 (B), IL-1
UC is a complex disease of the gastrointestinal tract, and its incidence continues to increase in China and worldwide. The inflammation-related colon cancer in UC is more challenging to treat than other types of colon cancer, with a higher mortality rate [18, 19]. The adverse reactions of glucocorticoids, sulfasalazine, and immunosuppressive agents in clinical practice are becoming more and more prominent, affecting the health status of patients [20]. Consequently, it is extremely vital to find effective and safe medicines for treating UC. PH-CC are traditional Chinese medicines. Previously, it has been reported that the extract of PH significantly improve the colon shortening induced by UC [21]. In addition, studies have found that CC can prevent intestinal barrier damage and alleviate the symptoms of UC by ameliorating gut dysbiosis and inhibiting the inflammatory response in rats [22]. Some active components of PH-CC were shown to have anti-inflammatory activity [13, 23, 24, 25, 26, 27]. However, the relationship between PH-CC and UC has not yet been systematically elucidated. Moreover, the complexity of traditional Chinese medicines with several components made it difficult to study the potential mechanism. Therefore, the effective ingredients and potential targets of PH-CC against UC were screened through network pharmacology. We also measured the potential mechanisms in animal experiments to provide a reference for future clinical studies.
By analyzing the “drug-active component-target” network, we found that
quercetin and kaempferol were the essential components of PH-CC against UC.
Quercetin is the active ingredient of PH-CC, and kaempferol is the main active
ingredient of PH. Twenty-three active ingredients and 191 targets of PH-CC were
examined. Study found that isorhamnetin has a therapeutic effect on mice with
inflammatory bowel disease through the metabolism of xenobiotics induced by
pregnane X receptor (PXR) was upregulated and nuclear factor kappa-B
(NF-
In this study, 141 co-targets of PH-CC against UC were obtained, and the top
five core targets (TNF-
Overexpression of LPS in intestinal mucosa can cause neutrophil infiltration and subsequent intestinal mucosal cell damage and inflammatory factor release [40, 41]. It is noteworthy that LPS is also an important activator of Caspase-1. LPS activates cytoplasmic lipid A, thereby activating Caspase-1 and triggering pyroptosis [42]. The abnormal activation of NLRP3 can cause uncontrollable inflammatory response and induce pyroptosis, and further leads to the occurrence and aggravation of UC. Therefore, blocking the activation of NLRP3 may alleviate UC. Animal experiments have confirmed that inhibiting the activation of Caspase-1 and NLRP3 in colitis mice can improve colon inflammation [43]. Pyroptosis induces cell enlargement and rupture, an inflammatory factor release, intestinal mucosal barrier damage, and infernal circle formation [44]. Sustained inflammation and pyroptosis are positively correlated with the severity of UC. However, whether PH-CC alleviates pyroptosis in intestinal mucosal cells remains unclear. Therefore, this study intends to further analyze the underlying mechanism of PH-CC against UC from the perspective of pyroptosis.
Pyroptosis, a novel mode of inflammatory programmed cell death, became a
research hotspot in the pathogenesis of UC [45]. NLRP3, an inflammatory complex
associated with inflammation, can be activated by bacterial, viral, fungal
infection, K
This study adopted a DSS-induced UC model in mice [49]. DSS-induced UC can destroy the colonic mucosal barrier, increase colon permeability, lead to the secretion imbalance of inflammatory and anti-inflammatory factors, Th1/Th2 imbalance, and gut dysbiosis [50, 51, 52, 53]. The inflammatory-immune response mechanism in DSS-induced UC is similar to the pathogenesis of human UC. Therefore, the DSS-induced UC model has been recognized and widely used in experimental studies of UC [54, 55, 56]. The DAI and colon length can help assess DSS-induced UC model whether it is successfully established [57, 58]. Our results showed that DSS-induced UC mice had hair discoloration, slow response, significant weight loss, bloody stool, and anal bleeding. Compared with the normal group, the DAI was significantly elevated, and colon length was shortened in UC mice. The histopathological assessment showed incomplete shedding of colon mucosa, derangement of intestinal glands, and severe crypt damage in the model group.
Immune-mediated inflammatory response is a main pathological feature of UC. Our
results indicated that the tissue expression of NLRP3, Caspase-1, and Gasdermin D
was increased in UC mice. Concomitantly, the expression of activated
IL-1
MPO is a neutrophil marker released into phagosomes to promote reactive oxygen species (ROS) generation and accelerate local intestinal inflammation [59]. Furthermore, MPO activity has a close relation with intestinal inflammation [60]. In the present study, MPO expression was obviously upregulated in UC mice and significantly decreased after treatment with PH-CC. These results demonstrated that DSS-induced UC mice increased neutrophil infiltration which exacerbated colonic mucosal inflammation. On the other hand, PH-CC inhibited the inflammatory response and improved the repair process by reducing neutrophil infiltration.
IL-1
Study proved that activated NLRP3 inflammasome enhances inflammatory factors expression in UC [62]. Research found that curcumin and Morus macroura Miq. fruit extract decreased ROS generation, downregulated activated NLRP3, and inhibited the expression of inflammatory factors to relieve intestinal lesions in the mice model of DSS and acetic acid-induced UC [46, 63]. Similarly, studies have shown that by inhibiting NLRP3, its downstream molecules, pyroptosis, cardamonin alleviated intestinal inflammation [64]. Our results are basically consistent with previous findings.
In conclusion, our study provides preliminary evidence supporting the efficacy of PH-CC against UC through network Pharmacology and in vivo experiments, thereby offering a novel perspective for further mechanistic investigations on the therapeutic action of PH-CC in UC. However, there are still certain limitations in this study. Firstly, a more comprehensive analysis of the synergy between the predicted multi-components and multi-targets is lacking. Additionally, the experimental verification method employed here appears relatively singular. Therefore, it is imperative to conduct further in-depth molecular biology experiments in order to provide a more precise experimental foundation for both the prediction results of this study and their potential clinical applications.
UC, Ulcerative colitis; PH-CC, Polygonum hydropiper L-Coptis chinensis;
NLRP3, NOD-like receptor protein domain 3; TCMSP, Traditional Chinese Medicines
for Systems Pharmacology; OMIM, Online Mendelian Inheritance in Man; STRING,
Search Tool for Recurring Instances of Neighbouring Genes; DAVID, Database for
Annotation, Visualization and Integrated Discovery; GO, Gene Ontology;
IL-1
The data used to support the findings of this study are available from the corresponding author upon request.
FZ and SR conceived and designed the experiments; FZ conducted the experimental work and analysis; YL analyzed network pharmacological data and assisted in animal experiment; FZ and YZ developed the animal models; FZ drafted the manuscript; HN made colon pathological sections and analyzed the pathological results; SR and HN provided major revisions and comments to the manuscript. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity. All authors read and approved the final manuscript. All authors contributed to editorial changes in the manuscript.
This study was approved by the Animal Ethics Committee of Hainan Medical College to pass the review of animal experiment ethics (HYLL-2022-365). The plants of Polygonum hydropiper L and Coptis chinensis were used in this study. Polygonum hydropiper L (No: 20191016) collected from Wuzhishan City (Wuzhishan, China) was identified by Prof. Niankai Zeng (the School of Pharmacy, Hainan Medical University, Haikou, China). Coptis chinensis (No: 220201) was purchased (Shijiazhuang Chengxin Traditional Chinese Medicine Co., Ltd., Shijiazhuang, China) and was identified by Prof. Niankai Zeng (the School of Pharmacy, Hainan Medical University, Haikou, China).
The authors thank Key Laboratory of Tropical Translational Medicine of Ministry of Education, Hainan Key Laboratory for Research and Development of Tropical Herbs, Haikou Key Laboratory of Li Nationality Medicine and Department of Pathology of First Affiliated Hospital of Hainan Medical University gave support to the lab research.
This work was supported financially by the National Natural Science Foundation of China (82260920).
The authors declare no conflict of interest.
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