Unveiling the Molecular Mechanisms and Active Components of Angong Niuhuang Pills in Stroke Treatment: Network Pharmacology, Molecular Docking, and Cellular Studies

Unveiling the Molecular Mechanisms and Active Components of Angong Niuhuang Pills in Stroke Treatment: Network Pharmacology, Molecular Docking, and Cellular Studies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Unveiling the Molecular Mechanisms and Active Components of Angong Niuhuang Pills in Stroke Treatment: Network Pharmacology, Molecular Docking, and Cellular Studies JU CHEN This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4759284/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Angong Niuhuang pills (ANPs) exhibit a curative effect in patients with stroke, but its main effective components remain unexplored. Here, we aims to elucidate the molecular mechanisms and active ingredients of ANPs against stroke through network pharmacology, molecular docking, and cellular experiments. Methods: The compounds and targets of each herb in the ANP were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) databases. The genes corresponding to the targets were retrieved from the GeneCards database. A traditional Chinese medicine (TCM) formulae-compound-disease-target network was constructed using Cytoscape. A protein-protein interaction network was constructed using the STRING database. The core targets of ANP were analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using the ClueGO plug-in of Cytoscape. The active ingredients of ANP were docked to the top three core targets. Finally, we explored the effects of coptisine on the oxygen-glucose deprivation/reperfusion (OGD/R) models of PC12 cells. Results: The TCM formulae-compound-disease-target network contained 81 active ingredients and 759 core targets. We identified 47 GO entries of ANP types for stroke, and 55 pathways were screened based on GO and KEGG pathway analyses. Core targets were mainly involved in biological processes (GO enrichment analysis, p < 0.05), including the regulation of heart contraction, muscle contraction, and steroid metabolic processes. In the KEGG pathway, the core targets were mainly involved in AGE-RAGE signaling pathway in diabetic complications, neurotrophin signaling pathway, and cGMP-PKG signaling pathway. Molecular docking results showed that norwogonin, coptisine, and musennin had a high affinity for ubiquitin C (UBC), E1A binding protein P300 (EP300), and cellular tumor antigen p53 (TP53), respectively. Coptisine alleviates OGD/R injury by regulating EP300 and LC3B expression. Conclusion: This study, using network pharmacology, molecular docking analysis, and cellular experiments, provides insights into the potential mechanisms and active ingredients of ANPs in stroke protection. The identification of core target genes and signaling pathways suggests that coptisine could be a promising candidate for treating cerebral ischemia-reperfusion injury. Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Biological sciences/Molecular biology Biological sciences/Plant sciences Angong Niuhuang pill Network pharmacology Molecular docking Stroke Coptisine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Stroke is the world's second most common cause of death and the third most common cause of disability ( 1 ). Although the age-adjusted mortality rate has declined in recent years, the total number of people affected by stroke-related disability and death has risen, with developing countries bearing the greatest burden( 2 ). The increasing prevalence of stroke in young people is alarming ( 3 ). Angong Niuhuang pill (ANP) is a Chinese patent medicine composed of Moschus , Margarita , Cinnabaris , Calculus bovis , Buffalo Horn , Radix Scutellariae Baicalensis , Fructus gradeniae , Realgar , Rhizoma coptidis , Borneolum synthcticum , and Radix curcumae ( 4 ). In China, ANP is generally prescribed for patients with cerebral diseases, such as hypertensive cerebral hemorrhage, hypoxic-ischemic encephalopathy, viral encephalitis, severe craniocerebral trauma, and cerebral paralysis ( 5 ). However, the molecular mechanisms and active ingredients of ANP against stroke have not been fully elucidated. In addition, the clinical application of ANP is limited because of the presence of two known toxic metalloids, arsenic and mercury, in ANP ( 6 , 7 ). Therefore, it is important to determine the active components and molecular mechanisms of ANP in the treatment of stroke. To understand traditional Chinese medicine (TCM) formulas ( 8 ) and identify potential new drugs or targets for specific diseases ( 9 – 11 ), network pharmacology combined with multidisciplinary technologies, such as systems biology and computational biology, is conducted. Molecular docking in TCM is a computer-based drug design technology that simulates the geometric structure of molecules and the interaction forces between molecules through stoichiometric calculation methods ( 12 ). In recent years, network pharmacology has been successfully used to predict the mechanisms of TCM in the treatment of a variety of diseases. Therefore, in this study, we aimed to use network pharmacology and molecular docking to explore the main active components of ANP, predict their potential targets and signaling pathways, and explore the association between the active ingredients of ANP and pathogenesis of stroke. Finally, the virtual results were preliminarily verified using oxygen glucose deprivation/reperfusion (OGD/R) modeling in PC12 cells. To the best of our knowledge, this is the first study to identify the molecular mechanisms and active ingredients of ANP against stroke using network pharmacology, molecular docking, and cell experiment. 2. Materials and methods 2.1 Identification of active ingredients and targets in ANP Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://lsp.nwu.edu.cn/tcmsp.php ) was used to authenticate active ingredients of the six Chinese medicinal herbs ( Calculus bovis , Radix Scutellariae Baicalensis , Fructus gradeniae , Rhizoma coptidis , Borneolum synthcticum , and Radix curcumae ) in ANP. TCMSP is a unique platform for analysis of active ingredients in TCM and their interaction in specific diseases. The oral bioavailability (OB) and drug-likeness (DL) are important pharmacokinetic parameters used to identify and screen active ingredients based on the absorption, distribution, metabolism, and excretion model. In this study, compounds with OB > 30% and DL > 0.18 were selected ( 13 ), and the relevant targets were identified. The active ingredients of the other four herbs ( Moschus , Margarita , Buffalo Horn , and Realgar ) were retrieved from the Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) database ( http://bionet.ncpsb.org/batman-tcm/ ), which is the first online bioinformatics analysis tool for screening of the molecular mechanisms of TCM ( 14 ). In this study, the compounds with a score ≥ 20 and P < 0.05 were selected, and the targets with correlative compounds were obtained. 2.2 Predicting the targets of stroke The GeneCards database ( https://www.genecards.org/ ) was used to obtain information on the stroke-associated target genes, which were identified by entering the keyword “stroke.” Finally, the target genes of ANP and stroke were intersected to obtain the common target genes. Venny 2.1, an online graphics software ( http://bioinfogp.cnb.csic.es/tools/venny/index.html ), was used to match and map the targets of ANP and stroke, and a Venn diagram of this analysis was constructed. 2.3 Construction of the TCM formulae-compound-disease-target regulation network The Cytoscape software (version 3.6.1, http://www.cytoscape.org ) was used to construct a TCM formulae-compound-disease-target regulation network. The formulae, compound, disease, and target regulation network nodes in the network represented the active ingredients, drugs, diseases, and key target genes, respectively. Edges were used to connect the drugs with the active ingredients, the active ingredients with the key target genes, and the diseases with the key target genes. The active ingredients of ANP shown in this network were related to stroke-associated targets as key compounds against stroke. The whole network showed a relationship between the drug, disease, active ingredients, and targets, and the mechanism of action of ANP against stroke was explored by constructing a regulatory network. 2.4 Protein-protein interaction (PPI) analysis and screening of core targets To clarify the interaction between ANP-related and stroke-associated targets, we screened for common targets that overlap between ANP-related and stroke target-related targets. The common targets of ANP were entered into STRING ( https://string-db.org/cgi/input.pl ) to construct the PPI network interaction by screening conditions with medium confidence (0.4) and defined species as Homo sapiens ( 15 ). The resulting files were input into the R language for calculation, and a bar diagram of the core target was obtained. 2.5 Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses To further reveal the potential mechanism of ANP in the treatment of stroke, we imported overlapping targets into the ClueGO plug-in of Cytoscape software to analyze the GO function and KEGG pathways with the following screening conditions: a kappa score = 0.4 and the species as Homo sapiens . Only pathways with a pV ≤ 0.05 were shown. The threshold for enrichment analysis was set to P ≤ 0.05. 2.6 Molecular docking simulation The protein crystal structures of the core genes was downloaded from the Protein Database Bank (PDB, https://www.rcsb.org/ ), and the proteins were selected with ligands to ensure docking accuracy. The 3D structure of the active ingredients were downloaded in PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ). Vina software (version 1.1.2) was used for the molecular docking analysis. Finally, the conformation with the lowest score was selected and plotted with PyMOL 2.4 for the analysis. 2.7 Cell culture PC12 cells were obtained from Tongpai Biotechnology Co., Ltd. (Shanghai, China). These PC12 cells were cultured in Dulbecco's modified eagle medium (DMEM) (Gibco, USA) containing 10% (v/v) fetal bovine serum (Gibco, USA) and placed in an incubator at 37℃ and 5% CO 2 . 2.8 OGD/R model The cell culture medium was removed, and the cells were washed three times with PBS and deoxygenated glucose-free DMEM for 1 h to induce the OGD. The cells were maintained in fetal bovine serum and deoxygenated glucose-free DMEM and cultured at 37℃ in an oxygen-free chamber with 95% N 2 and 5% CO 2 for 3 h. The medium was then replaced with glucose-containing DMEM containing 10% fetal bovine serum and incubated at 37℃ in a 5% CO 2 incubator for 24 h. 2.9 Cell viability assessment Cell viability was detected using the Cell Counting Kit-8 (CCK-8) (Beyotime, Beijing, China). The PC12 cells were cultured in 96-well plates at a density of 5 × 10 3 cells/well and incubated for 24 h. Coptisine (0, 2, 4, 8, and 16 µmol/L) was then added. After culturing for 24, 48, and 72 h, 10 µL CCK-8 reagent was added. The cells were then incubated at 37℃ for 2 h, and the optical density was measured at 450 nm using a microplate spectrophotometer. 2.10 Western blot analysis The PC12 cells were cultured in 96-well plates at a density of 5 × 10 3 cells/well and incubated for 24 h. After the OGD/R treatment, coptisine (1, 2, and 4 µmol/L) was added. The cells were incubated at 37℃ for 24 h. Cell viability was detected using the CCK-8 assay. The total protein was isolated using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The protein concentration was measured using a bicinchoninic protein assay kit (Pierce, Rockford, IL, USA). The total protein (50 µg) was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime, Shanghai, China) and transferred to a polyvinylidene fluoride membrane. The membrane was sealed with 5% skim milk powder for 2 h, followed by incubation with primary anti-EP300 (1:1000, Amyjet Scientific, China) and LC3B (1:1000, Cell Signaling, USA) at 4℃ overnight. β-actin (1:2000; Beyotime, China) was used as an internal reference. Goat anti-rabbit IgG (1:1000, Beyotime, China) was added to the membrane and incubated at room temperature (20–24 ℃) for 1 h. Finally, an enhanced chemiluminescence technique was used for the western blot assay kit (Beyotime, China). 2.11 Statistical analysis The Statistical Package of the Social Sciences software (version 16.0; SPSS, Inc., Chicago, USA) was used for statistical analysis of the experimental data, and the data are presented as the mean ± standard deviation (SD). The statistical significance of differences was determined using one-way analysis of variance. P < 0.05 was used to indicate statistically significant differences. 3. Results 3.1 Active ingredients and targets of ANP A total of 88 active ingredients and 1168 targets were retrieved from the TCMSP and BATMAN-TCM databases (after deduplication). These active ingredients primarily originated from Rhizoma coptidis (11 compounds and 90 targets), Radix Scutellariae Baicalensis (31 compounds and 53 targets), Calculus bovis (5 compounds and 4 targets), Radix curcumae (3 compounds and 29 targets), Fructus gradeniae (11 compounds and 59 targets), Borneolum synthcticum (2 compounds and 32 targets), Moschus (26 compounds and 1027 targets), Buffalo Horn (3 compounds and 41 targets), Realgar (1 compound and 67 targets), Margarita (5 compounds and 33 targets), and Cinnabaris (1 compound and 0 target). The main active ingredients of ANP are listed in Table 1 . Table 1 The results of screening active components and targets of ANP by TCMSP database and Batmann-TCM database TCM Number of component Number of target Rhizoma Coptidis 11 90 Radix Scutellariae Baicalensis 31 53 Calculus Bovis 5 4 Radix Curcumae 3 29 Fructus Gradeniae 11 95 Borneolum Synthcticum 2 32 Moschus 26 1027 Buffalo Horn 3 41 Realgar 1 67 Margarita 5 33 Cinnabaris 1 0 Total(deduplication) 88 1168 3.2 Construction of the TCM formulae-disease-target network In total, 7408 stroke-associated targets were identified. These 7408 targets were combined with 1168 targets of ANP to obtain a total of 759 core targets (Fig. 1 ). The 81 active ingredients of ANP mainly affected 759 stroke-associated targets, which may influence the occurrence and development of stroke (Fig. 2 ). 3.3 Construction of the PPI network and screening of core targets The interactions between the 759 targets of TCM formulae-disease network were analyzed using the STRING database. The PPI network showed strong correlations between the targets and a complex interlaced network (Fig. 3 A). The network exhibited the top 30 nodes that were the core targets of ANP against stroke (Fig. 3 B). Among these, ubiquitin C ( UBC ), E1A binding protein P300 ( EP300 ), cellular tumor antigen p53 ( TP53 ), mitogen-activated protein kinase 1 ( MAPK1 ), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA ) were considered to be the core genes (Fig. 3 B). 3.4 GO enrichment analysis To further understand the intersecting genes, GO enrichment analysis was performed. A total of 33 GO entries for ANP in stroke were identified (Fig. 4 C). The GO function of ANP against stroke was mainly involved as follows: regulation of heart contraction, response to catecholamines, response to the steroid biosynthetic process, positive regulation of the reactive oxygen species (ROS) metabolic process, regulation of systemic arterial blood pressure, response to vitamins, regulation of calcium ion transport into the cytosol, sodium channel activity, cellular response to steroid hormone stimulus, and regulation of the blood vessel diameter (Fig. 4 A). Ligand-gated ion channel activity accounted for 21.28% of all biological functions, regulation of heart contraction accounted for 19.15%, response to catecholamines accounted for 14.89%, and cellular response to steroid hormone stimulus accounted for 12.77% (Fig. 4 B). 3.5 KEGG pathway enrichment analysis KEGG pathway enrichment analysis was performed to determine the significant effects of ANP. A total of 55 pathways were screened based on the KEGG analysis (Fig. 5 C). The main pathways included the advanced glycation end products/receptor for advanced glycation end products (AGE-RAGE) signaling pathway, which is involved in diabetic complications; calcium signaling pathway, which includes gap junctions and is involved in insulin resistance; cGMP-PKG signaling pathway; and cAMP signaling pathway (Fig. 5 A). Among them, the AGE-RAGE signaling pathway accounted for 58.18%, neurotrophin signaling pathway accounted for 9.09%, and amphetamine addiction accounted for 7.27% of all signaling pathways (Fig. 5 B). 3.6 Molecular docking Molecular docking was used to verify the binding ability of the active ingredients in ANP to the core targets of stroke and to explore the accuracy of their binding modes. In this study, we conducted molecular docking analysis of the active ingredients of ANP with UBC (PDB ID 2CLW), EP300 (PDB ID 6V8K), and TP53 (PDB ID 4D1M). In this analysis, the Vina score (binding energy) indicates the binding activity between a protein and a compound. The results showed that UBC exhibited strong binding to norwogonin (Vina score = -6.0 kcal/mol) (Fig. 6 D). Norwogonin and UBC interacted via van der Waals, Pi-donor hydrogen, Pi-cation, conventional hydrogen bonds, and Pi-alkyl interactions (Fig. 6 E). Similarly, coptisine exhibited strong binding to EP300 (Vina score = -10.6) (Fig. 7 D). Coptisine and EP300 interacted via van der Waals, alkyl, conventional hydrogen bonds, Pi-alkyl, and Pi-sigma interactions (Fig. 7 E). Musennin exhibited strong binding to TP53 (Vina score = -9.3) (Fig. 8 D). They interacted via van der Waals forces, carbon hydrogen bonds, conventional hydrogen bonds, and alkyl chain interactions (Fig. 8 E). The surface properties of the receptors (aromaticity, charge, H-bond, hydrophobic, ionizability, and solvent accessibility) indicated that active compounds bound stably with the related receptors (Figs. 6 – 8 , F–K). Briefly, norwogonin, coptisine, and musennin can effectively bind to UBC, EP300, and TP53, respectively, with low binding energies and stable conformations. 3.7 Concentration of coptisine in PC12 cells was determined According to the results of the CCK-8 assay, the proliferation ability of PC12 cells was reduced by approximately 22.56% and 37.48% at 24 h after treatment with coptisine (8 and 16 µM, respectively). This proliferation ability was further reduced by coptisine in a time-dependent manner (48 and 72 h) ( P < 0.01; Fig. 9 ). 3.8 Coptisine alleviated OGD/R by regulating the expression of EP300 and LC3B As OGD/R reduced the viability of the PC12 cells, the mechanism for this effect may be through the induction of autophagy. Thus, the detection of apoptosis-related proteins was used to investigate the potential mechanism of cell autophagy. Western blot analysis showed that coptisine at different concentrations (1, 2, and 4 µmol/L) enhanced the expression of EP300 and inhibited the expression of LC3B ( P < 0.05; Fig. 10 ). 4. Discussion Stroke is a common disease that threatens the health of middle-aged and older individuals and often has a high rate of disability or death. However, there is currently no effective treatment for this condition. TCM has been used to treat stroke for millennia in clinical practice and can be mined as a valuable resource for novel drug discovery. ANP is a formulation composed of 11 herbs ( Moschus , Margarita , Cinnabaris , Calculus bovis , Buffalo Horn , Radix Scutellariae Baicalensis , Fructus gradeniae , Realgar , Rhizoma coptidis , Borneolum synthcticum , and Radix curcumae ). In China, ANP is generally prescribed for stroke patients, and it significantly improves their behavior and decreases the rate of disability or death. In this study, we aimed to explore the main active ingredients of ANP, predict their potential gene targets and the associated signaling pathways, and explore the association between the active compounds of ANP and stroke using network pharmacology and molecular docking analysis. Here, we screened 81 active ingredients and 759 core targets of ANP that are closely associated with stroke. In the molecular docking analysis, these active ingredients were combined with UBC, EP300, and TP53, which were the top three core targets identified in the PPI analysis. Results showed that norwogonin, coptisine, and musennin showed a strong affinity for UBC, EP300, and TP53, respectively. This indicates that norwogonin, coptisine, and musennin may be effective in the treatment of stroke. Studies have shown that ubiquitin-C-terminal hydrolase, which can hydrolyze UBC, is a novel biomarker for stroke and traumatic brain injury in rats ( 16 ). Norwogonin is a naturally occurring plant flavone that has potent anticancer activity in human colon cancer, and its anticancer effects are mediated via the induction of apoptosis, autophagy, and G2/M cell cycle arrest ( 17 ). Norwogonin has also been reported to induce different apoptotic effects in human leukemia cells via ROS stimulation ( 18 ). A study showed that flavonoid-enriched extracts from Scutellaria baicalensis roots (mainly norwogonin) ameliorated influenza virus-induced acute lung injury in mice by inhibiting the excessive activation of the complement system in vivo ( 19 ). One study showed that EP300 protects neurons from damage induced by oxygen deficiency ( 20 ). This indicated that EP300 is an essential target for stroke rehabilitation. Coptisine, an isoquinoline alkaloid originally isolated from TCM, has extensive pharmacological actions, including antimicrobial ( 21 – 23 ), anti-cancer ( 24 , 25 ), and antioxidative effects ( 26 ). There have been some studies on neuropathic diseases induced by coptisine. One study showed that coptisine ameliorated cognitive impairment by inhibited indoleamine 2,3-dioxygenase activity in a mouse model of Alzheimer's disease ( 27 ). TP53 is a tumor suppressor gene that has been previously identified to promote apoptosis, affect gene stability, and inhibit tumor formation. Later studies have shown that it has additional biological functions. It has been shown that the knockdown of TP53 expression might protect against focal ischemic injury in transgenic mice ( 28 ) and that TP53 can induce apoptosis and endothelial damage, aggravating brain injury ( 29 ). However, few studies have been conducted on musennin, but our results indicate that it has a potential therapeutic effect on stroke; however, further research is required to validate this finding. Briefly, it has been suggested that norwogonin, coptisine, and musennin are possible treatments for stroke. KEGG pathway enrichment analysis showed that the key targets were mainly concentrated in the AGE-RAGE signaling pathway, calcium signaling pathway, cGMP-PKG signaling pathway, cAMP signaling pathway, and neurotrophin signaling pathway, which is involved in aldosterone synthesis and secretion, morphine addiction, neuroactive ligand-receptor interaction, spinocerebellar ataxia, amphetamine addiction, serotonergic synapse, thyroid hormone signaling, and renin secretion. The results of the PPI network analysis showed that UBC , EP300 , TP53 , MAPK1 , and PIK3CA were the core genes. Based on these results, we hypothesized that ANP has a therapeutic effect on stroke mainly through the cAMP and calcium signaling pathways. The cAMP signaling pathway is crucial for the pathogenesis of stroke; therefore, identifying compounds that activate the cAMP/PKA/CREB signaling pathway may be an effective method for the treatment of stroke ( 30 , 31 ). Endoplasmic reticulum (ER) stress also plays a role in the pathogenesis of human diseases, including ischemia-reperfusion injury ( 32 ). Studies have suggested that ER stress can activate the cAMP/PKA pathway ( 33 ). Calcium is involved in the pathogenesis of stroke ( 34 , 35 ). Moreover, disruption of IP₃R2-mediated Ca²⁺ signaling pathway in astrocytes ameliorates neuronal death and brain damage after focal ischemic stroke ( 36 ). The GO enrichment analysis also showed that the major biological processes included regulation of calcium ion transport into the cytosol. In brief, the cAMP signaling pathway and calcium signaling pathway may be the main mechanisms by which ANP treats stroke. Excessive autophagy is an important factor in the pathogenesis of cerebral ischemia-reperfusion injury ( 37 ). ANP alleviates cerebral ischemia-reperfusion injury by inhibiting autophagy ( 38 ). Coptisine alleviates myocardial ischemia-reperfusion injury by regulating the expression of apoptotic proteins ( 39 ). Whether autophagy can alleviate ischemia-reperfusion injury has not been reported. The molecular docking results showed that coptisine had a good binding ability with EP300. As an acetyltransferase protein, EP300 plays an important role in regulating transcription by remodeling chromatin during cell proliferation and differentiation and is also considered an endogenous inhibitor of autophagy ( 40 ). Our results showed that coptisine alleviated OGD/R injury in PC12 cells by regulating EP300 and LC3B expression. 5. Conclusions As there are currently no effective treatment for stroke in clinical practice, we aimed to determine the molecular mechanisms and active ingredients of ANP against stroke. For this, we employed network pharmacology, molecular docking, and cell experiment. In summary, network pharmacology showed that the main active ingredients of ANP, particularly norwogonin, coptisine, and musennin, can act on multiple targets. We demonstrated that ANP has a therapeutic effect on stroke, mainly through the cAMP and calcium signaling pathways. Molecular docking analysis showed that norwogonin, coptisine, and musennin strongly bind to UBC, EP300, and TP53, respectively. Coptisine alleviated OGD/R injury by regulating EP300 and LC3B expression, indicating that coptisine may be effective as a treatment for stroke. Declarations Disclosure The authors declare no conflicts of interest in this work. Funding This work was supported by the postdoctoral Foundation of Guangdong Medical University (2020WTSCX031);Guangdong Provincial Department of Science and Technology ( 2023A1414020048 );Guangdong Medical University “Clinical + Basic” Project ( 4SG23281G ) Author Contribution J.C. and L.Z. conceived and designed the experiments; J.C. and W. L. wrote the paper; J.C. performed the experiments; C.H. and X.C. analyzed the data; J.C. and L.Z. were responsible for writing, reviewing and editing the manuscript, as well as conceptualization and funding acquisition for the research. All authors have read and agreed to the published version of the manuscript. Acknowledgements We are grateful for the financial support of the postdoctoral Foundation of Guangdong Medical University (2020WTSCX031);Guangdong Provincial Department of Science and Technology ( 2023A1414020048 );Guangdong Medical University “Clinical + Basic” Project ( 4SG23281G ). 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Yang et al, “The IDO inhibitor coptisine ameliorates cognitive impairment in a mouse model of Alzheimer's disease,” J Alzheimers Dis, vol.43, no.1, pp.291–302, 2015. R.C. Crumrine, A.L. Thomas, P.F. Morgan, “Attenuation of p53 expression protects against focal ischemic damage in transgenic mice,” J Cereb Blood Flow Metab, vol.14, no.6, pp.887–891, 1994. J. Li, G. Chen, X. Gao et al, “p53 participates in the protective effects of ischemic post-conditioning against OGD-reperfusion injury in primary cultured spinal cord neurons,” Neurosci Lett, vol. 638, pp.129–134, 2017. Y.-L. Wang, X.-L. Zhu, M.-H. Sun et al, “Effects of astaxanthin onaxonal regeneration via cAMP/PKA signaling pathway in mice with focal cerebral infarction,” Eur Rev Med Pharmacol Sci,vol.23, no.3 Suppl, pp.135–143, 2019. A.-R. Wang, M.-Z. Hu, Z.-L. Zhang et al, “Fastigial nucleus electrostimulation promotes axonal regeneration after experimental stroke via cAMP/PKA pathway,” Neurosci Lett,vol.699, pp.177–183, 2019. C. Yao, J. Zhang, G. Liu et al, “Neuroprotection by (-)-epigallocatechin-3-gallate in a rat model of stroke is mediated through inhibition of endoplasmic reticulum stress,” Mol Med Rep, vol. 9, pp.69–72, 2014. J. Deng, S. Liu, L. Zou et al, Lipolysis response to endoplasmic reticulum stress in adipose cells,” J Biol Chem, vol.287, no.9, pp. 6240–6249, 2012. P. Münzer, B. Walker-Allgaier, S. Geue et al, PDK1 Determines Collagen-Dependent Platelet Ca2 + Signaling and Is Critical to Development of Ischemic Stroke In Vivo,” Arterioscler Thromb Vasc Biol,vol.36, no.8, pp.1507–1516,2016. D. Stegner, S. Hofmann, M.K. Schuhmann et al, Loss of orai2-Mediated capacitative Ca2 + entry is neuroprotective in acute ischemic stroke,” Stroke, vol.50, no.11, pp.3238–3245,2019. H. Li, Y. Xie, N. Zhang et al, “Disruption of IP₃R2-mediated Ca²⁺ signaling pathway in astrocytes ameliorates neuronal death and brain damage while reducing behavioral deficits after focal ischemic stroke,” Cell Calcium, vol.58, no.6, pp.565–576, 2015. Mu Q, Zhou H, Xu Y, He Q, Luo X, Zhang W, Li H. NPD1 inhibits excessive autophagy by targeting RNF146 and wnt/β-catenin pathway in cerebral ischemia-reperfusion injury. J Recept Signal Transduct Res. 2020;40(5):456–463. Wu Chenghan, Shi Lin, Du Jiancheng, LIAO Yuansheng, Yang Ruiling, Li Shiju, GAO Lili, Wang Jianmin, Lin Jushan, Wu Songying. Effect of Angong Niuhuang Pill on the expression of autophagy protein LC3 in peripheral hematoma nerve cells of rats with intracerebral hemorrhage [A]. Chinese Medical Association, Neurology Branch of Chinese Medical Association. Proceedings of the 7th National Conference on Neurology of The Chinese Medical Association [C]. Chinese Medical Association, Neurology Branch of Chinese Medical Association: Chinese Medical Association,2014:1. Sun S, Wang P. Coptisine alleviates ischemia/reperfusion-induced myocardial damage by regulating apoptosis-related proteins. Tissue Cell. 2020;66:101392. Pietrocola F, Lachkar S, Enot DP, Niso-Santano M, Bravo-San Pedro JM, Sica V, Izzo V, Maiuri MC, Madeo F, Mariño G, Kroemer G. Spermidine induces autophagy by inhibiting the acetyltransferase EP300. Cell Death Differ. 2015;22(3):509–16. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4759284","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":332033285,"identity":"ada4074c-ac5e-45e3-91c1-32588eb87fe9","order_by":0,"name":"JU CHEN","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYDACCTC2YWA4AOKxEa8ljVQtDAyHSdDCP7v52APLtvN2fdfOGDB8KDsMFGkgYMmdY+kGkm23k2fezjFgnHHuMFDkAH4tBhI5ZhIgLQZALcy8bYeBIgmEtOR/A2o5B9HylzgtOWxALQfswFoYidEicSPNTELiXHKC5O20goM959J5JG4Q0MI/I/mZtESZnT3f7eSND36UWcvxzyCgBQSYgXGT2MAAiRoewuqBgPEDA4M9USpHwSgYBaNgZAIA3xlCC1pBhu4AAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"JU","middleName":"","lastName":"CHEN","suffix":""}],"badges":[],"createdAt":"2024-07-18 02:06:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4759284/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4759284/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62529032,"identity":"fd3fd9fb-ac40-46b6-81cb-cde58411dc31","added_by":"auto","created_at":"2024-08-15 12:03:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":306631,"visible":true,"origin":"","legend":"\u003cp\u003eTCM-disease target. Note: Drug represent ANP, Disease represent stroke.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/dcb1f7b2eb81af2d5bea4baa.png"},{"id":62529034,"identity":"8eafe108-1a18-4b5b-993a-1161568905dd","added_by":"auto","created_at":"2024-08-15 12:03:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":445662,"visible":true,"origin":"","legend":"\u003cp\u003eThe regulatory network of TCM formulae-ingredient-disease-target. The purple rectangle represents the common targets, the cyan oval represents the active ingredients, and the blue rhomboid represents the TCM.The purple rectangle represented the common targets, the cyan oval represented the active ingredients, and the blue rhomboid represented TCM.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/5e70c31b5434e26123f90621.png"},{"id":62529033,"identity":"77ece908-75b1-4700-8434-1ac582fc9ae0","added_by":"auto","created_at":"2024-08-15 12:03:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":336778,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of TCM formulae-disease target protein interaction (PPI) network and screening of core target. A. TCM formulae-disease target PPI network. B. Barplot of TCM formulae-disease core target. Note: Network nodes represented proteins, edges represented protein-protein associations, the thicker the line, the higher the edge confidence and the tighter the relationship between proteins (A). The left column represent gene and the right column represent the number of adjacent nodes (B).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/ca063629e7d74ea79c127fdb.png"},{"id":62529785,"identity":"a09e4c5f-c0dc-4642-9d4f-e4410875e216","added_by":"auto","created_at":"2024-08-15 12:11:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":952365,"visible":true,"origin":"","legend":"\u003cp\u003eGO functional enrichment analysis. A. GO functional enrichment analysis. B. The percentage of genes in each group. C. The percentage of genes in each term. Note: The GO function (P≤GO functional enrichment analysis. A. GO functio font; Red font represent target (A).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/f4690d8b083dbfd87da5547d.png"},{"id":62529786,"identity":"2e2a31e2-b1a3-42ed-b2d6-3b28bb202b0d","added_by":"auto","created_at":"2024-08-15 12:11:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":619311,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG pathway enrichment analysis. A. KEGG pathway enrichment analysis. B. The percentage of genes in each group. C. The percentage of genes in each term. Note: The KEGG pathway function (P≤0.05) was marked with different colors and large font; Red font represent target (A).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/b282c3112c0dbe3fe6d5aa48.png"},{"id":62529040,"identity":"094aee6a-d144-4650-93d4-957b928e35e1","added_by":"auto","created_at":"2024-08-15 12:03:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":776498,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking models of norwogonin with UBC. A. 3D structure of norwogonin; B. Cartoon pattern of UBC; C. Binding site of norwogonin with UBC; D. Molecular docking of norwogonin with UBC; E. 2D diagram of receptor-ligand interactions; F. Aromatic at receptor surfaces; G. H-Bonds at receptor surfaces; H. Interpolated charge at receptor surfaces; I. Hydrophobicity at receptor surfaces; J. Ionizability at receptor surfaces; K. SAS at receptor surfaces.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/8481898c04ca57cc111553e1.png"},{"id":62529038,"identity":"ac171a20-784e-4f2c-bab2-36f840c8e29a","added_by":"auto","created_at":"2024-08-15 12:03:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":978090,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking models of coptisine with EP300. A. Cartoon pattern of coptisine; B. Cartoon pattern of EP300; C. Binding site of coptisine with EP300; D. Molecular docking of coptisine with EP300; E. 2D diagram of receptor-ligand interactions; F. Aromatic at receptor surfaces; G. H-Bonds at receptor surfaces; H. Interpolated charge at receptor surfaces; I. Hydrophobicity at receptor surfaces; J. Ionizability at receptor surfaces; K. SAS at receptor surfaces.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/33357d5ead5e72de76be92d7.png"},{"id":62529042,"identity":"93b1a0a2-24ab-43ef-85fb-200c9ce9d1e9","added_by":"auto","created_at":"2024-08-15 12:03:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1059098,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking models of musennin with TP53. A. Cartoon pattern of musennin; B. Cartoon pattern of TP53; C. Binding site of musennin with TP53; D. Molecular docking of musennin with TP53; E. 2D diagram of receptor-ligand interactions; F. Aromatic at receptor surfaces; G. H-Bonds at receptor surfaces; H. Interpolated charge at receptor surfaces; I. Hydrophobicity at receptor surfaces; J. Ionizability at receptor surfaces; K. SAS at receptor surfaces.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/1aad3a45208605113f2a7fa1.png"},{"id":62529035,"identity":"ccfd60ed-beee-40ca-9857-adaea9f5f59d","added_by":"auto","created_at":"2024-08-15 12:03:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":85394,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of coptisine on PC12 cells. The cell proliferation of PC12 cell was respectively measured at 24, 48 and 72 h by CCK8, which affected with coptisine (2, 4, 8, and 16 μmol/L). \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, vs. coptisine ( 0 μmol/L) .\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/ae047f31f656023cd20afefa.png"},{"id":62529041,"identity":"aed49c38-e450-45c6-930f-8b1f6e351d61","added_by":"auto","created_at":"2024-08-15 12:03:54","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":193218,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of coptisine on autophagy. (A) The expression of EP300 and LC3B of PC12 cells (WB bands figure); (B) The cell proliferation of PC12 cell was respectively measured at 24h of OGD/R by CCK8, which affected with coptisine (1, 2, and 4 μmol/L). \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, vs. Model. (C) The expression of EP300 and LC3B of PC12 cells (Bar charts)\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/ba06bb0d88d37130f77450f6.png"},{"id":62531040,"identity":"b3325e39-d310-4a67-bcec-fb298802393d","added_by":"auto","created_at":"2024-08-15 12:27:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7209555,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4759284/v1/4c3533b5-7c7b-45b2-aed1-7e9ceec2dd56.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unveiling the Molecular Mechanisms and Active Components of Angong Niuhuang Pills in Stroke Treatment: Network Pharmacology, Molecular Docking, and Cellular Studies","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStroke is the world's second most common cause of death and the third most common cause of disability (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Although the age-adjusted mortality rate has declined in recent years, the total number of people affected by stroke-related disability and death has risen, with developing countries bearing the greatest burden(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The increasing prevalence of stroke in young people is alarming (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Angong Niuhuang pill (ANP) is a Chinese patent medicine composed of \u003cem\u003eMoschus\u003c/em\u003e, \u003cem\u003eMargarita\u003c/em\u003e, \u003cem\u003eCinnabaris\u003c/em\u003e, \u003cem\u003eCalculus bovis\u003c/em\u003e, \u003cem\u003eBuffalo Horn\u003c/em\u003e, \u003cem\u003eRadix Scutellariae Baicalensis\u003c/em\u003e, \u003cem\u003eFructus gradeniae\u003c/em\u003e, \u003cem\u003eRealgar\u003c/em\u003e, \u003cem\u003eRhizoma coptidis\u003c/em\u003e, \u003cem\u003eBorneolum synthcticum\u003c/em\u003e, and \u003cem\u003eRadix curcumae\u003c/em\u003e (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In China, ANP is generally prescribed for patients with cerebral diseases, such as hypertensive cerebral hemorrhage, hypoxic-ischemic encephalopathy, viral encephalitis, severe craniocerebral trauma, and cerebral paralysis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, the molecular mechanisms and active ingredients of ANP against stroke have not been fully elucidated. In addition, the clinical application of ANP is limited because of the presence of two known toxic metalloids, arsenic and mercury, in ANP (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Therefore, it is important to determine the active components and molecular mechanisms of ANP in the treatment of stroke.\u003c/p\u003e \u003cp\u003eTo understand traditional Chinese medicine (TCM) formulas (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) and identify potential new drugs or targets for specific diseases (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), network pharmacology combined with multidisciplinary technologies, such as systems biology and computational biology, is conducted. Molecular docking in TCM is a computer-based drug design technology that simulates the geometric structure of molecules and the interaction forces between molecules through stoichiometric calculation methods (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In recent years, network pharmacology has been successfully used to predict the mechanisms of TCM in the treatment of a variety of diseases. Therefore, in this study, we aimed to use network pharmacology and molecular docking to explore the main active components of ANP, predict their potential targets and signaling pathways, and explore the association between the active ingredients of ANP and pathogenesis of stroke. Finally, the virtual results were preliminarily verified using oxygen glucose deprivation/reperfusion (OGD/R) modeling in PC12 cells. To the best of our knowledge, this is the first study to identify the molecular mechanisms and active ingredients of ANP against stroke using network pharmacology, molecular docking, and cell experiment.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.1 Identification of active ingredients and targets in ANP\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTraditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://lsp.nwu.edu.cn/tcmsp.php\u003c/span\u003e\u003cspan address=\"http://lsp.nwu.edu.cn/tcmsp.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to authenticate active ingredients of the six Chinese medicinal herbs (\u003cem\u003eCalculus bovis\u003c/em\u003e, \u003cem\u003eRadix Scutellariae Baicalensis\u003c/em\u003e, \u003cem\u003eFructus gradeniae\u003c/em\u003e, \u003cem\u003eRhizoma coptidis\u003c/em\u003e, \u003cem\u003eBorneolum synthcticum\u003c/em\u003e, and \u003cem\u003eRadix curcumae\u003c/em\u003e) in ANP. TCMSP is a unique platform for analysis of active ingredients in TCM and their interaction in specific diseases. The oral bioavailability (OB) and drug-likeness (DL) are important pharmacokinetic parameters used to identify and screen active ingredients based on the absorption, distribution, metabolism, and excretion model. In this study, compounds with OB\u0026thinsp;\u0026gt;\u0026thinsp;30% and DL\u0026thinsp;\u0026gt;\u0026thinsp;0.18 were selected (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), and the relevant targets were identified. The active ingredients of the other four herbs (\u003cem\u003eMoschus\u003c/em\u003e, \u003cem\u003eMargarita\u003c/em\u003e, \u003cem\u003eBuffalo Horn\u003c/em\u003e, and \u003cem\u003eRealgar\u003c/em\u003e) were retrieved from the Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bionet.ncpsb.org/batman-tcm/\u003c/span\u003e\u003cspan address=\"http://bionet.ncpsb.org/batman-tcm/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which is the first online bioinformatics analysis tool for screening of the molecular mechanisms of TCM (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In this study, the compounds with a score\u0026thinsp;\u0026ge;\u0026thinsp;20 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were selected, and the targets with correlative compounds were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Predicting the targets of stroke\u003c/h2\u003e \u003cp\u003eThe GeneCards database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to obtain information on the stroke-associated target genes, which were identified by entering the keyword \u0026ldquo;stroke.\u0026rdquo; Finally, the target genes of ANP and stroke were intersected to obtain the common target genes. Venny 2.1, an online graphics software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinfogp.cnb.csic.es/tools/venny/index.html\u003c/span\u003e\u003cspan address=\"http://bioinfogp.cnb.csic.es/tools/venny/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), was used to match and map the targets of ANP and stroke, and a Venn diagram of this analysis was constructed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Construction of the TCM formulae-compound-disease-target regulation network\u003c/h2\u003e \u003cp\u003eThe Cytoscape software (version 3.6.1, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cytoscape.org\u003c/span\u003e\u003cspan address=\"http://www.cytoscape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to construct a TCM formulae-compound-disease-target regulation network. The formulae, compound, disease, and target regulation network nodes in the network represented the active ingredients, drugs, diseases, and key target genes, respectively. Edges were used to connect the drugs with the active ingredients, the active ingredients with the key target genes, and the diseases with the key target genes. The active ingredients of ANP shown in this network were related to stroke-associated targets as key compounds against stroke. The whole network showed a relationship between the drug, disease, active ingredients, and targets, and the mechanism of action of ANP against stroke was explored by constructing a regulatory network.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.4 Protein-protein interaction (PPI) analysis and screening of core targets\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo clarify the interaction between ANP-related and stroke-associated targets, we screened for common targets that overlap between ANP-related and stroke target-related targets. The common targets of ANP were entered into STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/cgi/input.pl\u003c/span\u003e\u003cspan address=\"https://string-db.org/cgi/input.pl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to construct the PPI network interaction by screening conditions with medium confidence (0.4) and defined species as \u003cem\u003eHomo sapiens\u003c/em\u003e (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The resulting files were input into the R language for calculation, and a bar diagram of the core target was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses\u003c/h2\u003e \u003cp\u003eTo further reveal the potential mechanism of ANP in the treatment of stroke, we imported overlapping targets into the ClueGO plug-in of Cytoscape software to analyze the GO function and KEGG pathways with the following screening conditions: a kappa score\u0026thinsp;=\u0026thinsp;0.4 and the species as \u003cem\u003eHomo sapiens\u003c/em\u003e. Only pathways with a pV\u0026thinsp;\u0026le;\u0026thinsp;0.05 were shown. The threshold for enrichment analysis was set to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.6 Molecular docking simulation\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe protein crystal structures of the core genes was downloaded from the Protein Database Bank (PDB, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the proteins were selected with ligands to ensure docking accuracy. The 3D structure of the active ingredients were downloaded in PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Vina software (version 1.1.2) was used for the molecular docking analysis. Finally, the conformation with the lowest score was selected and plotted with PyMOL 2.4 for the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell culture\u003c/h2\u003e \u003cp\u003ePC12 cells were obtained from Tongpai Biotechnology Co., Ltd. (Shanghai, China). These PC12 cells were cultured in Dulbecco's modified eagle medium (DMEM) (Gibco, USA) containing 10% (v/v) fetal bovine serum (Gibco, USA) and placed in an incubator at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 OGD/R model\u003c/h2\u003e \u003cp\u003eThe cell culture medium was removed, and the cells were washed three times with PBS and deoxygenated glucose-free DMEM for 1 h to induce the OGD. The cells were maintained in fetal bovine serum and deoxygenated glucose-free DMEM and cultured at 37℃ in an oxygen-free chamber with 95% N\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e for 3 h. The medium was then replaced with glucose-containing DMEM containing 10% fetal bovine serum and incubated at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cell viability assessment\u003c/h2\u003e \u003cp\u003eCell viability was detected using the Cell Counting Kit-8 (CCK-8) (Beyotime, Beijing, China). The PC12 cells were cultured in 96-well plates at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well and incubated for 24 h. Coptisine (0, 2, 4, 8, and 16 \u0026micro;mol/L) was then added. After culturing for 24, 48, and 72 h, 10 \u0026micro;L CCK-8 reagent was added. The cells were then incubated at 37℃ for 2 h, and the optical density was measured at 450 nm using a microplate spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western blot analysis\u003c/h2\u003e \u003cp\u003eThe PC12 cells were cultured in 96-well plates at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well and incubated for 24 h. After the OGD/R treatment, coptisine (1, 2, and 4 \u0026micro;mol/L) was added. The cells were incubated at 37℃ for 24 h. Cell viability was detected using the CCK-8 assay. The total protein was isolated using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The protein concentration was measured using a bicinchoninic protein assay kit (Pierce, Rockford, IL, USA). The total protein (50 \u0026micro;g) was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime, Shanghai, China) and transferred to a polyvinylidene fluoride membrane. The membrane was sealed with 5% skim milk powder for 2 h, followed by incubation with primary anti-EP300 (1:1000, Amyjet Scientific, China) and LC3B (1:1000, Cell Signaling, USA) at 4℃ overnight. β-actin (1:2000; Beyotime, China) was used as an internal reference. Goat anti-rabbit IgG (1:1000, Beyotime, China) was added to the membrane and incubated at room temperature (20\u0026ndash;24 ℃) for 1 h. Finally, an enhanced chemiluminescence technique was used for the western blot assay kit (Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe Statistical Package of the Social Sciences software (version 16.0; SPSS, Inc., Chicago, USA) was used for statistical analysis of the experimental data, and the data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The statistical significance of differences was determined using one-way analysis of variance. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was used to indicate statistically significant differences.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1 Active ingredients and targets of ANP\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA total of 88 active ingredients and 1168 targets were retrieved from the TCMSP and BATMAN-TCM databases (after deduplication). These active ingredients primarily originated from \u003cem\u003eRhizoma coptidis\u003c/em\u003e (11 compounds and 90 targets), \u003cem\u003eRadix Scutellariae Baicalensis\u003c/em\u003e (31 compounds and 53 targets), \u003cem\u003eCalculus bovis\u003c/em\u003e (5 compounds and 4 targets), \u003cem\u003eRadix curcumae\u003c/em\u003e (3 compounds and 29 targets), \u003cem\u003eFructus gradeniae\u003c/em\u003e (11 compounds and 59 targets), \u003cem\u003eBorneolum synthcticum\u003c/em\u003e (2 compounds and 32 targets), \u003cem\u003eMoschus\u003c/em\u003e (26 compounds and 1027 targets), \u003cem\u003eBuffalo Horn\u003c/em\u003e (3 compounds and 41 targets), \u003cem\u003eRealgar\u003c/em\u003e (1 compound and 67 targets), \u003cem\u003eMargarita\u003c/em\u003e (5 compounds and 33 targets), and \u003cem\u003eCinnabaris\u003c/em\u003e (1 compound and 0 target). The main active ingredients of ANP are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of screening active components and targets of ANP by TCMSP database and Batmann-TCM database\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTCM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of component\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of target\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRhizoma Coptidis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRadix Scutellariae Baicalensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCalculus Bovis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRadix Curcumae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFructus Gradeniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBorneolum Synthcticum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMoschus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBuffalo Horn\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRealgar\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMargarita\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCinnabaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal(deduplication)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1168\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Construction of the TCM formulae-disease-target network\u003c/h2\u003e \u003cp\u003eIn total, 7408 stroke-associated targets were identified. These 7408 targets were combined with 1168 targets of ANP to obtain a total of 759 core targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The 81 active ingredients of ANP mainly affected 759 stroke-associated targets, which may influence the occurrence and development of stroke (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Construction of the PPI network and screening of core targets\u003c/h2\u003e \u003cp\u003eThe interactions between the 759 targets of TCM formulae-disease network were analyzed using the STRING database. The PPI network showed strong correlations between the targets and a complex interlaced network (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The network exhibited the top 30 nodes that were the core targets of ANP against stroke (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Among these, ubiquitin C (\u003cem\u003eUBC\u003c/em\u003e), E1A binding protein P300 (\u003cem\u003eEP300\u003c/em\u003e), cellular tumor antigen p53 (\u003cem\u003eTP53\u003c/em\u003e), mitogen-activated protein kinase 1 (\u003cem\u003eMAPK1\u003c/em\u003e), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (\u003cem\u003ePIK3CA\u003c/em\u003e) were considered to be the core genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 GO enrichment analysis\u003c/h2\u003e \u003cp\u003eTo further understand the intersecting genes, GO enrichment analysis was performed. A total of 33 GO entries for ANP in stroke were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The GO function of ANP against stroke was mainly involved as follows: regulation of heart contraction, response to catecholamines, response to the steroid biosynthetic process, positive regulation of the reactive oxygen species (ROS) metabolic process, regulation of systemic arterial blood pressure, response to vitamins, regulation of calcium ion transport into the cytosol, sodium channel activity, cellular response to steroid hormone stimulus, and regulation of the blood vessel diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Ligand-gated ion channel activity accounted for 21.28% of all biological functions, regulation of heart contraction accounted for 19.15%, response to catecholamines accounted for 14.89%, and cellular response to steroid hormone stimulus accounted for 12.77% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 KEGG pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eKEGG pathway enrichment analysis was performed to determine the significant effects of ANP. A total of 55 pathways were screened based on the KEGG analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The main pathways included the advanced glycation end products/receptor for advanced glycation end products (AGE-RAGE) signaling pathway, which is involved in diabetic complications; calcium signaling pathway, which includes gap junctions and is involved in insulin resistance; cGMP-PKG signaling pathway; and cAMP signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Among them, the AGE-RAGE signaling pathway accounted for 58.18%, neurotrophin signaling pathway accounted for 9.09%, and amphetamine addiction accounted for 7.27% of all signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Molecular docking\u003c/h2\u003e \u003cp\u003eMolecular docking was used to verify the binding ability of the active ingredients in ANP to the core targets of stroke and to explore the accuracy of their binding modes. In this study, we conducted molecular docking analysis of the active ingredients of ANP with UBC (PDB ID 2CLW), EP300 (PDB ID 6V8K), and TP53 (PDB ID 4D1M). In this analysis, the Vina score (binding energy) indicates the binding activity between a protein and a compound. The results showed that UBC exhibited strong binding to norwogonin (Vina score = -6.0 kcal/mol) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Norwogonin and UBC interacted via van der Waals, Pi-donor hydrogen, Pi-cation, conventional hydrogen bonds, and Pi-alkyl interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Similarly, coptisine exhibited strong binding to EP300 (Vina score = -10.6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Coptisine and EP300 interacted via van der Waals, alkyl, conventional hydrogen bonds, Pi-alkyl, and Pi-sigma interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Musennin exhibited strong binding to TP53 (Vina score = -9.3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). They interacted via van der Waals forces, carbon hydrogen bonds, conventional hydrogen bonds, and alkyl chain interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). The surface properties of the receptors (aromaticity, charge, H-bond, hydrophobic, ionizability, and solvent accessibility) indicated that active compounds bound stably with the related receptors (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, F\u0026ndash;K). Briefly, norwogonin, coptisine, and musennin can effectively bind to UBC, EP300, and TP53, respectively, with low binding energies and stable conformations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Concentration of coptisine in PC12 cells was determined\u003c/h2\u003e \u003cp\u003eAccording to the results of the CCK-8 assay, the proliferation ability of PC12 cells was reduced by approximately 22.56% and 37.48% at 24 h after treatment with coptisine (8 and 16 \u0026micro;M, respectively). This proliferation ability was further reduced by coptisine in a time-dependent manner (48 and 72 h) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Coptisine alleviated OGD/R by regulating the expression of EP300 and LC3B\u003c/h2\u003e \u003cp\u003eAs OGD/R reduced the viability of the PC12 cells, the mechanism for this effect may be through the induction of autophagy. Thus, the detection of apoptosis-related proteins was used to investigate the potential mechanism of cell autophagy. Western blot analysis showed that coptisine at different concentrations (1, 2, and 4 \u0026micro;mol/L) enhanced the expression of EP300 and inhibited the expression of LC3B (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eStroke is a common disease that threatens the health of middle-aged and older individuals and often has a high rate of disability or death. However, there is currently no effective treatment for this condition. TCM has been used to treat stroke for millennia in clinical practice and can be mined as a valuable resource for novel drug discovery. ANP is a formulation composed of 11 herbs (\u003cem\u003eMoschus\u003c/em\u003e, \u003cem\u003eMargarita\u003c/em\u003e, \u003cem\u003eCinnabaris\u003c/em\u003e, \u003cem\u003eCalculus bovis\u003c/em\u003e, \u003cem\u003eBuffalo Horn\u003c/em\u003e, \u003cem\u003eRadix Scutellariae Baicalensis\u003c/em\u003e, \u003cem\u003eFructus gradeniae\u003c/em\u003e, \u003cem\u003eRealgar\u003c/em\u003e, \u003cem\u003eRhizoma coptidis\u003c/em\u003e, \u003cem\u003eBorneolum synthcticum\u003c/em\u003e, and \u003cem\u003eRadix curcumae\u003c/em\u003e). In China, ANP is generally prescribed for stroke patients, and it significantly improves their behavior and decreases the rate of disability or death. In this study, we aimed to explore the main active ingredients of ANP, predict their potential gene targets and the associated signaling pathways, and explore the association between the active compounds of ANP and stroke using network pharmacology and molecular docking analysis.\u003c/p\u003e \u003cp\u003eHere, we screened 81 active ingredients and 759 core targets of ANP that are closely associated with stroke. In the molecular docking analysis, these active ingredients were combined with UBC, EP300, and TP53, which were the top three core targets identified in the PPI analysis. Results showed that norwogonin, coptisine, and musennin showed a strong affinity for UBC, EP300, and TP53, respectively. This indicates that norwogonin, coptisine, and musennin may be effective in the treatment of stroke. Studies have shown that ubiquitin-C-terminal hydrolase, which can hydrolyze UBC, is a novel biomarker for stroke and traumatic brain injury in rats (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Norwogonin is a naturally occurring plant flavone that has potent anticancer activity in human colon cancer, and its anticancer effects are mediated via the induction of apoptosis, autophagy, and G2/M cell cycle arrest (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Norwogonin has also been reported to induce different apoptotic effects in human leukemia cells via ROS stimulation (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). A study showed that flavonoid-enriched extracts from \u003cem\u003eScutellaria baicalensis\u003c/em\u003e roots (mainly norwogonin) ameliorated influenza virus-induced acute lung injury in mice by inhibiting the excessive activation of the complement system \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). One study showed that EP300 protects neurons from damage induced by oxygen deficiency (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). This indicated that EP300 is an essential target for stroke rehabilitation. Coptisine, an isoquinoline alkaloid originally isolated from TCM, has extensive pharmacological actions, including antimicrobial (\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), anti-cancer (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and antioxidative effects (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). There have been some studies on neuropathic diseases induced by coptisine. One study showed that coptisine ameliorated cognitive impairment by inhibited indoleamine 2,3-dioxygenase activity in a mouse model of Alzheimer's disease (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). \u003cem\u003eTP53\u003c/em\u003e is a tumor suppressor gene that has been previously identified to promote apoptosis, affect gene stability, and inhibit tumor formation. Later studies have shown that it has additional biological functions. It has been shown that the knockdown of TP53 expression might protect against focal ischemic injury in transgenic mice (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and that TP53 can induce apoptosis and endothelial damage, aggravating brain injury (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). However, few studies have been conducted on musennin, but our results indicate that it has a potential therapeutic effect on stroke; however, further research is required to validate this finding. Briefly, it has been suggested that norwogonin, coptisine, and musennin are possible treatments for stroke.\u003c/p\u003e \u003cp\u003eKEGG pathway enrichment analysis showed that the key targets were mainly concentrated in the AGE-RAGE signaling pathway, calcium signaling pathway, cGMP-PKG signaling pathway, cAMP signaling pathway, and neurotrophin signaling pathway, which is involved in aldosterone synthesis and secretion, morphine addiction, neuroactive ligand-receptor interaction, spinocerebellar ataxia, amphetamine addiction, serotonergic synapse, thyroid hormone signaling, and renin secretion. The results of the PPI network analysis showed that \u003cem\u003eUBC\u003c/em\u003e, \u003cem\u003eEP300\u003c/em\u003e, \u003cem\u003eTP53\u003c/em\u003e, \u003cem\u003eMAPK1\u003c/em\u003e, and \u003cem\u003ePIK3CA\u003c/em\u003e were the core genes. Based on these results, we hypothesized that ANP has a therapeutic effect on stroke mainly through the cAMP and calcium signaling pathways. The cAMP signaling pathway is crucial for the pathogenesis of stroke; therefore, identifying compounds that activate the cAMP/PKA/CREB signaling pathway may be an effective method for the treatment of stroke (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Endoplasmic reticulum (ER) stress also plays a role in the pathogenesis of human diseases, including ischemia-reperfusion injury (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Studies have suggested that ER stress can activate the cAMP/PKA pathway (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Calcium is involved in the pathogenesis of stroke (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Moreover, disruption of IP₃R2-mediated Ca\u0026sup2;⁺ signaling pathway in astrocytes ameliorates neuronal death and brain damage after focal ischemic stroke (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The GO enrichment analysis also showed that the major biological processes included regulation of calcium ion transport into the cytosol. In brief, the cAMP signaling pathway and calcium signaling pathway may be the main mechanisms by which ANP treats stroke.\u003c/p\u003e \u003cp\u003eExcessive autophagy is an important factor in the pathogenesis of cerebral ischemia-reperfusion injury (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). ANP alleviates cerebral ischemia-reperfusion injury by inhibiting autophagy (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Coptisine alleviates myocardial ischemia-reperfusion injury by regulating the expression of apoptotic proteins (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Whether autophagy can alleviate ischemia-reperfusion injury has not been reported. The molecular docking results showed that coptisine had a good binding ability with EP300. As an acetyltransferase protein, EP300 plays an important role in regulating transcription by remodeling chromatin during cell proliferation and differentiation and is also considered an endogenous inhibitor of autophagy (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Our results showed that coptisine alleviated OGD/R injury in PC12 cells by regulating EP300 and LC3B expression.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eAs there are currently no effective treatment for stroke in clinical practice, we aimed to determine the molecular mechanisms and active ingredients of ANP against stroke. For this, we employed network pharmacology, molecular docking, and cell experiment. In summary, network pharmacology showed that the main active ingredients of ANP, particularly norwogonin, coptisine, and musennin, can act on multiple targets. We demonstrated that ANP has a therapeutic effect on stroke, mainly through the cAMP and calcium signaling pathways. Molecular docking analysis showed that norwogonin, coptisine, and musennin strongly bind to UBC, EP300, and TP53, respectively. Coptisine alleviated OGD/R injury by regulating EP300 and LC3B expression, indicating that coptisine may be effective as a treatment for stroke.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDisclosure\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest in this work.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the postdoctoral Foundation of Guangdong Medical University (2020WTSCX031);Guangdong Provincial Department of Science and Technology (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2023A1414020048\u003c/span\u003e);Guangdong Medical University \u0026ldquo;Clinical\u0026thinsp;+\u0026thinsp;Basic\u0026rdquo; Project (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4SG23281G\u003c/span\u003e)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.C. and L.Z. conceived and designed the experiments; J.C. and W. L. wrote the paper; J.C. performed the experiments; C.H. and X.C. analyzed the data; J.C. and L.Z. were responsible for writing, reviewing and editing the manuscript, as well as conceptualization and funding acquisition for the research. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe are grateful for the financial support of the postdoctoral Foundation of Guangdong Medical University (2020WTSCX031);Guangdong Provincial Department of Science and Technology (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2023A1414020048\u003c/span\u003e);Guangdong Medical University \u0026ldquo;Clinical\u0026thinsp;+\u0026thinsp;Basic\u0026rdquo; Project (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4SG23281G\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization, \u0026ldquo;Health statistics and information systems,\u0026rdquo; 2019, available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/healthinfo/global_burden_disease/en/\u003c/span\u003e\u003cspan address=\"https://www.who.int/healthinfo/global_burden_disease/en/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR.V. 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Geue et al, PDK1 Determines Collagen-Dependent Platelet Ca2\u0026thinsp;+\u0026thinsp;Signaling and Is Critical to Development of Ischemic Stroke In Vivo,\u0026rdquo; Arterioscler Thromb Vasc Biol,vol.36, no.8, pp.1507\u0026ndash;1516,2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. Stegner, S. Hofmann, M.K. Schuhmann et al, Loss of orai2-Mediated capacitative Ca2\u0026thinsp;+\u0026thinsp;entry is neuroprotective in acute ischemic stroke,\u0026rdquo; Stroke, vol.50, no.11, pp.3238\u0026ndash;3245,2019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Li, Y. Xie, N. Zhang et al, \u0026ldquo;Disruption of IP₃R2-mediated Ca\u0026sup2;⁺ signaling pathway in astrocytes ameliorates neuronal death and brain damage while reducing behavioral deficits after focal ischemic stroke,\u0026rdquo; Cell Calcium, vol.58, no.6, pp.565\u0026ndash;576, 2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMu Q, Zhou H, Xu Y, He Q, Luo X, Zhang W, Li H. NPD1 inhibits excessive autophagy by targeting RNF146 and wnt/β-catenin pathway in cerebral ischemia-reperfusion injury. J Recept Signal Transduct Res. 2020;40(5):456\u0026ndash;463.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Chenghan, Shi Lin, Du Jiancheng, LIAO Yuansheng, Yang Ruiling, Li Shiju, GAO Lili, Wang Jianmin, Lin Jushan, Wu Songying. Effect of Angong Niuhuang Pill on the expression of autophagy protein LC3 in peripheral hematoma nerve cells of rats with intracerebral hemorrhage [A]. Chinese Medical Association, Neurology Branch of Chinese Medical Association. Proceedings of the 7th National Conference on Neurology of The Chinese Medical Association [C]. Chinese Medical Association, Neurology Branch of Chinese Medical Association: Chinese Medical Association,2014:1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun S, Wang P. Coptisine alleviates ischemia/reperfusion-induced myocardial damage by regulating apoptosis-related proteins. Tissue Cell. 2020;66:101392.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePietrocola F, Lachkar S, Enot DP, Niso-Santano M, Bravo-San Pedro JM, Sica V, Izzo V, Maiuri MC, Madeo F, Mari\u0026ntilde;o G, Kroemer G. Spermidine induces autophagy by inhibiting the acetyltransferase EP300. Cell Death Differ. 2015;22(3):509\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Angong Niuhuang pill, Network pharmacology, Molecular docking, Stroke, Coptisine","lastPublishedDoi":"10.21203/rs.3.rs-4759284/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4759284/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction: \u003c/strong\u003eAngong Niuhuang pills (ANPs) exhibit a curative effect in patients with stroke, but its main effective components remain unexplored. Here, we aims to elucidate the molecular mechanisms and active ingredients of ANPs against stroke through network pharmacology, molecular docking, and cellular experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The compounds and targets of each herb in the ANP were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) databases. The genes corresponding to the targets were retrieved from the GeneCards database. A traditional Chinese medicine (TCM) formulae-compound-disease-target network was constructed using Cytoscape. A protein-protein interaction network was constructed using the STRING database. The core targets of ANP were analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using the ClueGO plug-in of Cytoscape. The active ingredients of ANP were docked to the top three core targets. Finally, we explored the effects of coptisine on the oxygen-glucose deprivation/reperfusion (OGD/R) models of PC12 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe TCM formulae-compound-disease-target network contained 81 active ingredients and 759 core targets. We identified 47 GO entries of ANP types for stroke, and 55 pathways were screened based on GO and KEGG pathway analyses. Core targets were mainly involved in biological processes (GO enrichment analysis, p \u0026lt; 0.05), including the regulation of heart contraction, muscle contraction, and steroid metabolic processes. In the KEGG pathway, the core targets were mainly involved in AGE-RAGE signaling pathway in diabetic complications, neurotrophin signaling pathway, and cGMP-PKG signaling pathway. Molecular docking results showed that norwogonin, coptisine, and musennin had a high affinity for ubiquitin C (UBC), E1A binding protein P300 (EP300), and cellular tumor antigen p53 (TP53), respectively. Coptisine alleviates OGD/R injury by regulating EP300 and LC3B expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThis study, using network pharmacology, molecular docking analysis, and cellular experiments, provides insights into the potential mechanisms and active ingredients of ANPs in stroke protection. The identification of core target genes and signaling pathways suggests that coptisine could be a promising candidate for treating cerebral ischemia-reperfusion injury.\u003c/p\u003e","manuscriptTitle":"Unveiling the Molecular Mechanisms and Active Components of Angong Niuhuang Pills in Stroke Treatment: Network Pharmacology, Molecular Docking, and Cellular Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-15 12:03:48","doi":"10.21203/rs.3.rs-4759284/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"19341f21-82e4-45bc-b49a-809e4d5eb3f0","owner":[],"postedDate":"August 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35156453,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":35156454,"name":"Biological sciences/Drug discovery"},{"id":35156455,"name":"Biological sciences/Molecular biology"},{"id":35156456,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2024-08-15T12:03:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-15 12:03:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4759284","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4759284","identity":"rs-4759284","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}