Ilex kudingcha and common hypertension-related targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted, and molecular docking was performed to verify molecular dynamic simulations. Six active components in Ilex kudingcha were identified; furthermore, 123 target genes common to hypertension were identified. Topological analysis revealed the strongly associated proteins, with RELA, AKT1, JUN, TP53, TNF, and MAPK1 being the predicted targets of the studied traditional Chinese medicine. In addition, GO enrichment analysis revealed significant enrichment of biological processes such as oxidative stress, epithelial cell proliferation, cellular response to chemical stress, response to xenobiotic stimulus, and wound healing. Furthermore, KEGG enrichment analysis revealed that the genes were particularly enriched in lipid and atherosclerosis, fluid shear stress and atherosclerosis, and other pathways. Molecular docking revealed that the key components in Ilex kudingcha exhibited good binding potential to the target genes RELA, AKT1, JUN, TP53, TNF, and IL-6. Our study results suggest that Ilex kudingcha plays a role in hypertension treatment by exerting hypolipidemic, anti-inflammatory, and antioxidant effects and inhibiting the transcription of atherosclerosis-related genes.Herein, network pharmacology was used to identify the active components in  Globally, approximately 1 billion individuals suffer from hypertension; it is directly responsible for 13% of all fatalities . The useIlex species exhibit a broad geographic range, and some species have been used to develop daily herbal tea blends. Ilex kudingcha, a Chinese herbal tea, possesses many health-improving properties  to screen the active ingredients in Ilex kudingcha. The DrugBank database (https://www.drugbank.ca/) and peer-reviewed literature were used to identify the expected targets of the tested compounds. Furthermore, UniProt (https://www.uniprot.org/) was used to standardize gene names and compare target information. The Ilex kudingcha–ingredient–target regulatory network was developed using Cytoscape 3.9.1 software.The parameters oral bioavailability (OB) of 30% and drug-like (DL) characteristic of 0.18 p were used in the Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform (2.2https://genecards.org). The targets with relevance scores of ≥1 were chosen based on the keyword “hypertension.” The hypertension-related targets were identified after weight removal and consolidation.The hypertension-related targets with high relevance were chosen using GeneCards . The species was limited to humans and a minimum interaction score of 0.9 was used. The key targets were imported into the Cytoscape program. The interaction network diagram of the target proteins of the active ingredients in Ilex kudingcha and hypertension-related target proteins was developed by sorting based on degree value.The intersection targets of 2.5https://www.kegg.jp/) was also used (doi: 10.18129/ http://b9.bioc.org.Hs.eg.db). For the parameters of the two functions, species was set to “has,” and the filter values  were set to 0.05. A bubble graph was prepared to visualize the top 10 enrichment findings, and Cytoscape 3.9.1 was used to develop the KEGG regulatory network.R 4.2.2 with the “colorspace,” “stringi,” and “ggplot2” packages was installed. The “DOSE,” “clusterProfiler,” and “annotationHub” Bioconductor packages were used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. For GO enrichment analysis, the function “enrichGO” was employed. For KEGG enrichment analysis, the database org.Hs.eg.db and the “enrich-KEGG” tool were used. The KEGG database (2.6https://www.rcsb.org) for the target genes implicated in the first eight protein–protein interaction (PPI) findings. The primary active ingredients in Ilex kudingcha were retrieved in a two-dimensional (2D) structure format from the PubChem website. The sdf files were converted into the pdb file format using Discovery Studio 2019 software. PyMOL 4.3.0 software was used to separate the original ligand and protein structures and to dehydrate and remove the organics. Furthermore, AutoDock Vina 1.1.2 software was used to process the proteins as follows: non-polar hydrogen was added, the Gasteiger charge was calculated, the AD4 type was assigned, and the flexible bonds of small molecules/ligands were set to be rotatable. Based on the original ligand coordinates, the docking box was adjusted to include all protein structures. Furthermore, the receptor protein was set to a semiflexible butt joint, and the Lamarckian genetic algorithm was selected. The docking results were obtained by running autogrid4 and autodock4; as a result, the binding energies were revealed. PyMOL 4.3.0 and Discovery Studio 2019 software were used to analyze and visualize the forces in 3D and 2D.Three-dimensional (3D) protein conformations with a crystal resolution of <3, as determined using X-ray crystal diffraction, were gathered by searching the Protein Data Bank (PDB)  simulation. The AMBER force field was used to describe the proteins and ligand molecules. The water model was set to the SPC water model. The system temperature was set to a vacuum environment of 300 K, and the simulation time was 100 ns. First, the energy balance of the simulated system was determined by using the steepest gradient algorithm under the absolute vacuum environment. After energy balance, MD simulation was completed; thereafter, root mean square deviation (RMSD) analysis of the simulation results was conducted to analyze the relative binding stability between the two chains in the simulation process. Furthermore, root mean square fluctuation (RMSF), which demonstrates the structural adaptability of each protein residue, was used to analyze the flexibility and intensity of movement of the amino acid residues of the protein throughout the simulation. The radius of gyration (Rg) was used to characterize the compactness of the protein structure and changes in the looseness of the peptide chain during the simulation. VMD software was used to analyze the changes in the number of hydrogen bonds formed between the two chains over time as well as to observe whether the bond was stable from the interaction point of view.33.1Ilex kudingcha. They were screened using an OB of ≥30% and a DL of 0.18. Subsequently, six active ingredients were obtained , cellular component (CC), and molecular function (MF). BP was mainly associated with response to oxidative stress, epithelial cell proliferation, cellular response to chemical stress, response to xenobiotic stimulus, and wound healing. CC was mainly associated with the membrane raft, membrane microdomain, vesicle lumen, secretory granule lumen, cytoplasmic vesicle lumen, and plasma membrane raft. MF was mainly associated with DNA-binding transcription factor binding, RNA polymerase II-specific DNA-binding transcription factor binding, signaling receptor activator activity, receptor–ligand activity, cytokine receptor binding, and cytokine activity Figure 6Ilex kudingcha was mainly concentrated in lipid and atherosclerosis, fluid shear stress and atherosclerosis, the AGE-RAGE signaling pathway in diabetic complications, human cytomegalovirus infection, the TNF signaling pathway, chemical carcinogenesis receptor activation, hepatitis B, prostate cancer, and Chagas disease  and kaempferol (PubChem CID: 5280863) were downloaded from PubChem. Furthermore, the 3.6TNF primarily via the formation of hydrophobic forces and hydrogen bond forces. It binds to the amino acid residues VAL-17, PRO- 20, and ARG- 32 of the receptor protein via four hydrophobic bonds and to ALA- 18, ARG- 32, GLY- 148, GLN- 149, VAL- 150, and amino acid residues of the receptor protein via seven hydrogen bonds /AKT via human T-lymphotropic virus type 1 signaling pathways. The leukemia/lymphoma virus inhibits the production of several proteins that are the hallmarks of epithelial–mesenchymal transition, such as Slug, N-cadherin, E-cadherin, and Snail and indicators of metastasis, including matrix metallopeptidase 2 , leading to obesity and insulin resistance, followed by artery dysfunction and hypertension (TNF and IL-6 are two proinflammatory cytokines associated with atherosclerosis, plaque development, and increased cardiovascular risk (The core genes were obtained based on the PPI network, and the main targets were rtension , 31. TNFlar risk . In the Ilex kudingcha were exerted via signaling cytokine receptor binding, cytokine activity, receptor–activator activity, and receptor–ligand activity binding of transcription factors, which may have affected membrane rafts, membrane microdomains, cytoplasmic vesicle lumens, secretory granule lumens, plasma membrane rafts, and vesicle lumens, thus contributing to cellular responses to oxidative stress, epithelial cell proliferation, and biological processes including wound healing, reaction to xenobiotic stimulation, and chemical stress. The KEGG enrichment analysis indicated that the action mechanism of Ilex kudingcha in non-hypertensive atherosclerosis was primarily associated with lipids, fluid shear stress, and human cytomegalovirus infection, the AGE–RAGE signaling pathway in diabetes complications, and TNF activation of the chemical carcinogenesis signaling pathway. The lipid and atherosclerosis map (Ilex kudingcha acted on many targets and formed an interactive relationship with atherosclerosis, fluid shear stress, and MAPK, TNF, and PI3K/AKT signaling pathways, all of which helped regulate oxidative stress and vascular hardness. TNF inhibits inflammatory responses while reducing blood pressure and excreting salt through the kidneys (PI3K/AKT signaling pathway controls the cytoskeletal rearrangement and phenotypic transformation of arterial smooth muscle cells, affects the excitability of sympathetic nerves and the function of vascular endothelial cells, and antagonizes angiotensin II (in- vivo experiments have shown that the Ilex kudingcha extract reduces atherosclerosis in apoE-deficient mice by decreasing cholesterol buildup in macrophages (Ilex kudingcha may possess considerable therapeutic value for treating hypercholesterolemia and atherosclerosis (Ilex kudingcha exerts potent anti-inflammatory effects on LPS-induced inflammatory responses by inhibiting NF-κB and MAPK pathways (Dioscorea opposita Thunb., a common staple food in China, exerts antihypertensive effects by inhibiting the endothelin-converting enzyme and antioxidant activity in 2K1C hypertensive rats (TNF with quercetin and kaempferol was −8.8 kcal/mol, followed by −8.4 kcal/mol of MARKI with quercetin. The docking result was less than −7.0 kcal/mol, indicating that the ligand and receptor exhibited strong binding activity (TNF and quercetin. MD simulation has been widely used in the biomedical field to study conformational transformations caused by protein mutations or ligand binding/debinding. It provides some findings that are difficult to obtain in traditional biochemical or pathological experiments, such as the detailed effects of mutations on protein structures and protein–protein/ligand interactions at the atomic level (GO enrichment was performed to identify disease–drug intersection genes. The findings revealed that the biological effects of the active ingredients of osis map Figure 8 kidneys . The PI3ensin II , 37. Dysensin II  and induensin II –41. The ensin II . A consiensin II . Severalrophages . Consideclerosis . Ilex kupathways . Dioscorive rats . Furtheractivity . The MD ic level . MD simuic level .5Ilex kudingcha can play a role in lowering blood pressure via multiple components, targets, and pathways. This study provides insights into how the hypolipidemia-, antioxidation-, and atherosclerosis-related gene inhibitory effects of Ilex kudingcha are associated with its mode of action in treating hypertension. The action targets include RELA, AKT1, TNF, IL-6, and MARK1. Studies that are pertinent to these findings rely on data from already-existing databases and lack experimental validation. Therefore, further studies are required to verify the accuracy of the present findings in vitro and in vivo.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.FL wrote and revised the manuscript and constructed the tables and figures. MY and RH revised the manuscript. YQ and YH conceived the study and revised the manuscript. All authors have read and approved the final manuscript. Data authentication is not applicable.


IL1B, GSK3B, NOS3, RELA and CDK4. GO analysis results suggested that the anti-colorectal cancer effect of geniposide by regulating oxidative stress is possibly related to the influence of multiple biological processes, including response to temperature stimulus, response to alkaloid, nitric oxide biosynthetic process, nitric oxide metabolic process, reactive nitrogen species metabolic process, cellular response to peptide, etc. KEGG enrichment analysis results indicated that the PI3K–Akt signaling pathway, IL-17 signaling pathway, p53 signaling pathway, NF-κB signaling pathway and NOD-like receptor signaling pathway are likely to be the significant pathways. Molecular docking results showed that the geniposide had a good binding activity with the hub genes. This study demonstrates that geniposide can regulate oxidative stress in CRC, and induction of oxidative stress is one of the possible mechanisms of anti-recurrence and metastasis effects of geniposide against CRC.This study aims to identify the mechanism of geniposide regulating oxidative stress in colorectal cancer (CRC) through network pharmacology and bioinformatics analysis. Targets of geniposide, oxidative stress-related targets and targets related to CRC were applied from databases. The hub genes for geniposide regulating oxidative stress in CRC were identified with the protein–protein interaction (PPI) network. Furthermore, we applied Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment to analyze the hub genes from a macro perspective. We verified the hub genes by molecular docking, GEPIA, HPA and starBase database. We identified five hub genes:  Colorectal cancer is one of the top three malignancies with the highest mortality rate today . ClinicaGardenia jasminoides Ellis (GjE), belonging to the family Rubiaceae [17H24O10), a well-known iridoid glycoside compound, is one of the main bioactive components of FG [Fructus Gardeniae (FG) is the dried fruit of ubiaceae . Geniposts of FG . Previouts of FG . Geniposts of FG  and gastts of FG . Though Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and the body’s antioxidant defenses. This imbalance contributes to chronic inflammation, which can lead to precancerous conditions. ROS can influence cellular functions, including gene regulation and apoptosis, making oxidative stress a significant factor in cancer development ,11,12. NIn this study, network pharmacology was used to identify the pharmacologic target network of geniposide, and bioinformatics was used to compile information on the relevant targets of colorectal cancer. The intersection targets were subjected to GO and KEGG enrichment analysis. To clarify the pathophysiology of colorectal cancer and offer a fresh perspective on clinical intervention therapy, we verified the hub genes by using molecular docking, GEPIA, HPA, and the starBase database. The study flowchart is shown in We retrieved 102, 473, 24 and 7 geniposide-related targets, respectively, through the Swiss Target Prediction, TargetNet, CTD (Comparative Toxicogenomics Database) and TCMSP databases . After merging and deduplicating the related targets of the four databases, 548 geniposide-related targets in total were obtained. Subsequently, we used the Cytoscape software to construct a network diagram of geniposide and its related targets, as shown in To identify the hub gene modules associated with CRC, we used the WGCNA algorithm to establish co-expression networks and modules in healthy people and CRC patients. We calculated the variance of the expression of each gene in GSE44076 and then selected the top 25% of genes with the most significant variance for further analysis. Co-expressed gene modules were identified when the soft power value was set to 10, and the scale-free R2 equaled 0.9, as illustrated in DNMT1, HSP90AA1, PTPN1, ESR1, PTK2, RELA, CDK4, CDK5, ADRB1, MAPKAPK2, TRPV1, CDK2, NOS3, JAK2, MIF, GSK3B, ACHE, MMP3, NR3C1, HSP90AB1, ADRB3, MMP1, BCL2, ELANE, HDAC2, RAC1, PRKCB, BCL2L1, GAPDH, HSPA8, HSPA5, CHEK1, FOXO1, IL1B. Then, we constructed a compound–disease–targets network diagram using the Cytoscape software, as illustrated in In order to explore the role of geniposide regulating oxidative stress against colorectal cancer, we intersected the targets of geniposide with the most critical module genes and oxidative stress-related genes in CRC, and obtained a total of 34 core genes as shown in We imported the above 34 core genes into the String database to obtain the PPI network between the core genes, as shown in p-value < 0.05), and the main category was the biological process (BP), cell composition (CC), and molecular function (MF), as illustrated in Through the “cluster profiler” package of the R language software, the GO function enrichment analysis of the 34 core genes of geniposide anti-colorectal cancer by regulating oxidative stress was carried out. A total of 1582 GO entries were obtained  in which geniposide interfered with CRC, mainly involving pathways in the cancer signaling pathway, PI3K–Akt signaling pathway, IL-17 signaling pathway, p53 signaling pathway, NF-κB signaling pathway, and NOD-like receptor signaling pathway, as shown in By utilizing the “cluster profiler” package of the R language software, the KEGG pathway enrichment analysis of the 34 core genes of geniposide on CRC was carried out. The results showed 87 signal pathways . As shown in IL1B, GSK3B, NOS3, RELA, and CDK4) are above 15, which were selected as hub genes for subsequent analysis. The functional enrichment of GO and KEGG suggested that the mechanism of geniposide’s anti-colorectal cancer regulating oxidative stress may be related to cell pyroptosis, and oxidative stress is one of the vital induction methods of cell pyroptosis. In order to explore the correlation between hub genes and pyroptotic genes, we conducted a correlation analysis between hub genes and pyroptotic hotspot genes . The results showed that 1L1B, CDK4 and RELA are closely related to pyroptosis hotspot genes, as shown in As shown in GSK3B. In the ceRNA network, 11 lncRNAs can combine with miRNA hsa-miR-495-3p, hsa-miR-326, and hsa-miR-4303 to regulate the hub gene IL1B. miRNA hsa-miR-4318, hsa-miR-4280, hsa-miR-103b, hsa-miR-506-3p, hsa-miR-198, hsa-miR-568, hsa-miR-149-3p, and hsa-miR- 486-5p bind to regulate the hub gene CDK4. miRNA hsa-miR-4318, hsa-miR-4254, hsa-miR-761, hsa-miR-4297, hsa-miR-3202, hsa-miR-1303, hsa-miR-4324, and hsa-miR-214-3p bind to regulate the hub gene NOS3. In addition, 18 lncRNAs could combine with has-miR-156-5p, hsa-miR-858-3p and hsa-miR-7-5p to regulate the hub gene RELA.We constructed a ceRNA network based on hub genes with starBase, the miRDB database and the miRnada database. The network includes 319 nodes  . In detap-value < 0.05, as shown in IL1B high expression group was statistically significant, as illustrated in IL1B gradually decreased while the mRNA levels of NOS3 gradually increased, as shown in IL1B, which had no corresponding data in the database  between the two groups. Among them, Eosinophil, Macrophages M2, Plasma cells, T cells CD4 memory resting, T cells CD8, T cells gamma delta, T cells regulatory (Tregs) were the most invasive immune cells in the CRC group. The M1/M2 ratio imbalance had significance in the formation of malignancies, immune escape, and subsequent metastatic treatment resistance. The relationship between the hub genes and immune cells is shown in The difference in immune microenvironment between the normal group and the CRC group was analyzed by CYBERSORT. The difference in immune cell content between the normal group and the CRC group can be visually seen in IL1B_1I1B: The binding energy between the small molecule and the receptor was −6.2kcal/mol, which demonstrated that it had a good interaction force and formed hydrogen bond associations mainly with GLY A49 and LYS A103, as shown in GSK3B_1J1B: The binding energy between the small molecule and the receptor was −7.5 kcal/mol, which demonstrated that it had a good binding effect. Small molecules interacted with receptor proteins, mainly forming hydrogen bonds with ASN A152, GLNA151, ARGA107, ILEA28, and LYS A103, as shown in NOS3_3E7S: The binding energy between the small molecule and the receptor was −7.0 kcal/mol, which indicated that it has a good binding effect. Small molecules interacted with receptor proteins, mainly with GLUA284, ARGA106, TRPA370, and TRPA279, and obvious hydrogen bond interactions were found, as shown in RELA_1NFI: The binding energy of the small molecule to the receptor was −6.3 kcal/mol, which indicated that it had a good binding effect. Small molecules interacted with receptor proteins mainly through the formation of hydrogen bonds and hydrophobic bond forces, including hydrogen bonds THRA164, ASNA131, and GLUE92, as shown in CDK4_3G33: The binding energy of the small molecule to the receptor was −6.4 kcal/mol, which indicated that it had a good binding effect. Small molecules interacted with receptor proteins, including hydrogen bonds formed by GLUB74 and GLUD175, as shown in Results of The prevalence of colorectal cancer has been rising annually in recent years because of changes in human living habits, and the age at which it occurs is getting younger. In the past 10 years, more and more natural products have been confirmed to have anti-tumor effects. Geniposide is the primary active component in the herbal remedy Gardenia jasminoides used in traditional Chinese medicine. It has activities in anti-inflammatory, antimicrobial, anti-tumor, anti-oxidation, and immunological regulatory, according to preliminary research. Oxidative stress is produced when the ratio of reactive oxygen species (ROS) production to antioxidant effectiveness is out of balance. Excessive ROS synthesis can cause cellular damage and is one of the factors resulting in many disorders, including cancer. This study aims to explore the molecular mechanism of geniposide in the treatment of colorectal cancer by regulating oxidative stress and the bioinformatics analysis of its hub genes to provide a reference for the development of new treatment strategies. Through the Swiss Target Prediction, TargetNet, CTD and TCMSP databases, we collected 548 targets of geniposide. Then, we searched the CRC dataset in the GEO database and used the WGCNA algorithm to establish the co-expression network and modules of healthy people and CRC patients and a collection of 5157 genes with positive correlations among different CRC samples was identified. Since the occurrence of oxidative stress is closely related to the occurrence and development of malignant tumors, we need to explore the potential mechanism of whether geniposide can treat CRC by regulating oxidative stress. We employed the GSEA website to collect 855 oxidative stress-related genes and took the intersection of the three to obtain the hub genes of geniposide treatment on CRC.In addition, GO enrichment analysis showed that the biological functions mainly involved positive regulation of response to temperature stimulus, response to alkaloid, nitric oxide biosynthetic process, nitric oxide metabolic process, reactive nitrogen species metabolic process, cellular response to peptide, and so on. From the enrichment results of GO, the synthesis and metabolism of nitric oxide and the metabolic process of reactive nitrogen were significantly enriched. Nitric oxide (NO) is synthesized by many cell types involved in immunity and inflammation. It also controls the functional activity, growth, and death of a wide range of immunological and inflammatory cell types, including Macrophages, T lymphocytes, antigen-presenting cells, Mast cells, Neutrophils, and natural killer cells . Inflammp-value < 0.05) in which geniposide-regulated oxidative stress intervened in CRC, mainly involving pathways in the cancer signaling pathway, PI3K–Akt signaling pathway, IL-17 signaling pathway, p53 signaling pathway, NF-κB signaling pathway and NOD-like receptor signaling pathway. Despite the fact that the PI3K–AKT signaling pathway governs various cellular processes such as differentiation, metabolism, survival, and apoptosis, its abnormal activation contributes to the development of colorectal cancer malignancy [KEGG pathway enrichment analysis showed that there were 87 signaling pathways  is a transcription factor in the nuclear factor κB (NF-κB) family [CDK4 is one of the cell cycle regulators that controls the G1 to S phase of the cell cycle. Because multiple cancer treatments may reduce CDK4 expression, this protein has been regarded as a critical target protein in a variety of malignancies [CDK4 is overexpressed in cancer [CDK4 overexpression improves clinical treatment efficacy for breast cancer, melanoma, liposarcoma, and mantle cell lymphoma [NOS3 is known to be involved in NO synthesis, primarily in endothelial cells, and is linked to cardiovascular disorders such as hypertension, atherosclerosis, and diabetes mellitus [NOS3 inhibits apoptosis and promotes angiogenesis, proliferation, invasiveness, and immunosuppression in malignant tumors [NOS3 levels were found to be inversely associated with progression-free survival and overall survival (OS) in patients with metastatic colorectal cancer [Our PPI network analysis results showed that g tumors . Conside general . In contlignancy . RELA(P6) family ,33. NF-κgnancies . Severaln cancer ,36 and ilymphoma , which slymphoma . NOS3 ismellitus . It has t tumors . Furtherl cancer .IL-1B is produced and secreted by a range of tumor cell types, including immune cells, fibroblasts, and cancer cells. It has also been connected to a wide range of physiological activities. It has the capacity to control cytokine production and gene expression as well as cellular adhesion and migration, angiogenesis, and immune response [IL-1B often encourages the growth and invasion of cancer cells as well as neo-angiogenesis and immune cells that infiltrate tumors [IL-1B and IL-1A [IL1B as a potential method to treat colorectal cancer, it is important to further examine the correlations between IL1B polymorphism and prognosis. In summary, in this study, we employed four databases—Swiss Target Prediction, Target Net, CTD, and TCMSP—to identify genes and targets associated with geniposide. Each of these databases has its unique strengths and coverage, and their combined use ensures a more comprehensive and accurate target identification. GO and KEGG enrichment analyses provided useful insights into the biological processes and signaling pathways these genes may be involved in. Notably, enrichment results in cancer signaling pathways and the PI3K–Akt signaling pathway may suggest potential mechanisms of geniposide’s anti-CRC effects. Molecular docking results indicated possible interaction mechanisms between geniposide and its primary targets, providing direction for subsequent in vitro and in vivo experiments. However, these docking results are predictive and will require experimental validation through techniques such as X-ray crystallography or NMR in the future. Our data showed a significant increase in certain immune cells, such as M2 Macrophages and Tregs, in the CRC group. These cells are generally associated with immune suppression and cancer progression. These results suggest that geniposide may exert its anti-cancer effects by modulating the immune response in the tumor microenvironment. Despite the promising findings, our study has several limitations that should be acknowledged. First, the target identification relies heavily on in silico databases, which might not encompass all possible interactions or biological activities of geniposide. Second, our work is predominantly computational and lacks experimental validation. Molecular docking results are purely predictive and should be confirmed through in vitro and in vivo experiments. Third, while our study points to alterations in immune cell populations within the CRC tumor microenvironment, the mechanistic relationship between geniposide and immune modulation remains unclear and warrants further investigation. Lastly, our analyses were based on publicly available datasets, the quality and comprehensiveness of which we could not control. Future work should aim to address these limitations, perhaps by employing a wider variety of databases, conducting laboratory experiments for validation, and exploring the impact of geniposide on immune cells through more targeted assays.Hub gene response . Additioe tumors . Furthernd IL-1A . Furthernd IL-1A . Inhibitnd IL-1A . Therefop-value < 0.05). With the evolution of pathological stages (Stage I to Stage IV), the mRNA expression of IL1B gradually decreased, while the mRNA expression of NOS3 gradually increased. The above analysis results were basically consistent with literature reports. Geniposide displayed high binding activity with the hub genes, as demonstrated in molecular docking results. We discovered that the mRNA levels of five hub genes were significantly expressed in CRC tissues, and the prognostic value of ILIB was significantly different  to search for geniposide-related SMILES numbers and used Swiss Target Prediction databases  and TargetNet database  [https://ctdbase.org/, accessed on 2 July 2023) [https://old.tcmsp-e.com/tcmsp.php, accessed on 2 July 2023) [http://www.uniprot.org/, accessed on 2 July 2023) [We utilized PubChem (ly 2023)  with thely 2023)  and the ly 2023)  with “gely 2023)  was usedhttp://www.ncbi.nlms.nih.gov/geo, accessed on 3 July 2023) [We took “Colorectal cancer” as keywords in Gene Expression Omnibus   to retriTo explore whether the potential mechanism of geniposide in the treatment of CRC is related to oxidative stress, we collected 855 oxidative stress- related genes from previous literature . Then, whttps://cn.string-db.org/, version 11.5, accessed on 5 July 2023). The protein–protein interaction network (PPI) was constructed with a minimum required interaction score ≥ 0.4 as the screening condition. The PPI network diagram and tsv file were downloaded. To further explore the biological functions of core genes, GO gene enrichment analysis and KEGG signaling pathway analysis were performed and visualized on core genes by using the “clusterProfiler” , “org.Hs.eg.db” , and “ggplot2”  packages in R software to explore the main molecular biological processes and signaling pathways of potential targets of geniposide. Finally, the compound–disease–therapeutic targets-core pathways network diagram was constructed using Cytoscape software  with nodes in the network representing compound, disease, targets, and pathways. Nodes were connected by edges that represented different meanings depending on the ways the network was built.The above-screened core genes were imported into the String database . Finally, a correlation analysis was performed on hub genes and hotspot pyroptosis-related genes.After 34 core genes were imported into Cytoscape software, the degree value was calculated, and hub genes were selected according to their degree value. To further understand the potential role of geniposide in the treatment of hub genes against CRC, we analyzed the correlations between hub genes and hotspot genes of pyroptosis. First, we collected 44 pyroptosis-related genes from previous study  Subsequehttp://starbase.sysu.edu.cn, accessed on 5 July 2023), miRDB database  and miRanda database  and miRNA–lncRNAs were screened to obtain the ceRNA network of mRNA–miRNA–lncRNAs. Among them, the Cytoscape software was used for visual analysis.StarBase database (uly 2023 ) were ushttps://string-db.org/, accessed on 7 July 2023) and used “minimum required interaction score ≥ 0.4” as the screening condition to construct a PPI network, download and save the PPI network diagram and tsv format file. We used the “limma” package  in R software to analyze the differential expression of hub genes in CRC. Then, the hub genes were input into the online tool GEPIA  [https://www.proteinatlas.org/, accessed on 7 July 2023) [We imported the hub genes screened above into the String database (ly 2023)  to verifly 2023) .http://cibersortx.stanford.edu, accessed on 8 July 2023) [We utilized the CIBERSORT (ly 2023)  algorithhttps://www.rcsb.org/, accessed on 10 July 2023), corresponding proteins were downloaded as PDB format files and imported into PyMol software  to remove water molecules and small molecules. The protein was imported into the autodocktool software  for hydroprocessing and saved as a PDBQT format file. Finally, PyMol and MOE software  were used for molecular docking and visualization analysis [Geniposide was imported into Chem3D software  to construct its chemical structure formula and minimize its energy, then saved as a PDB format file. The hub genes were brought into the PDB database  were most likely to be involved in the effects of oxidative stress brought on by geniposide in the prevention of colorectal cancer. The underlying mechanisms through which geniposide curbs colorectal cancer effects were the regulation of the multiple BP, including response to temperature stimulus, response to alkaloid, nitric oxide biosynthetic process, nitric oxide metabolic process, reactive nitrogen species metabolic process, cellular response to peptide, and so on. We identified six critical pathways  involved in the treatment of colorectal cancer with geniposide by regulating oxidative stress. In this research, we successfully determined the molecular pathways of geniposide-regulated oxidative stress anti-colorectal cancer therapy. This study also demonstrated that hub genes (Hence, our findings showed a synergistic effect between various anti-colorectal cancer core targets, numerous molecular pathways, and geniposide treatment for colorectal cancer. The findings of molecular docking revealed that geniposide had a regulatory effect on the anti-colorectal cancer core targets. Further bioinformatic analysis showed the impact of hub genes on disease prognosis and other aspects. Furthermore, the results from molecular docking, bioinformatics, and network pharmacology screening were all consistent, proving the validity of network pharmacology in this study. These results, therefore, provide a basis for the future development of geniposide-based anti-colorectal cancer medications.


Aonyx cinereus (A. cinereus), from Yogyakarta, Indonesia. Six domesticated A. cinereus adults were obtained from a local otter breeder in Yogyakarta, with no regard to sex. The animals were acclimated to the laboratory for one day, following this, the animals underwent macroscopy identification and scanning electron microscopy (SEM) and light microscopy (LM) analysis. Macroscopically, the tongue of domesticated A. cinereus is divided into three parts: the apex, corpus, and radix. The apex is the shortest part and can move freely. A median groove is bent along the corpus. Typically, the radix contains circumvallate papillae and the epiglottic valleculae. The SEM and LM observations revealed that the lingual morphology of A. cinereus consisted of two types of papillae: mechanical papillae  and gustatory papillae . The lingual glands consisted of Weber’s glands and von Ebner’s glands secreting acid and neutral mucins. Collagen fibres are found in the lamina propria and muscular layer. In conclusion, the papillae of the Asian short-clawed otter have the same structure as those of other Mustelidae family members.This study aimed to observe the lingual morphology of the domesticated Asian small-clawed otter,  Aonyx cinereus (A. cinereus), more commonly known to locals as berang-berang, is a semi-aquatic animal that is an excellent swimmer. This animal is found in Indonesia and in the south-eastern and eastern regions of Asia. A. cinereus belongs to the order Carnivora. A. cinereus has recently been adopted as a pet in Indonesia to replace dogs and cats due to its physical characteristics, which are similar to those of cats. This phenomenon increases the changes in the habitual behaviour of these animals and shifts the life pattern of A. cinereus. Originally a wild animal, it is becoming domesticated. Interestingly, this change in habitat encourages changes in the dietary patterns of A. cinereus. As the entrance organ to the digestive tract, the tongue of A. cinereus may be significantly impacted by changes in habitat  have been performed. Therefore, this study aimed to describe the morphological lingual surface of the domesticated A. cinereus in Yogyakarta, Indonesia.Recently, the large variation in the structures of the oral cavity, mainly in the lingua of the carnivora, has been related to adaptation to feeding methods, different types of food as well as the climate and habitat. Many studies concerning the dorsal structure of the lingual have been performed on carnivores, including golden Japanese weasels , sea ottA. cinereus adults were obtained from local specialised otter breeders in Yogyakarta, Indonesia, with no regard to sex. The animals were obtained from a pet pond using the fish trap method and transported to the laboratory in an air-conditioned car equipped with ad libitum water. The animals were then run through a health check-up and acclimated for one day before being euthanised. Each animal was anaesthetised using 10 mg/kg body weight (b.w.) ketamine  and 2 mg/kg b.w. xylazine , after which perfusion was performed. The species analysis was performed in the Laboratory of Animal Systems, Faculty of Biology, Gadjah Mada University.Six domesticated A. cinereus used in this study do not appear on any conservation list in Indonesia and are not protected by the national laws of the Republic of Indonesia.The This research study was approved by the Ethics Clearance Committee of the Faculty of Veterinary Medicine, Gadjah Mada University (No. 0146/EC-FKH/Int/2019).The tongue of each animal was taken by first disjoining the articulatio temporomandibularis. The basis of the tongue was cut through the frenulum and the epiglottis. The tongue samples were rinsed with 0.9% NaCl . Macroscopic observations were conducted on the entire sample using a Canon EOS 7000 digital camera .Four tongue samples were then fixed with 0.5% glutaraldehyde , 1.5% formaldehyde, and 100 g HEPES  in 100 ml phosphate buffered saline for 24–48 hours. The samples were then rinsed with 0.9% NaCl  and dehydrated using a graded series of ethanol, vacuum dried  and sputter coated  with platinum before being examined using Scanning Electron Microscopy (SEM)  at an accelerating voltage of 15 kV.To measure the length of the papillae, ImageJ software v1.53t was used . The SEM images were opened in the software; then, the measurement scale was equalised to the scale bar. Subsequently, a sample of papillae was selected, and a straight line was drawn from the tip to the base of the papilla to measure its length. To measure the width and diameter of the papilla, a straight line was drawn from end to end on the widest side of the papilla. The same steps were undertaken with a total sample of 20 papillae, measurements were performed a minimum of 3 times per papilla to minimise the error; then, the mean and standard deviation of each result was calculated. The measurements of each type of papillae are presented in Two organ samples were fixed in a 4% paraformaldehyde solution  at a pH 7.4 for 48 h, and then trimmed accordingly to the apex, body and radix of the tongue. Subsequently, the tissue was sequentially processed by dehydration using graded ethanol ; which was then cleared and embedded in a paraffin solution  and then cut, using a rotary microtome , with a thickness of 8 μm. The sample slides were then separately stained with histology staining using a haematoxylin and eosin (H&E) staining kit  and a Masson-Goldner trichrome staining kit , as well as histochemical staining using alcian blue (AB) staining at a pH 2.5  and periodic acid-Schiff (PAS) .Observations of the staining results of the samples were made using light microscopy (LM) with the microscope connected to Optilab software . For the LM analysis, × 40 magnification was applied to all the samples.A. cinereus is divided into three regions: the apex, corpus, and radix  and periodic acid-Schiff (PAS) can illuminate this. The AB staining of von Ebner’s glands showed a weak positive (+) result . Weber’sThe PAS staining showed positive results in all the glands found in the radix of the tongue. The von Ebner’s glands were coloured purple, which showed a moderate positive reaction (++) . Weber’sA. cinereus tongue samples, the collagen intensity from the apex to the radix did not change.The collagen present in the connective tissue produces a blue-green colour in the Masson-Goldner trichrome staining. Collagen is abundant in the lamina propria under the tongue’s papillae and the textus muscularis striatus syncytialis tissue between the muscle fibres with a lower intensity . In the In mammals, the feeding behaviour is an important factor determining the vertebrates’ successful adaptation to their environment. Mammalian feeding has been the subject of intense study from comparative, functional, and biomechanical perspectives . From thThe tongue morphology, in its macroscopic structure  in vertebrates, varies to fit their diets  as well as their environments and habitats, which may have played a partial role in the evolution of the tongue’s structure . Such anA. cinereus, has been widely used as a pet. Since A. cinereus became a pet, many changes have occurred, primarily related to the shape and type of food eaten by this animal. In the wild, A. cinereus usually consumes fresh fish, but when it is adopted as a pet, it also eats other foods, such as meat, cat food, and enrichment food . This may contribute to structural changes in the types of papillae found on the dorsal surface of the tongue. Previous SEM studies have been conducted on a variety of carnivores; the present study reported numerous variations in the type of lingual papillae. The study explored the papillae and lingual gland structures using the tongues of domesticated A. cinereus derived from a local breeder in Yogyakarta, Indonesia.Recently, in Indonesia, the Asian short-clawed otter, A. cinereus consisted of three parts: the apex, corpus and radix  and conical papillae. The gustatory papillae are divided into fungiform and circumvallate papillae. Fungiform papillae were found scattered along the dorsal surface of the tongue from the apex to the radix. Foliate papillae were not found in this study. Instead, the function and position of the foliate papillae are replaced by large numbers of conical papillae. Unlike foliate papillae, conical papillae do not have taste buds.The scanning electron microscopy of the dorsal surface of The observations of the apex revealed three types of filiform papillae. Interestingly, on the apex tip, we found horny filiform papillae; on another area of the apex, we found leaf-like filiform papillae and fungiform papillae . Recent On the corpus, the SEM observations revealed numerous filiform papillae in the anterior and posterior corpora. In the anterior corpus, there were bifid filiform, trifid filiform, and cornflower filiform papillae. In the posterior corpus, there were elongated leaf-like and triangular papillae . FurtherIn the radix, we found six circumvallate papillae, with a “V” formation pointing to the larynx . The SEMA. cinereus in Japan   . Severalapillae) . In contapillae) . There wapillae) . Five toapillae) . Seven capillae) . This vaapillae) .In the radix, we observed long conical and short conical papillae positioned laterally and medially to the circumvallate papillae . These pA. cinereus  has fungiform papillae with 5–7 taste buds on the LM that are club-shaped from the apex to the anterior radix and vary in shape on the posterior radix. This resembles the fungiform papillae on the tongue of the sea otter ; in the posterior radix, there are mucous dominant glands (Weber’s glands); in between, von Ebner’s glands are located medially and Weber’s glands are located laterally. The dispersal pattern is considered to support the ingestion of food boluses and is associated with the protection of the oral cavity  is a major component of the connective tissue, and collagen is a critical element of the ECM . The den tightly .A. cinereus samples were not clearly observed in this study. This is presumably due to the size of the tongue, which is large and thick, so a strong collagen layer is required from the apex to the radix to support the main building of the tongue, which is composed of muscles.Variations in the collagen intensity between parts of the tongue in the A. cinereus) from Yogyakarta, Indonesia, consist of numerous types of filiform papillae, including horny filiform papillae and gustatory papillae. Lingual glands secrete neutral and acid mucins, which aid in the processing and swallowing of food. Thus, both the lingual morphological structure and the lingual glands of domestic A. cinereus are similar to those of other members of the Mustelidae family.In conclusion, structurally, the papillae of the Asian short-clawed otter (


Ketamine, a racemic mixture of esketamine (S-ketamine) and arketamine (R-ketamine), has received particular attention for its rapid antidepressant and antisuicidal effects. NMDA receptor inhibition has been indicated as one of the main mechanisms of action of the racemic mixture, but other pharmacological targets have also been proposed. This study aimed to explore the possible multiple targets of ketamine enantiomers related to their antidepressant and antisuicidal effects. To this end, targets were predicted using Swiss Target Prediction software for each ketamine enantiomer. Targets related to depression and suicide were collected by the Gene Cards database. The intersections of targets were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Network pharmacology analysis was performed using Gene Mania and Cytoscape software. Molecular docking was used to predict the main targets of the network. The results indicated that esketamine and arketamine share some biological targets, particularly NMDA receptor and phosphodiesterases 3A, 7A, and 5A but have specific molecular targets. While esketamine is predicted to interact with the GABAergic system, arketamine may interact with macrophage migration inhibitory factor (MIF). Both ketamine enantiomers activate neuroplasticity-related signaling pathways and show addiction potential. Our results identified novel, poorly explored molecular targets that may be related to the beneficial effects of esketamine and arketamine against depression and suicide.  Major depression disorder (MDD) is the leading cause of disability and the largest contributor to suicide deaths in the world . AccordiKetamine is a racemic mixture of S-ketamine (esketamine) and R-ketamine (arketamine) that has been used due to its anesthetic and analgesic properties since the 1970s. Recently, esketamine was approved by the FDA for the treatment of refractory depression and suicidal ideation because of its rapid, potent, and sustained effect, which differs substantially from the antidepressant profile of conventional antidepressants. The pharmacological properties of ketamine are mainly attributed to the inhibition of N-methyl-D-aspartate receptors (NMDAr). However, ketamine has multiple pharmacological targets, such as gamma aminobutyric acid (GABA), dopamine, serotonin, opioids, and cholinergic receptors . A singlOne possibility to study the multitarget potential of ketamine is through bioinformatics tools, which allow screening of depression and suicide molecular targets rapidly and in an early approach that can guide future higher cost experimental studies. Bioinformatics is an area with great potential applications for the study of diseases and drug design and discovery. Pharmacological target network analysis, molecular docking, and gene ontology (GO) enrichment analyses may allow the understanding of the polypharmacological action of molecules, investigating their potential targets, and allowing a more rational use of animal models in scientific research ,14,15,16In this study, we used some of these bioinformatics tools to investigate the potential molecular targets of ketamine enantiomers in depression and suicide. Ketamine has been reported to exhibit polypharmacological action, and there may be differences between esketamine and arketamine in terms of their potency and biological effects, which may be due to their ability to interact with different molecular targets ,21. TherAccording to the SwissTargetPrediction database, 105 potential targets of arketamine and 105 potential targets of esketamine were found. Importantly, although most of the target genes were shared by the enantiomers (91 genes), there were differential genes that were unique targets for arketamine (14 genes), and others unique to esketamine (14 genes).As a result of the search for depression-related genes and suicide-related genes in the GeneCards database, we obtained a total of 13,914 genes and 2409 genes, respectively. Of note, suicide and depression shared 1983 genes, according to the prediction. PDE4B, KDM4C, MIF, NOS2, CREBBP, HMOX1), and 6 genes intersected by depression, suicide, and esketamine . According to the Venn diagram, no overlapping genes were observed between arketamine and suicide or between esketamine and suicide. However, the CA12 gene that codes for carbonic anhydrase 12 was shared by esketamine, arketamine, and suicide. Considering that ketamine has been used for treating severe depression with suicide ideation [The Venn diagram illustrated in ideation , we focuHomo sapiens was performed in GeneMANIA. The obtained network was imported and analyzed in Cytoscape V. 3.9.1. software . As a result, a network with 73 nodes, 891 connections, and a clustering coefficient equal to 0.349 was obtained. The colors of the edges (connections) reflected the type of interaction analyzed. The network had 891 connections, of which 34.85% were physical interactions (green), 28.20% were co-expressed (yellow), 25.94% represented shared protein domains (pink), 9.09% were co-localized (blue), 7.20% were predictions (gray), 2.84% were genetic interactions (purple), and 1.88% were metabolic pathways (black). The network construction of esketamine, arketamine, suicide, and depression overlapping genes is shown in KCNN4, NQO1, AOC2, VEGFD, VEGFC, CA3, AOC1, CA12, GABRA3, PDE5A, MTNR1B, GABRB2, CA13, GRIN2C, CA8, GABRA6, GABRA4, GRIN3A, PDE4B, and PDE3B. Subsequently, the network was filtered considering degree > 30 and betweenness centrality between 0.017 and 0.044. From these data, 11 genes were found to have the highest number of connections (Degree > 30) and the shortest paths  in the network. The genes found were GRIN2B, GRIN2A, GRM4, EGFR, KDR, GABRB3, GABRB2, GABRA3, KCNN1, PDE5A, and MTNR1B. The proteins encoding for these genes were as follows: NMDA receptor (GRIN2B and GRIN2A), metabotropic glutamate receptor (GRM4), GABA receptor subunits β2, β3 and α3 subunits , epithelial growth factor receptor (EGFR), melatonin receptor 1β (MTNR1B), kinase insertion domain receptor (KDR), member 1 of the N subfamily of calcium-potassium activated channel (KCNN1), and the enzyme phosphodiesterase 5A (PDE5). The filtered network of the targets obtained is illustrated in The search for the ketamine enantiomers, suicide, and depression overlapping genes in SIRT1, PIK3CA, I-CAM1, GABRA1, GABRB2, and PRF1. As a result, we obtained a network with 26 nodes, 242 connections, and a clustering coefficient equal to 0.714. GABRA1, GABRB1, GABRA4, GABRG2, GABRB2), but genes encoding sirtuin 1 (SIRT1), integrin subunit alpha L (ITGAL), phosphatase (FOSPI3K) and kinase (PI3K) subunits, thrombopoietin receptor (MPL), transcription factor (BCL11A), and perforin 1 (PRF1) were also identified.In the second step of the network analysis, we obtained potential targets shared by esketamine, suicide, and depression. Therefore, the green diagram was built by searching for the genes CREBBP, NOS2, HMOX1, KDM4C, PDE4B, and MIF. As a result, we obtained a network with 26 nodes, 189 connections, and a clustering coefficient equal to 0.293. When we performed the pharmacological network analysis of only esketamine, the result was a cluster of GABAA receptors , highlighting the genes coding for some specific subunits, such as GABRA1, GABRA4, GABRB1, GABR2, and GABRG2. Most interactions were physical interactions (80.30%), followed by predicted interactions (9.48%), genetic interactions (4.28%), co-expression (3.72%), metabolic pathways (1.12%), co-localization (0.74%), and shared protein domains (0.37%). In the network construction, we retrieved 20 genes. After applying degree (DC > 16) and betweenness centrality (BC) filters between 0.039 and 0.157, we obtained the 10 most important genes of the arketamine, depression, and suicide network, as illustrated in CREB1, CREBBP, and COPS5), enzyme-related genes , genes related to immune responses (MIF and CD74), and cytoskeleton (ACTN4). The third network, represented in ROCK1, AKR1B1, JAK2, DHODH, TYK2, PDE4C, TGFBR1, TNKS2, GABRA4, CCND1, PDE4D, OPRD1, RPS6KA1, F13A1, MTNR1B, PDE5A, IGF1R, BRD4, PDE3B, DYRK1A, JAK1, ADORA1, SYK, CSNK1G1, ADORA2B, TNKS, AURKB, EPHB3, CA13, GRK6, and ADORA3, which are the 31 target genes shared by esketamine, arketamine, and depression. As a result, a network with 51 nodes, 698 connections, and a clustering coefficient equal to 0.584 was obtained. This high degree of clustering was composed of several types of phosphodiesterases. Most interactions in this network were shared protein domains (42.12%), followed by co-expression (26.24%), metabolic pathways (17.70%), physical interactions (6.43%), co-localization (5.77%), genetic interactions (0.98%), and predicted interactions (0.76%). In the network construction, we retrieved 20 genes. After applying degree (DC > 11) and betweenness centrality (BC) filters between 0.038 and 0.079, we obtained the 8 most important genes, as illustrated in The purple network represented in We used GO and KEGG for a global evaluation of ketamine enantiomers, suicide, and depression overlapping genes. According to biological process (BP) analysis of GO, the top five terms were chemical synaptic transmission, anterograde trans-synaptic signaling, trans-synaptic signaling, synaptic signaling, and regulation of membrane potential A. FurtheWe used GO and KEGG enrichment analysis to investigate the functions and biochemical pathways of esketamine, suicide, and depression overlapping genes. According to BP analysis of GO, the top five terms were GABA signaling pathway, chloride transmembrane transport, inorganic anion transmembrane transport, GABAergic synaptic transmission, and regulation of postsynaptic membrane potential A. MoreovTo investigate the biological functions and biochemical pathways involved in the action of arketamine in depression and suicide, GO and KEGG analyses were performed. According to BP, the top five terms were positive regulation of cytokine production, cytokine production, regulation of cytokine production, positive regulation of gene expression, and positive regulation of multicellular organism processes A. AdditiWe used GO and KEGG for a global evaluation of overlapping ketamine enantiomers and depression genes. The top five BP terms were cyclic nucleotide catabolic process, ribonucleotide catabolic process, purine-containing compound catabolic process, purine ribonucleotide catabolic process, and cyclic nucleotide metabolic process A. MoreovGRIN2B and GRIN2A genes, respectively), which were consolidated pharmacological targets for both ketamine enantiomers, being considered our positive control in this docking study. Therefore, the main pharmacological targets of esketamine and the respective affinity values were GluN2B , PDE7A , PDE5A , JAK1 , PDE3A , GluN2A , and GABRG2 . The main pharmacological targets of arketamine and the respective affinity values were as follows: PDE3A , MIF , GluN2B , PDE7A , GABRB2  PDE5A , JAK1 , and GluN2A . To perform docking, the targets of each filtered network were selected by the parameters DC and BC, plus the top five highest values of closeness centrality (CC). After selecting the top 5 biological targets from each network, molecular docking was performed according to the structures and parameters described in In the computational prediction, MIF (1GCZ structure) had one of the highest docking scores for arketamine  and interacted in the center of the protein, as illustrated in According to docking scores, arketamine was predicted to have a higher affinity for PDE3A and PDE5A  than esketamine , while the affinity of esketamine and arketamine was similar for PDE7A . In molecular docking simulations, we found hydrogen bond interactions between PDE3A Leu910 and esketamine B, PDE7A GABRB2, GABRB3, and GABRA3 genes were potential targets shared by both enantiomers of ketamine. In the molecular docking analyses, the proteins obtained from PDB were 6D6T (GABAAα1β2γ2 receptor), 6DW1 (GABAAα1β1γ2 receptor), and 7QNA (GABAAα4β3γ2 receptor). We also found that the Aα1β2γ2 receptor, showing the neurosteroid (left) and benzodiazepine (right) binding sites. In molecular docking simulations, we found two hydrogen bond interactions between GABAA receptor Arg141 and esketamine in the neurosteroid binding site , the benzodiazepine site , and the neurosteroid site  were potential targets for interaction with ketamine. ing site I, and a ing site J.The main purpose of this study was to explore the potential molecular targets of ketamine enantiomers in suicide and MDD by employing network pharmacology and molecular docking simulations. Our workflow involved a sequential analysis composed of the following steps: (a) identification of molecular targets of ketamine enantiomers in SwissTargetPrediction; (b) collection of genes targets of MDD and suicide in GeneCards; (c) creation of a Venn diagram to obtain the overlapping genes; (d) construction of the network of overlapped genes; (e) implementation of network filter parameters to obtain the highest closeness, betweenness, and degree centrality; and (f) running molecular docking simulations. Our bioinformatics analysis uncovered that both enantiomers of ketamine, esketamine, and arketamine share biological targets that may be implicated in their antidepressant and antisuicide properties. However, due to the large number of pharmacological targets found in the network analyses, we limited the molecular docking analyses to the nodes that had the best topological parameters. The main targets obtained are NMDA receptors (GluN2 subunits), GABA receptors , macrophage migration inhibitory factor (MIF), and various subtypes of phosphodiesterases . However, it is important to consider that other targets not analyzed may be relevant to the antidepressant and antisuicidal effects of ketamine enantiomers.GRIN1), glutamate-binding GluN2 , and glycine-binding GluN3 subunit (GRIN3A and GRIN3B). Regarding GluN2, four different subunits (GluN2A-D) are present throughout the central nervous system [GO and KEGG functional analyses pointed out relevant targets for both ketamine enantiomers and were useful in directing subsequent studies. In GO enrichment analysis related to molecular function, NMDA receptor activity was pointed out as one of the main targets. In agreement, ketamine is a well-known, noncompetitive open-channel NMDAr antagonist . The GRIs system . Of notes system .We found that GluN2 subunits of NMDAr are potential targets for the action of both ketamine enantiomers, particularly GluN2B, using docking analyses. Interestingly, a study by Zhang and co-workers  reportedAlthough NMDAr is the key target of esketamine and arketamine, other targets may be implicated in their antidepressant properties. Indeed, it has been discussed whether the inhibition of NMDAR is critical for the mood-enhancing effects of ketamine enantiomers, and a hypothesis that has been raised is that their antidepressant and antisuicidal effects may be dependent on their interaction with multiple molecular targets . RegardiMIF) as a central node. Subsequently, our study identified MIF, to our knowledge, for the first time, as a potential molecular target of arketamine by molecular docking. MIF is a cytokine originally characterized by its ability to prevent migration of macrophages, which is now recognized as a key mediator of the innate immune system and adaptive immunity [MIF gene polymorphism may be related to attempted suicide in patients with bipolar disorder, other studies failed to indicate a relationship between MIF levels or MIF gene polymorphism and suicide [MIF-794CATT5–8 microsatellite or MIF-173G/C single-nucleotide polymorphism (SNP) on the MIF gene promoter. In addition, no significant alterations in MIF levels in the dorsolateral prefrontal cortex were observed in suicide completers as compared to control individuals [Network construction of overlapping arketamine, suicide, and depression genes identified macrophage migration inhibitory factor  may be molecular targets of ketamine enantiomers. PDEs are classified into 11 subtypes based on sequence homology and structural and enzymatic features mainly related to sensitivity to modulators . These ePDE3 occurs as two groups of isoenzymes (PDE3A and 3B) that are involved in the hydrolysis of cGMP, mainly cAMP ,43. AlthPDE7 comprises 2 subclass members: PDE7A and B. This enzyme, abundant in the brain, is responsible for the hydrolysis of cAMP. The highest levels of PDE7A have been reported in the olfactory bulb, olfactory tubercle, hippocampus, cerebellum, medial habenula nucleus, pineal gland, area postrema, and choroid plexus . NotablyPDE5A gene), namely PDE5A1, PDE5A2, and PDE5A3 [PDE5A single nucleotide polymorphism (SNP) may be associated with MDD [PDE5 hydrolyzes cGMP, which is a critical component of the cGMP-protein kinase G (PKG) intracellular signaling in neurons. There are three isoforms of PDE5A, encoded by a unique gene  family. These receptors have R1 subunits where orthostatic ligands bind (encoded by the GABBR1 gene) and R2 coupled to G protein (GABBR2) [Ar are pentamer chloride channels and have 19 genes that encode for subunits of the GABAA receptor, which is organized into classes  and isoforms  [GABRA1 gene encodes the GABA Type A Receptor Subunit Alpha1(GABAAα1) protein, the GABRB3 gene has GABA Type A Receptor Subunit Beta3 (GABAAβ3) as its product, and the GABRG2 gene encodes the GABA Type A Receptor Subunit Gamma2 (GABAAG2) protein. The assembly of GABAAr as heteropentamers produces complex heterogeneity in their structure, and different subunit compositions are possible. A typical GABAA receptors are made up of two alpha subunits, two beta subunits, and one gamma subunit, and the interaction sites lie at the connections between these subunits. The GABA interaction site is located between the interfaces of the alpha and beta subunits, the neurosteroid site is located in the transmembrane domain between the alpha and beta subunits, and the benzodiazepine site is located at the interface between the alpha and gamma subunits [GABRB2, GABRB3, and GABRA3 genes are potential targets shared by both enantiomers of ketamine, while GABAAG2 coded by GABRG2 is predicted to mainly interact with esketamine. Network pharmacology, GO, KEGG analysis, and molecular docking pointed out that GABA genes are also potential targets for both enantiomers but mainly for esketamine. The GABA receptor (GABAr) family is divided into metabotropic and ionotropic receptors. GABA(GABBR2) . GABAAr nd ρ1–3) . Thus, tsubunits . In our Ar) reported that this compound recovered hedonic behavior in mice and that this behavior was prevented using the benzodiazepine antagonist flumazenil, suggesting the involvement of the benzodiazepine site of GABAAr in anhedonia symptoms [Ar may be involved in the mechanism of action of ketamine in improving anhedonic symptoms in MDD.The benzodiazepine site of GABAr is closely implicated in anhedonic behavior. Anhedonia is one of the symptoms of major depressive disorders that is closely related to suicide risk . Of notesymptoms . A clinisymptoms . These dAr, have potential use as antidepressants and anesthetics [A receptor to rapid antidepressant responses, brexanolone and zuranolone, positive allosteric modulators of the neurosteroid site of this receptor elicit rapid and sustained antidepressant effects in postpartum depression and MDD [Neurosteroid analogs, molecules that bind to the allosteric site of neurosteroids on GABAsthetics . Reinfor and MDD ,59,60. Ketamine is known for its dissociative properties and abuse potential , used evOne of the limitations of our study is that we cannot predict the effect of gender on the findings for each ketamine enantiomer. The availability of software able to predict sex differences between esketamine and arketamine enantiomers in depression and suicide would be welcome to provide information on this issue. However, it has been reported in preclinical studies that females are more sensitive to the antidepressant effect of ketamine ,65,66, bAlthough our study suggests novel and poorly explored proteins as possible targets for the antidepressant and antisuicidal effects of ketamine enantiomers, future experimental studies are needed to confirm the role of these targets in the effects of ketamine enantiomers. The list of biological targets of arketamine and esketamine was obtained from SwissTargetPrediction   by searcHomo sapiens. The network was imported and analyzed in Cytoscape V. 3.9.1 [The list of genes obtained from the crossover was searched in GeneMANIA  using default settings , and a nV. 3.9.1 . TopologV. 3.9.1 . It is cV. 3.9.1 ,16,77. TGO and KEGG enrichment analyses were performed in ShinyGO v.0.76.3   for eachThe molecular docking analysis was carried out to predict the binding affinity between arketamine or esketamine and the potential biological targets using the DockThor 2.0 software   in a simIn summary, our study uncovered that ketamine had a polypharmacological profile, with many biological targets involved in suicide and MDD. The findings indicated GRIN2A, GRIN2B, PDE7A, PDE5A, and PDE3A as the most likely targets predicted for both ketamine enantiomers. Regarding S-ketamine, it was predicted that the main targets were GABRB2 and GABRG2. In the case of arketamine, it was predicted that MIF was the main target. These new targets pointed out in our study have not yet been explored with regard to their involvement in the antidepressant and antisuicidal effects of ketamine and therefore deserve attention in future experimental studies.


Ischemic stroke (IS) is affected by a wide range of factors and has certain treatment limitations. Studies have reported that compound musk injection (CMI) is effective in the treatment of IS, however, its mechanism of action is still unclear.The main active ingredients in CMI were retrieved from HERB, TCMSP and BATMAN databases, and the relevant targets were predicted by Swiss Target Prediction platform. MalaCards, OMIM, DrugBank, DisGeNET, Genecards and TTD databases were used to obtain the genes related to IS. The intersection of drugs and disease targets was used to construct protein–protein interaction networks, and gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed. AutoDock Vina software was used for molecular docking, and cell experiments were conducted to verify the results. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression level of relative mRNA in cells.Network analysis and molecular docking results showed that the key targets of CMI in the treatment of IS were SRC, TP53, PIK3R1, MAPK3, PIK3CA, MAPK1, etc. KEGG pathway enrichment analysis mainly involved PI3K/Akt signaling pathway, Rap1 signaling pathway and MAPK signaling pathway. The molecular docking results all showed that the key ingredients were strong binding activity with the key targets. The quantitative RT-PCR results indicated that CMI may increase the expression of PIK3CA, MAPK3 mRNA and decrease the expression of SRC mRNA.CMI can treat IS by regulating pathways and targets related to inflammatory response and apoptosis in a multi-component manner. IS known as “cerebral infarction” in modern medicine, is a kind of irreversible damage or necrosis of local brain tissue caused by ischemia and hypoxia due to local blood supply disorders caused by a variety of factors. Clinical manifestations included sudden fainting, unconsciousness, slanting tongue, poor language, hemiplegia, etc. IS has a high mortality and disability rate, which brings huge psychological and economic burden to patients. Although thrombolysis or mechanical thrombectomy can improve the condition of patients to a certain extent, due to the limitation of treatment time window and the high risk of hemorrhagic metastasis, only 2%–10% of patients in the world can benefit from it, and 1.2%–3.8% of patients in China can benefit from it.,3 There are certain limitations in treatment, which causes the occurrence of alternative and complementary therapy. Therefore, it is critical to seek more effective measures to prevent and treat stroke.Stroke is an acute cerebrovascular disease, including ischemic and hemorrhagic stroke, of which ischemic stroke (IS) accounts for more than 80% of all strokes. In modern clinical application, CMI can treat acute cerebral infarction, cerebral hemorrhage, severe craniocerebral injury, pneumonia toxic encephalopathy, vertebrobasilar ischemic vertigo, etc. Musk volatile components can improve motor function after cerebral ischemia by up-regulating the relative levels of AKT1, PI3KA and VEGFA through HIF1A pathway. Studies have shown that borneol can inhibit cell death by inhibiting calcium overload and ferroptosis,,8 and can also alleviate brain edema by changing the permeability of the blood-brain barrier. Radix Curcumae, one of the core drugs for the treatment of stroke, inhibits the activation of NLRP3 inflammasome through the NF-κB pathway, thereby reducing the expression levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Moreover, it may play a protective role in neurons by inhibiting PRV virus-induced neuronal damage through BDNF/Trk B pathway. Alpha-asarone in acorus tatarinowii can inhibit neuronal calcium overload, regulate glutamate receptors, maintain glutamate concentration, and eliminate glutamate excitability and neurotoxicity. Flavonoids are composed of a group of phenylpropanoids, which can be divided into 12 subclasses according to the degree of oxidation of heterocycles: chalcones, astragalus, flavonoids, flavanones, flavones, isoflavones, phlobaphenes, dihydroflavonols, flavonols, colorless anthocyanins, proanthocyanidins, and anthocyanins.,14 They show a wide range of health benefits, including antibacterial, anticancer, anti-osteoporosis and antiviral activities. However, the pharmacological effects of CMI in the treatment of IS are not fully understood.With the continuous improvement of the level of traditional Chinese medicine, a series of Chinese patent medicines for the treatment of IS have been developed through continuous optimization and reform of important prescriptions in clinical practice. Compound Musk injection (CMI) is a kind of medicine widely used in clinical medicine, which includes 6 Chinese herbs: Acori tatarinowii rhizoma, musk, Borneol, Radix Curcumae, Pogostemon cablin and menthol. It is mainly used for stroke coma caused by internal occlusion of phlegm and heat. We constructed a network of drug and disease targets through network analysis, identifying potential mechanisms and key targets. Molecular docking technology was used to simulate the interaction of ligand-receptor proteins, and 6 genes with the highest correlation with the disease were screened. RT-PCR was used to detect the mRNA expression levels of the 3 genes. In order to provide a theoretical basis for the subsequent research of CMI.Network pharmacology is helpful to reveal the mechanism of action of drugs, and the integration strategy of multi-direction pharmacology is helpful to expand the available target space of drugs, increase the success rate of clinical trials, and reduce the cost of finding new drugs. , Bioinformatics Analysis tool of Molecular mechanism of traditional Chinese medicine  and herbal database  were used to search the active components of CMI.Traditional Chinese medicine systems pharmacology database and analysis platform Active ingredients were obtained by setting “Score cutof” at 20 and P value at .05 based on the predicted score of drug-target interaction prediction during the BATMAN database search. Because the algorithms in each database are different and the names of intellectual chemistry are different, the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) retrieval of each component of the Canonical SMILES number or a 3D structure, and its input Swiss Target Prediction (http://swisstargetprediction.ch/) platform predict related targets. The target was further standardized in UniProtKB database (https://www.uniprot.org/).The TCMSP database screened active ingredients based on drug similarity (DL) ≥ 0.18 and oral bioavailability ≥ 30%.https://www.omim.org/), DisGeNET (http://www.disgenet.org), MalaCards , Genecards (https://www.genecards.org), DrugBank (https://www.drugbank.ca/) and TTD (https://db.idrblab.net/ttd/). The intersection of drug targets and disease targets was selected as the potential therapeutic targets of CMI in the treatment of IS. Targets for drugs and diseases import Venny website (https://bioinfogp.cnb.csic.es/tools/venny/) to get Wayne Figure Using “ischemic stroke” as the key word, the genes related to IS were collected from the following 6 disease-related databases: OMIM (https://www.string-db.org/), and the species was selected as “Homo sapiens,” the minimum required interaction score was set as “Combined score > 0.4,” and the nodes with hidden disconnections were set. Cytoscape software (3.9.1) was used to perform topological analysis and construct the PPI network of key therapeutic targets. The potential therapeutic target genes were ranked according to the degree value, and the targets and active ingredients with higher degree values were screened.The intersection targets were imported into the STRING database (Http://metascape.org) database for Gene ontology (GO) functional enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. A threshold of P < .05 was used to screen the enrichment results with significant differences, and bar charts and bubble charts were made according to the enrichment results.The 353 intersection genes were entered into the Metascape (https://www.rcsb.org/) database and imported into PyMOL 2.5.2 software to remove water molecules and small molecule ligands, and added hydrogen atoms in AutoDockTools 1.5.7 software, and converted to pdbqt file format. Molecular docking was performed using AutoDock Vina (v.1.1.2). Based on the evaluation index of binding energy, it is generally believed that the binding energy < 0.0 kcal/mol indicates that the ligand has binding activity with the target, and the lower the binding energy, the more stable the binding. The docking results were visualized by PyMOL 2.5.2 software.The most important 6 key targets and active ingredients were first selected. The 2D structures of active ingredients were downloaded from PubChem database and imported into Chem Bio 3D Ultra 14.0.0.117 software to obtain the 3D structures, which were saved as mol2 file format and converted to pdbqt file format in AutoDockTools 1.5.6 software. The 3D structure of the protein crystal of the target gene was downloaded from PDB  for 12 hours to prepare the OGD/R model. After oxygen-glucose deprivation, the cells in each group were replaced by high-glucose DMEM medium containing 10% FBS, and the cells in the drug group were added with the corresponding concentration of Compound Musk Injection, and cultured in the normal incubator for 12 hours.The digested and resuspended HT-22 cells were randomly divided into Control group (Control), model group (OGD/R), CMI high, medium and low concentration groups . The cell concentration was adjusted to 80,000 cells/mL and seeded in 96-well plates with 100uL of cell suspension per well. After being cultured in the cell incubator for 24 hours, the Control group and OGD group were treated with 10% fetal bovine serum (FBS) high-glucose DMEM medium, and the drug group was treated with an equal volume of CMI  + 10% FBS high-glucose DMEM medium for 8 hours, respectively. After the pretreatment, the Control group was replaced with 10% FBS high-glucose DMEM medium, the OGD/R group was replaced with serum-free and sugar-free DMEM medium, and the drug group was replaced with equal volume of CMI  + serum-free and sugar-free DMEM medium. The Control group was put back into the normal cell incubator at 37°C, 5% COSerum-free high-glucose DMEM medium was mixed with CCK8 test solution at 10:1, added to the cells (in the dark), 100 μL per well, and returned to the incubator for further treatment for 2 hours. At the end of the treatment, the Optical Density (OD) value of each well was measured at the wavelength of 490 nm by microplate reader. The cell growth survival rate was calculated according to the OD value and cell survival rate formula. Cell survival rate (%) =  /  × 100 %.HT-22 cells treated with 2.6.1 were collected and total RNA was extracted using TRIzol kit. Cells were first resuspended with 1 mL TRIzol test, transferred to 1.5 mL enzyme-free EP tubes, and allowed to stand for 5 minutes at room temperature. Then 200 μL chloroform was added to each centrifuge tube and centrifuged at 10,000 rpm and 4°C for 10 minutes. Finally, 0.5 mL of isopropanol was added to the EP tube containing total RNA, an appropriate amount of 75% ethanol was added to clean the isopropanol by centrifugation, and 100 μL of DEPC water was added to dissolve the RNA precipitate.−∆∆Ct method.The primer sequences were reviewed in the NCBI database as shown in Table After eliminating the duplicate items, 136 active ingredients were obtained, including 20 for musk, 1 for menthol, 54 for radix cureumae, 39 for patchouli, 37 for tatarinoa, and 18 for borneol , which included 489 nodes  and 3039 edges. The degree value reflects the number of nodes directly linked to the compound and is used to assess the importance of the compound in the network. The top 20 active ingredients were screened by topological analysis , 183 items for cell composition (CC), and 375 items for molecular function. Ranked by ion Fig. . Among tP value, we selected the top 20 signaling pathways for bubble plots . Compared with the model group, the CMI groups had a tendency to improve the cell survival rate, as shown in Figure P > .05).Cell experiments showed that the survival rate of HT-22 cells was significantly decreased after oxygen-glucose deprivation/reoxygenation, and the difference was statistically significant compared with the blank group (P > .05). Compared with the Model group, the expression of PIK3CA and MAPK3 mRNA in the CMI group  tended to increase, and the expression of SRC mRNA tended to decrease , thereby damaging cells in the ischemic semidark zone. The MAPK signaling pathway involves can be grouped into 3 main families of intracellular serine/threonine protein kinases that include extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). Activated MAPK, which phosphorylates and activates downstream ERK kinases ERK-1 and ERK-2, ultimately transmits cell proliferation and differentiation signals to the nucleus by regulating the activity of transcription factors.,35 MAPK contributes to the maintenance of neural stem cells and can be activated by phosphorylation of threonine and tyrosine residues in response to cerebral ischemia. MAPK phosphorylation can promote neuronal survival in the dentate gyrus region. In the present study, CMI increased MAPK mRNA expression in OGD/R model cells, indicating that CMI could act on MAPK targets. PIK3CA is the catalytic subunit encoding PI3K, and PIK3R1 is the regulatory subunit of phosphatidylinositol-3-kinase (PI3Ks). In the process of stroke, PIK3CA and PIK3R1 are the main genes that promote cell survival and reduce cell apoptosis.,38Src is a non-receptor protein tyrosine kinase, which can regulate extracellular signal-regulated kinase in ischemic brain tissue, and then play a protective role in brain.,40 Studies have demonstrated the neuroprotective effects of Danhong injection in rats with ischemia-reperfusion injury and suggest that these effects on the brain are in part due to activation of the PI3K-Akt signaling pathway. The PI3K inhibitor LY294002 decreased Akt activity, which partially inhibited the protective effect on infarct size reduction, and increased Akt activity correlates with changes in glycogen synthase kinase 3β (GSK-3β) phosphorylation shortly after cerebral ischemia and changes in β-catenin phosphorylation patterns in the ischemic core and penumbra. Fucoidan may protect cerebral ischemia-reperfusion injury by inhibiting MAPK pathway. OXA may play a neuroprotective role in CIRI by inhibiting NF-κB, MAPK/ERK and MAPK/p38 signaling pathways mediated by OX1R, and inhibiting astrocyte apoptosis, activation and proinflammatory cytokine production. Endogenous inhibition of p38 MAPK rescues hippocampal apoptosis, reduces ischemic penumbra, and ameliorates neurobehavioral deficits. cAMP/PKA signaling pathway plays an important role in inhibiting neuronal apoptosis. Once the cell is stimulated, the signaling molecule binds to the receptor on the cell membrane to form a complex, which then activates adenylate cyclase. Eventually cAMP is produced, which enters the nucleus and directly activates RNA polymerase to promote mRNA transcription of the target gene.,47 Lidocaine may improve the neural function and inhibit the neuronal apoptosis in CIRI rats by activating cAMP/PKA signaling pathway. It has been found that cAMP can participate in many signaling pathways initiated by neurotransmitters, among which cAMP can directly act on the specific guanylate exchange factor EPAC of Rap1 to induce its activation, and activated EPAC can directly activate the downstream Rap1. Inhibition of Epac pathway by Rap1 inhibitor GGTI298 and Rac1 inhibitor NSC23766 can aggravate blood-brain barrier injury and brain injury, suggesting that activation of Epac/Rap 1 signaling pathway has neuroprotective effect on cerebral ischemia-reperfusion injury.PI3K-Akt signaling pathway is a key pathway in the pathological mechanism of ischemic brain injury, which mainly regulates cell survival, proliferation, metabolism, neuroscience, movement and cancer progression.This study lays a foundation for further study on the mechanism of CMI, provides a theoretical basis for further systematic experiments, and provides new ideas for the targeted therapy of IS. However, this study still has some limitations. Due to the limited data in the database and related literature, the therapeutic targets, related pathways and further mechanisms of action predicted in this paper need to be carried out in relevant systematic experimental empirical work.In this study, network pharmacological analysis, molecular docking technology and cell experiments were used to explore the mechanism of CMI in the treatment of IS. The results showed that Apigenin, Quercetin, Morin and other components in CMI may act on targets such as SRC, TP53, PIK3R1, MAPK3, PIK3CA, MAPK1 and other targets through PI3K/AKT and MAPK signaling pathways. Thus, it can reduce reperfusion injury, inhibit cell apoptosis and anti-inflammation. It provides some theoretical basis for further determining the mechanism of action of traditional Chinese medicine in the prevention and treatment of IS.Conceptualization: Xiaoqing Li, Ya Yan, Qian Wang, Dexiao Wang.Data curation: Xiaoqing Li, Hua Yang, Jianjie Cheng, Hairong Zhao, Qian Wang, Dexiao Wang.Formal analysis: Xiaoqing Li, Jianjie Cheng, Hairong Zhao, Qian Wang, Dexiao Wang.Methodology: Xiaoqing Li, Hairong Zhao.Project administration: Guangming Wang.Software: Xiaoqing Li, Qian Wang, Dexiao Wang.Supervision: Guangming Wang, Hua Yang, Jianjie Cheng, Ya Yan.Validation: Hua Yang.Visualization: Xiaoqing Li.Writing – original draft: Xiaoqing Li.Writing – review & editing: Guangming Wang, Hairong Zhao.


Progression of today’s world has been given setback due to the adversity of a novel, viral, deadly outbreak COVID 19, which raised the concerns of the scientists, researchers and health related officials about the inherent and adaptive immune system of the living body and its relation with healthy diet balanced with pharma foods. Now world is coming out of the destructive pandemic era, the choice of right food can help to build and boost adaptive immunity and pumpkin due to excellent profile of functional and nutraceutical constituents could be the part of both infected and non-infected person’s daily diet. Vitamins like A, C and E, minerals like zinc, iron and selenium, essential oils, peptides, carotenoids and polysaccharides present in pumpkin could accommodate the prevailing deficiencies in the body to fought against the viral pathogens. In current post COVID 19 scenario adequate supply of healthy diet, balanced with pharma foods could play a basic role in boosting immune system of the populations. This review covers the pharmacological activities of pumpkin functional constituents in relation with COVID 19 pandemic. Pumpkins are well equipped with nutraceuticals and functional bioactives like tocopherols, polyphenols, terpenoids and lutein therefore, consumption and processing of this remarkable vegetable could be encouraged as pharma food due to its antihyperlipidemic, antiviral, anti-inflammatory, antihyperglycemic, immunomodulatory, antihypertensive, antimicrobial and antioxidant potential. Need of healthy eating in current post COVID 19 period is very crucial for healthy population, and medicinal foods like pumpkin could play a vital role in developing a healthy community around the globe. The COVID 19, a pandemic declared by World Health Organization, is usually caused by a novel corona virus, which normally attacks the people with fragile immune response. Population with weak immunity is more vulnerable towards novel coronavirus. Plant based foods rich in vitamins, minerals and proteins play crucial role in boosting immunity as they promote beneficial bacteria in human body. Food combinations containing immunity boosting nutrients are needed to be explored and marketed in current post COVID 19 period, which can play vital role against common viruses and novel corona virus improving the immunity all around the world , whereas total phenolic contents in pumpkin seed oils ranged from 24.71 to 50.93 mg/gallic acid equivalent/kg oil. Pumpkin seed oils oxidative stability was calculated about 4 h and antioxidant capacity was found 62%, measured by DPPH radical scavenging method  is an herbaceous vine attached, edible and heat sensitive vegetable, well-known due to its food and medicinal value, which has been found associated with macro and micro nutrients present abundantly, and relatively low amounts of antinutrients . This protein exhibits a strong cytotoxicity to three cancer cell lines of both murine and human origin beside rRNA and N-glycosidase activity.Peptides from pumpkin seeds after digestion in digestive tract act as anti-COVID 19 agents, therefore these can be consumed on daily basis for the prevention and management of this outbreak. Molecular docking study has revealed that from seed proteins during digestion,1593 peptides were released out of which, 36 were of high gastrointestinal absorption. These bioactive peptides could bind the catalytic sites of the viral proteins. Pumpkin seed protein derived peptides possessed more strong affinities toward binding of COVID 19 spike proteins (Wong et al. Botrytis cinereal, Fusarium oxysporum and Mycosphaerella arichidicola at a dose of 375 µg (Vassiliou et al. Fusarium oxysporum at a concentration of greater than 2 Mm in an agar disc plate media. The same protein exhibited a synergistic effect with nikkomycin, a chitin synthase inhibitor, as a growth inhibitor of Candida albicans (Ng et al. coli was not inhibited by MAP2 and MAP4 (Cheong et al. 0C but no inhibitory effect towards growth of E. coli and Staphylococcus aureus. From all these data it can be concluded that pumpkin can be consumed by humans as an antimicrobial agent as it will protect human beings from infectious diseases caused by pathogenic micro-organisms.A peptide with molecular weight 8 kDa from pumpkin seeds was isolated and observed to inhibit In case of intoxication resulted from carbon tetra chloride the administration of pumpkin seed proteins activity levels of alanine transaminase, aspartate transaminase, lactate dehydrogenase and alkaline phosphatase were significantly reduced, so it was concluded that administration of these proteins was effective in protein malnutrition condition and its adverse effects (Nkosi et al. Steiner-Asiedu et al.  made res.Pumpkin seed oil extracts in the form of microemulsion, when used for the treatment of COVID 19 patients expressed promising results due to their anti-inflammatory, antioxidant and immunomodulatory effects. Nanostructure of squalene has been tested in clinical trials for its efficacy on COVID 19 patients and at the end of trials blood tests revealed that no adverse effects of squalene were induced on blood cells, suggesting the potential use of squalene for treatment of COVID 19 (Ebrahimi et alPumpkin seeds oil are good source of edible oil and protein as they contain 41.59% fat and 25.4% protein contents. Further proximate analysis of pumpkin seeds reported moisture 5.2%, crude fiber 5.34%, total ash 2.49% and carbohydrates 25.19% (Ardabili et al. Karanja et al.  proved tFurther clinical work is required to be undertaken before those isolated, purified compounds can be marketed. However, in the developing countries, to cut down costs, intake of pumpkin fruit in form of vegetable should be encouraged that also shown clinically effective. To maintain healthy body with properly working immune system consumption of pumpkin on daily basis must be encouraged in this current period of post COVID 19.Carotenoids especially β carotene is thought to prevent diseases mainly due to its antioxidant role as it can be converted in to Vitamin A in human body. These carotenoids cannot be synthesized by our body and only source is diet or supplements. In current COVID 19 outbreak diet rich in carotenoids should be appraised for people of all age of groups in the world (Khalil et al. 2 have been reported in pumpkin fruit along with carotenes and phenolic compounds (Chanwitheesuk et al.Pumpkin is an important source of healthy nutrients which includes minerals, dietary fiber, ascorbic acid, carotenoids, tocopherols and phenolics. β-carotene present in pumpkin is an anti-inflammatory agent as it decreases the skin cancer which occurs due to sunlight whereas α-carotene is considered responsible to prevent aging, growth of tumor and preventing the chance of developing cataracts in eye. (Jacobo-Valenzuela et al. Pumpkin fruits are an important source of carotenoids and these carotenoids in the form of pro-vitamin A play important role in human nutrition. Skin diseases, eye disorders and cancer can be prevented by using pumpkin-based diets as these diets are rich source of carotenoids. Incorporation of pumpkin as an ingredient and source of β-carotene in a variety of food products is considered very effective towards vitamin A related health disorders and it is also very much cost-effective approach. For the development of nutraceuticals and value-added food products incorporation of pumpkin has gained great interest due to its anti-inflammatory, antioxidant, antidiabetic and anticarcinogenic activities. Pumpkin fruit could be consumed as a source of different bio-actives promoting human health (Dar et al. Along with allopathic drugs and vaccines, healthy eating compromised of pharma foods should be the key strategy to counter the attack of diseases especially the COVID 19. Populations with poor eating habits have been badly affected by the attack of novel coronavirus due to their weaker immune system. Pumpkin has been proved an excellent pharma food, possessed with remarkable amounts of functional and nutraceutical constituents responsible for multifunctional roles in human body. Proteins, polysaccharides, oils, vitamins, minerals and phenolic compounds present in pumpkin fruits exhibits immunomodulatory, anti-inflammatory, antioxidant, antimicrobial and antiviral activities. Need of the time is to incorporate these constituents in humans’ daily diet to maintain a well-balanced body equipped with boosted immune system.