Ischemic stroke (IS) is a common neurological disorder associated with high disability rates and mortality rates. At present, recombinant tissue plasminogen activator (r-tPA) is the only US(FDA)-approved drug for IS. However, due to the narrow therapeutic window and risk of intracerebral hemorrhage, r-tPA is currently used in less than 5% of stroke patients. Natural compounds have been widely used in the treatment of IS in China and have a wide range of therapeutic effects on IS by regulating multiple targets and signaling pathways. The keywords “ischemia stroke, traditional Chinese Medicine, Chinese herbal medicine, natural compounds” were used to search the relevant literature in PubMed and other databases over the past five years. The results showed that JAK/STAT, NF-κB, MAPK, Notch, Nrf2, and PI3K/Akt are the key pathways, and SIRT1, MMP9, TLR4, HIF-α are the key targets for the natural compounds from traditional Chinese medicine in treating IS. This study aims to update and summarize the signaling pathways and targets of natural compounds in the treatment of IS, and provide a base of information for the future development of effective treatments for IS. Stroke is associated with the second leading cause of death and the third leading cause of disability among human diseases , and IS IS is a complex pathological cascade reaction involving various pathological factors, including oxidative stress, inflammation, apoptosis, autophagy, and BBB damage. Oxidative stress, inflammation, and apoptosis are the critical factors in cerebral ischemic injury ,9. An imTraditional Chinese medicine (TCM) has been practiced in China for thousands of years, and has gained wide clinical application , numerouThe JAK/STAT signaling pathway is involved in various physiological processes, such as cell proliferation, differentiation, and apoptosis. The JAK protein tyrosine kinase family consists of JAKl, JAK2, JAK3, and Tyk2. To date, seven members of the STAT family have been ascertained: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6 . ExtraceSophora flavescens Aiton., has been shown to reduce the expression of the p-JAK2 and p-STAT3 proteins and the number of apoptotic cells in the brain tissue of Middle Cerebral Artery Occlusion (MCAO) rats, and plays a neuroprotective role by inhibiting the activation of JAK-STAT signaling pathway and reducing the inflammatory response [Carthamus tinctorius L., it can significantly down-regulate the expression of JAK2-mediated signaling due to ischemic injury, while significantly promoting the expression of SOCS3, which is a negative regulator of STAT3. By modulating the cross between JAK2/STAT3, Hydroxy saffron yellow A can confer neuroprotection against focal cerebral ischemia [Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C. A. Mey., has multiple pharmacological activities, it can increase blood flow in ischemic brain tissues of MCAO rats, upregulate EPO and EPOR expression, promote STAT3 phosphorylation and inhibit VEGF mRNA expression, thus improving blood supply to ischemic brain tissues, reducing vascular permeability and promoting angiogenesis through the JAK2/STAT3 signaling pathway [Carthamus tinctorius L., can increase the protein expression level of Bcl-2 and downregulate the expression of p-JAK2, p-STAT3, caspase-3 and Bax, and inhibit the JAK2/STAT3 signaling pathway to alleviate apoptosis caused by cerebral ischemia-reperfusion injury (CIRI) [Matrine, an alkaloid that is extracted from response . Hydroxyischemia . Catalpo pathway . Nicotify (CIRI) . Additioy (CIRI) ,26. The The NF-κB signaling pathway is a classic signal transduction pathway mediated by cytokines. It plays an important role in several physiological and pathological activities, including inflammation, oxidative stress, endothelial cell injury, and cell death . NF-κB iScutellaria baicalensis Georgi, exerts protective effects against cerebral ischemia by inhibiting TLR4/NF-ĸB signaling pathway and suppressing mitochondrial apoptosis, inflammation, and oxidative stress [Eleutherococcus senticosus (Rupr. & Maxim.) Maxim., can promote FOXO3a phosphorylation and inhibit NF-κB nuclear translocation, which in turn reduces the levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and MPO, and exerts a protective effect against ischemic brain injury by reducing the inflammatory response through the FOXO3a/NF-κB signaling pathway [Schisandra chinensis (Turcz.) Baill., can inhibit TLR4 expression and NF-κB activation and reduces TNF-α, IL-6 and IL-1β levels, exerts protective effects against cerebral ischemia by inhibiting TLR4/NF-κB signaling pathway [Ephedra sinica Stapf, has been shown to decrease oxidative stress, prevent inflammation, increase immunological function, and decrease CIRI, which may be due to their suppression of NF-κB-NLRP3 signaling [Salvia miltiorrhiza Bunge, inhibits NF-κB activation and inflammatory factor release mediated by HMGB1-TLR4 signaling and attenuates HMGB1-mediated inflammatory response by inhibiting TLR4/MyD88/NF-κB signaling pathway [Artesunate, a derivative of artemisinin, reduces tissue damage caused by traumatic brain injury and protects MCAO mice from inflammatory injury by inhibiting NF-κB, releasing the pro-inflammatory cytokines IL-1β and TNF-α, reducing neutrophil infiltration, and inhibiting microglia activation . Skullcae stress . Syringi pathway . Schisan pathway . Ephedriignaling . Salvian pathway . Furthor pathway ,42,43,44Recent research demonstrated that the MAPK pathway plays an essential role in the initiation and progression of IS . The MAPCitrus reticulata Blanco, can reduce ischaemic/reperfusion-induced brain apoptosis by upregulating Bcl-2 expression, downregulating Bax and caspase-3 expression, and reducing the levels of pro-inflammatory factors TNF-α and IL-6 and the expression of p-p38 and MAPAP-2 in MCAO rats. This mechanism is related to the MAPK signaling pathway [Rheum palmatum L., can induce the expression of Bcl-2 and GLT-1 through the ERK-1/2 signaling pathway, inhibits neuronal apoptosis and ROS production, reduces glutamate toxicity, and alleviates nerve cell injury in a rat model of MCAO [Nobiletin, a flavonoid extracted from  pathway . Coriolu pathway . Scrophu pathway . Emodin, of MCAO . Further of MCAO ,57,58. TNotch is a highly conservative signaling pathway that plays a critical role in cell proliferation, differentiation, and apoptosis , it is aAstragalus penduliflorus subsp. mongholicus (Bunge) X. Y. Zhu significantly reduced the infarct area in MCAO rats, and promoted cell proliferation and duct formation, which in turn promoted angiogenesis and had a protective effect against cerebral ischemic injury, which was closely related to the upregulation of miRNA-210 expression, induction of HIF-VEGF-Notch signaling pathway activation and inhibition of target gene ewitinA3 expression [Cnidium monnieri (L.) Cusson., can significantly reduce the volume of cerebral infarction, reduce apoptosis, increase the expression of target proteins Notch1, Hes-5 and NICD by acting on the Notch pathway, and play a protective role for neurons [Astragaloside IV, the main saponin isolated from roots of pression . Osthole neurons . The NotThe Nrf2 signaling pathway plays a significant role in the occurrence and development of IS, and it can regulate the ability of cells to resist oxidative stress and protect brain tissue . Nrf2 beTrifolium pratense L., promotes the nuclear translocation of Nrf2 and induces the expression of HO-1 by regulating the Nrf2/HO-1 signaling pathway, it protects the rat brain from ischemic injury through antioxidant and anti-inflammatory effects [Coptis chinensis Franch., can reduce oxidative stress, inflammatory response, and neuronal apoptosis in MCAO mice by activating the AMPK/ Nrf2 pathway [Taraxacum mongolicum Hand.-Mazz., can significantly inhibit the generation of ROS and MDA in hippocampal neurons induced by OGD/R, leading to a decrease in caspase-3 and Bcl-2 expression, and a concurrent increase in the expression of Bax, HO-1, NQO-1, and GPX-3. Taraxasterol can protect hippocampal neurons from OGD/R-induced injury by activating the Nrf2 signaling pathway [Biochanin A, the main flavonoid component of  effects . Rosmari effects , can up- effects . Adenosi pathway . Taraxas pathway . In addi pathway ,77. The There are many experimental studies on the regulatory role of the PI3K/Akt signaling pathway in IS ,79,80. TReynoutria japonica Houtt. and Vitis vinifera L., it reduces the expression of IL-1β, COX-2 and TNF-α by stimulating the PI3K/Akt signaling pathway as well as decreasing infiltration of neutrophils, thereby reducing the inflammatory response in rats with ischemic stroke [Ligusticum chuanxiong Hort., can significantly increase the levels of p-Akt and p-eNOS in the brain tissue of MCAO rats, and play a neuroprotective role on the brain of ischaemic/reperfusion injury rats by stimulating the PI3K/Akt pathway [Polygala tenuifolia Willd., can downregulate the expression of Bcl-2/Bax and caspase-3 in PC12 cells and prevent OGD/R-induced injury by stimulating the PI3K/Akt signaling pathway [Puerariae Lobata (Willd.) Ohwi, can significantly increase the expression of Akt1, GSK-3β, and MCL-1 p62 as well as decrease caspase-3 expression levels in MCAO rats. These findings indicate that puerarin can regulate the neuroprotective mechanism of autophagy via the PI3K/Akt1/GSK-3β/MCL-1 signaling pathway [Resveratrol is a natural polyphenol isolated from plants such as c stroke . Ligustr pathway . Polygal pathway . Puerari pathway . In addi pathway ,92. The Magnolia officinalis Rehd. Et Wils) and Salvianolic acid B  can regulate brain injury induced by cerebral ischemia by activating SIRT1, deacetylating to inhibit Ac-FOXO1 expression, and suppressing inflammatory cytokines and apoptosis [O-β-D-glucoside, a flavonoid isolated from Astragalus penduliflorus subsp. mongholicus var. dahuricus (Fisch. ex DC.) X. Y. Zhu, can attenuate OGD/R-induced oxidative stress and neuronal apoptosis by activating SIRT1 and upregulating FOXO1 and PGC-1 α expression [SIRT1 is a nicotinamide adenine dinucleotide-dependent histone deacetylase with deacetylation of various histones and non-histones , which cpoptosis ,98. Calypression . MoreoveEpimedium brevicornu Maxim.) and ursolic acid  R. J. Wang and Actinidia chinensis Planch.) could further inhibit neuronal apoptosis by regulating the balance of MMP9/TIMP1, thereby significantly improving the ischemia-reperfusion induced BBB disruption in MCAO rats, preventing cerebral ischemia-reperfusion injury [Astragalus penduliflorus subsp. mongholicus var. dahuricus (Fisch. ex DC.) X. Y. Zhu) and oxymatrine  can reduce the expression of MMP9 protein by downregulating the expression of CAV1, thereby improving the integrity of the BBB after CIRI [MMP9 is a member of the zinc-dependent protein hydrolase family and can degrade extracellular matrix, including collagen IV, laminin, and fibronectin . MMP9 exn injury ,105. Calter CIRI ,107.Gentiana scabra Bunge., can inhibit and attenuate the expression of TLR4, MyD88 mRNA, and nuclear translocation of NF-κB in brain tissue, as well as the levels of IL-1β, TNF-α, and IL-6 in serum, suggesting that gentianine may reduce brain tissue injury due to ischemia/reperfusion by inhibiting TLR4 pathway-mediated inflammatory response [TLR4, also known as CD284, is a transmembrane protein in the Toll-like receptor family . During response . Procyanresponse .2+ generated during cerebral ischemia-reperfusion, thereby reducing brain cell apoptosis [Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C. A. Mey.) and Cardamonin  activates the HIF-1α/VEGF signaling pathway in rats with ischemia-reperfusion injury, and upregulate the protein expression of HIF-1α and VEGF, thereby increasing cerebral microvascular density and promoting intracerebral revascularization, and promoting angiogenesis, neural repair and functional recovery in MCAO rats [HIF-1α is a transcription factor that is widely distributed in mammals under hypoxic conditions and can activate a variety of hypoxia-response genes (HRGs) expression to regulate the oxygen homeostasis and energy metabolism balance of cells and organism . HIF-1α-poptosis , and canpoptosis . CatalpoCAO rats ,119.IS is a serious life-threatening disease associated with high rates of disability and mortality. Due to the rapid onset of the disease, there are many delayed factors in the treatment process, making early thrombolysis challenging to implement, thus affecting the outcome of neural health and functioning post-stroke. In recent years, a large amount of literature has shown that TCM can significantly improve IS with few side effects, demonstrating TCM’s potential benefits and significant potential for future development. TCM can regulate the signaling pathways to treat IS, such as JAK/STAT, NF-κB, MAPK, Notch, Nrf2, PI3K/Akt, which maintain BBB function, decrease brain edema, regulate energy metabolism, promote antioxidation, anti-inflammatory, and anti-apoptosis, reducing excitatory amino acid toxicity, enhancing neurogenesis, angiogenesis, and synaptogenesis.By summarizing and analyzing the natural compounds of traditional Chinese medicine that treat IS, we found that there are mainly flavonoids, alkaloids, polysaccharides, saponins, polyphenols, and terpenoids. The mechanism is mainly related to anti-oxidation, anti-inflammation, anti-apoptosis, and improving the permeability of BBB. The regulation effect of natural compounds from TCM on IS-related signaling pathways is shown in Despite the positive therapeutic effect of TCM on IS, there are still numerous challenges to overcome. Most of the research on TCM in IS models is still in the primary experimental stage, with few clinically applicable products. Furthermore, only known signaling pathways and related protein targets have been shown to play an important role in IS. However, there may be other pathway targets that have not yet been discovered. A comprehensive strategy combining metabolomics and network pharmacology is an effective tool to discover the targets and signaling pathways of TCM against cerebral ischemia-reperfusion. This strategy has been applied to TCM research, including drug target discovery, efficacy evaluation and mechanism research ,138,139.


Objective: Hedyotis diffusa-Scutellaria barbata herb pair (HS) has therapeutic effects on a variety of cancers, and this study aims to systematically explore the multiple mechanisms of HS in the treatment of colorectal cancer (CRC). Methods. The active ingredients of HS were obtained from TCMSP, and the potential targets related to these ingredients were screened from the STITCH, SuperPred, and Swiss TargetPrediction databases. Targets associated with CRC were retrieved by Drugbank, TTD, DisGeNET, and GeneCards. We used a Venn diagram to screen the intersection targets and used Cytoscape to construct the herb-ingredient-target-disease network, and the core targets were selected. The Go analysis and KEGG pathway annotation were performed by R language software. We used PyMol and Autodock Vina to achieve molecular docking of core ingredients and targets. Results: A total of 33 active ingredients were obtained from the HS, and 762 CRC-related targets were reserved from the four databases. We got 170 intersection targets to construct the network and found that the four ingredients with the most targets were quercetin, luteolin, baicalein, and dinatin, which were the core ingredients. The PPI analysis showed that the core targets were STAT3, TP53, MAPK3, AKT1, JUN, EGFR, MYC, VEGFA, EGF, and CTNNB1. Molecular docking results showed that these core ingredients had good binding potential with core targets, especially the docking of each component with MAPK obtained the lowest binding energy. HS acts simultaneously on various signaling pathways related to CRC, including the PI3K-Akt signaling pathway, proteoglycans in cancer, and the MAPK signaling pathway. Conclusions: This study systematically analyzed the active ingredients, core targets, and central mechanisms of HS in the treatment of CRC. It reveals the role of HS targeting PI3K-Akt signaling and MAPK signaling pathways in the treatment of CRC. We hope that our research could bring a new perspective to the therapy of CRC and find new anticancer drugs. Colorectal cancer (CRC) is a common digestive tract tumor, which has a high incidence rate, a high mortality rate, rapid progress, and easy spread . The incHedyotis diffusa belongs to the genus Hedyotis of Rubiaceae, and it is bitter, light, and cold in nature [Scutellaria barbata, belonging to Labiatae, is sour in taste and cold in nature [Hedyotis diffusa and Scutellaria barbata herb pair (HS) are widely used to treat cancer, and their combined effects are more than a single use of drugs. Studies have shown that HS can inhibit the proliferation of human breast cancer cells, and the combination of CTX treatment can significantly inhibit breast cancer model mice [TCM believes that cancer toxin is the core of tumor pathogenesis. After cancer toxin is produced, it can induce pathological products such as phlegm turbidity, qi stagnation, and blood stasis . Blood sn nature . Scutelln nature . They han nature , 13. Then nature , 15. Heddel mice , 17. In del mice . The mixdel mice .Network pharmacology emphasizes the combination of bioinformatics, system biology, and pharmacology, which not only explains the interaction between TCM and diseases but also conforms to the systematic and holistic view of TCM , 21. Nethttps://tcmspw.com/tcmsp.php). This study used oral bioavailability (OB) and ingredient drug-likeness (DL) as screening conditions for active ingredients.With the common name of a single drug as a keyword, all chemical ingredients of the drug were retrieved by TCMSP  (https:/https://pubchem.ncbi.nlm.nih.gov) to search and export the chemical structure data of active ingredients. Since the targets of ingredients without accurate structural information could not be predicted successfully, we decided to remove these ingredients after deleting the duplicate data. The active ingredients were predicted through the STITCH [http://stitch.embl.de/), SuperPred [http://prediction.charite.de/), and Swiss TargetPrediction [http://www.swisstargetprediction.ch/) databases to obtain the corresponding known or predicted targets. The duplicate data had been eliminated, and only the human targets were retained.We used PubChem  (https:/e STITCH  (http://uperPred  (http://ediction  (http://https://www.uniprot.org).The selected active ingredients were imported into the STITCH database for putative target prediction, and the targets with a confidence ≥0.7 were assumed as potential targets. The potential targets of drugs can also be obtained by inputting SMILES into the SuperPred database. The Canonical SMILES of the main active ingredients were uploaded to the Swiss TargetPrediction database, and the probability of each potential target was determined to be greater than or equal to 0.1. The retrieved targets were converted into standardized abbreviations by UniProt  (https:/https://www.drugbank.ca), TTD [http://db.idrblab.net/ttd/), DisGeNET [http://www.disgenet.org/), and GeneCards [https://www.genecards.org). Then the retrieval results of these databases were merged, and only one repeated target was reserved.Targets associated with CRC were collected from Drugbank  (https:/ca), TTD   platform for retrieval. Protein interaction data with high confidence (score >0.7) were selected and saved in a TSV format file. The information of node1, node2, and combined score in the file was imported into the software of Cytoscape to construct a PPI network, and the hub gene was screened by the cytohubba plug-in. The Sankey diagram revealed the relationship between herbs, core ingredients, and hub genes was drawn in R language version 4.1.1.The intersection targets related to HS and CRC were input into the STRING  , removed ions and water molecules through PyMol 2.4.0 [https://swift.cmbi.umcn.nl/servers/html/model.html) and prepared the receptor file in Autodock Vina. Then, the molecular docking simulation was carried out by using Autodock Vina software and visualized by PyMol 2.4.0. The 3D and 2D diagrams of molecular docking models were displayed by PyMol 2.4.0 and PROTEINS PLUS (https://proteins.plus/), respectively.We downloaded the SDF format 3D structure file of key ingredients from the PubChem database and converted it to PDB format through Open Babel. We also downloaded the 3D crystal structure of the hub genes through the PDB database , beta-sitosterol (MOL000358), and stigmasterol (MOL000449). The basic information of active ingredients in HS is shown in 37 ingredients were found in Hedyotis diffusa and 589 targets of Scutellaria barbata were obtained.The structures of these active ingredients were obtained in PubChem. We removed six ingredients named MOL001646, MOL000953, MOL005869, MOL012248, MOL012250, and MOL012270, which had no accurate structural information for predicting the target. The structures of the active ingredients were imported into STITCH, SuperPred, and Swiss Target Prediction databases for target prediction. A total of 490 potential targets of 77, 104, 353, and 390 CRC targets were obtained from four databases, including Drugbank, TTD, DisGeNET, and GeneCards, respectively, shown in the Venn diagram . A totalHedyotis diffusa and CRC, and 161 genes were shared by Scutellaria barbata and CRC. Then, the two herbs, 27 active ingredients, 170 intersection targets, and CRC disease were imported into Cytoscape 3.8.0 to construct the herb-ingredient-target-disease network  . The cytBased on GO enrichment and KEGG analysis, we determined how these intersecting targets function biologically. As a result, the bar plot of the top 10 terms of biological process (BP), cellular component (CC), and molecular function (MF) terms were displayed . In the In this study, we conducted molecular docking of 10 hub genes  with four core ingredients  to evaluate the protein-ligand binding potential. The results showed that the four ingredients had the best docking effect with MAPK, and the binding energies were -8.2 kcal/mol, -8.3 kcal/mol, -8.6 kcal/mol, and -8.0 kcal/mol, respectively .Quercetin and luteolin also achieved quite good docking results with TP53, and the docking results were -8.0 kcal/mol and -8.0 kcal/mol, respectively. The 3D diagrams of molecular docking models were displayed by PyMol 2.4.0, which showed the interaction of MAPK with quercetin, luteolin, baicalein, and dinatin . At the Hedyotis diffusa and Scutellaria barbata are heat-clearing and detoxicating TCMs. Hedyotis diffusa can also activate the blood circulation and relieve pain, while Scutellaria barbata can remove blood stasis and diuresis [Hedyotis diffusa is mainly achieved by enhancing immune function, interfering with the energy metabolism of tumor cells, inducing tumor cell apoptosis, and influencing the mitochondrial pathway [Scutellaria barbata include inhibiting tumor cell growth, inducing tumor cell apoptosis, inhibiting tumor angiogenesis and metastasis, regulating immunity, and reversing drug resistance of tumor cells [Both diuresis . They codiuresis . The ant pathway , 41. Howor cells , 43.γ and IL-2 levels, and decrease serum TNF-α level [2+, followed by Hedyotis diffusa, and the extract of Scutellaria barbata was relatively weak. It is suggested that the herb pair may inhibit or resist tumor formation by chelating the transition metal ions and blocking the lipid peroxidation chain reaction. In addition, HS can provide the most substantial DNA protection, which may be why it works [In other words, some of their functions are the same, especially in inducing apoptosis, enhancing cell immunity, and reducing telomerase activity . They al-α level . It is rit works .In this study, the network pharmacology method was used to analyze the active ingredients, targets, and potential mechanisms of HS in CRC treatment. Through the construction of an herb-ingredient-target-disease network and a PPI network of intersecting targets, the mechanism of action of HS on CRC was systematically analyzed.Hedyotis diffusa, and the other three are the main ingredients of Scutellaria barbata. It has been reported that quercetin can not only directly inhibit the proliferation of tumor cells [In the herb-ingredient-target-disease network, quercetin, luteolin, baicalein, and dinatin have more targets than other ingredients, which may be the core active ingredients of HS. Quercetin is one of the main ingredients of or cells , 46, butor cells  and actior cells  and inhior cells , 50. Lutor cells , 52. In or cells –55. Baicor cells , 57. It or cells , 59. Dinor cells –62. Moreor cells .In the PPI network，we identified ten hub genes, namely, STAT3, TP53, MAPK3, AKT1, JUN, EGFR, MYC, VEGFA, EGF, and CTNNB1, which may be the targets of HS in the treatment of CRC. The activation of STAT3 could be observed in various tumors, which was closely related to inflammation and immunity and promoted tumor progression as an oncogene . The expIn the pathway enrichment, the PI3K-Akt signaling pathway hsa04151) has been identified as the critical target of tumor-targeted therapy, which plays a vital role in regulating the proliferation, migration, and apoptosis of tumor cells –77. Phos1 has beeIn this study, we screened four core ingredients  from 33 HS active ingredients and obtained 170 intersection targets related to CRC. The top 10 hub genes were STAT3, TP53, MAPK3, AKT1, JUN, EGFR, MYC, VEGFA, EGF, and CTNNB1, which may be the core targets of HS. Our study shows that HS acts simultaneously on a variety of signaling pathways related to CRC, such as the PI3K-Akt signaling pathway, proteoglycans in cancer, and the MAPK signaling pathway, which provides a reference for the research on the specific mechanism of the drug in the treatment of CRC. However, the limitation of this study is that network pharmacology ignores the content of each ingredient in the drug. In addition, the current research methods ignore the possible production of new compounds in the process of drug decoction. At the same time, another limitation is that network pharmacology has a high false-positive rate. It is necessary to illustrate the molecular level of HS on the treatment of CRC in the future. However, this screening technology, combined with network pharmacology and molecular docking, saves a lot of scientific research resources and helps researchers screen drug ingredients and core targets efficiently. In the follow-up research, on the one hand, we could continue to study the effect of HS on the treatment of CRC at the molecular level. On the other hand, we need to constantly optimize the technology related to drug screening to achieve more accurate drug screening and reduce the false-positive rate. We hope that our research can bring a new perspective to the therapy of CRC and find new anticancer drugs.


Ardisia gigantifolia Stapf. (AGS), a Chinese folk medicine widely grows in the south of China and several studies reported that AGS could inhibit the proliferation of breast cancer, liver cancer, and bladder cancer cell lines. However, little is known about its anti-colorectal cancer (CRC) efficiency.In the present study, a combination of MTT assay, network pharmacological analysis, bioinformatics, molecular docking, and molecular dynamics simulation study was used to investigate the active ingredients, and targets of AGS against CRC, as well as the potential mechanism.50 values of 197.24, 264.85, 15.45 µg/mL on HCT116 cells, and 523.6, 323.59, 150.31 µg/mL on SW620 cells, respectively. Eleven active ingredients, including, 11-O-galloylbergenin, 11-O-protocatechuoylbergenin, 11-O-syringylbergenin, ardisiacrispin B, bergenin, epicatechin-3-gallate, gallic acid, quercetin, stigmasterol, stigmasterol-3-o-β-D-glucopyranoside were identified. A total of 173 targets related to the bioactive components and 21,572 targets related to CRC were picked out through database searching. Based on the crossover targets of AGS and CRC, a protein-protein interaction network was built up by the String database, from which it was concluded that the core targets would be SRC, MAPK1, ESR1, HSP90AA1, MAPK8. Besides, GO analysis showed that the numbers of biological process, cellular component, and molecular function of AGS against CRC were 1079, 44, and 132, respectively, and KEGG pathway enrichment indicated that 96 signaling pathways in all would probably be involved in AGS against CRC, among which MAPK signaling pathway, lipid, and atherosclerosis, proteoglycans in cancer, prostate cancer, adherens junction would probably be the major pathways. The docking study verified that AGS had multiple ingredients and multiple targets against CRC. Molecular dynamics (MD) simulation analysis showed that the binding would be stable via forming hydrogen bonds.MTT assay showed that three kinds of fractions from AGS, including the n-butanol extract (NBAGS), ethyl acetate fraction (EAAGS), and petroleum ether fraction (PEAGS) significantly inhibited the proliferation of CRC cells, with the ICOur study showed that AGS had good anti-CRC potency with the characteristics of multi-ingredients, -targets, and -signaling pathways.The online version contains supplementary material available at 10.1186/s12906-022-03822-8. Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide which occurs in the colon or rectum . The incIn the clinic, the conventional treatments of CRC mainly include surgery, chemotherapy/radiotherapy, and targeted drug therapy. The CRC drugs currently used are 5-fluorouracil (5-FU), oxaliplatin, and irinotecan, and monoclonal antibodies newly developed including bevacizumab  and cetuximab . However, the 5-year survival rate of CRC patients in the advanced stages remains poor, being 18.5% in the United States and 27.7% in Europe, respectively . Hence, Ardisia gigantifolia Stapf. (AGS) is a kind of TCM that widely grows in the south of China. It was first recorded in the book of Sheng-Cao-Yao-Xing-Bei-Yao in the Qing dynasty and was commonly used for treating rheumatism, the pain of bones and muscles, and traumatic injury [c injury . Previouc injury , 13. AGSc injury . Severalc injury , Bel-740c injury , and EJ c injury . HoweverNetwork pharmacology, an emerging methodology and a useful bioinformatics tool to investigate the complex effects and mechanisms between drugs and diseases at molecular, cellular, tissue, and biologic levels from a systems-level perspective, is now widely used in TCM investigation . In thisThe roots and rhizomes of AGS were purchased from Guangzhou Zhixin medicinal herbs Co., Ltd  and identified by Professor Suying Tian . The ethanol, petroleum ether , ethyl acetate, and n-butanol were obtained from Xilong Chemical Co., Ltd . Diatomite was obtained from Dingshengxin Chemical Co., Ltd . McCoy’s 5 A medium was purchased from Procell Life Science&Technology Co., Ltd . Leibovitz’s L-15 medium was purchased from Dalian Meilun Biotechnology Co., Ltd . Fetal bovine serum, phosphate-buffered saline, 5% trypsin-EDTA were purchased from Gibco . Penicillin-streptomycin (P/S) and 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Beyotime Biotechnology .The roots and rhizomes of AGS were powdered by a high-speed multifunctional crusher  to obtain a granulometry of 2 ~ 5 mm. 3 kg of dry powder was combined with 70% ethanol in a round glass flask and extracted by a reflux method for 2 h. This process was repeated three times with the ratio of powder/ethanol of 1:8, 1:6, and 1:4 (w/v), respectively. All the extracted products were collected and dried under a vacuum freeze dryer . The extraction yield is 11.28%. Afterward, the ethanolic extracts were dissolved in distilled water and mixed well with diatomite. Dry it in an automatic program-controlled oven with the temperature setting at 70 ℃ and then grind the mixture into a fine powder . The mix2.Human colorectal cancer cell lines (HCT-116 and SW620) were commercially purchased from the cell resource center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , and cultured in McCoy’s 5 A medium and Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, respectively, at 37 ℃ in a humidified incubator with 5% CO3 / well) and SW620 cells (4.5 × 103 / well) were seeded in 96-well plates and allowed to grow for 24 h, and then treated with different concentrations of PEAGS, EAAGS, NBAGS (0 ~ 1000 µg/mL) for 48 h. After treatment, 20 µL of MTT solution (5 mg/mL) was added to each well and co-incubated for another 4 h at 37 °C. Finally, the supernatant was removed and 150 µL of DMSO was added to each well to dissolve the purple formazan crystals. A microplate reader  was used to measure the optical density (OD) of each well at a wavelength of 570 nm. Each experiment was performed in triplicate.HCT-116 cells (2.5 × 10https://www.cnki.net/), Blyun database (http://www.blyun.com/), and Pubmed database (https://pubmed.ncbi.nlm.nih.gov/). The chemical structures of these active ingredients were drawn using the ChemDraw software and were input into the PharmMapper database (http://www.lilab-ecust.cn/pharmmapper/) to obtain the PDB IDs, target names, and fit scores. Then, the potential target set was further summarized according to the fit scores (> 0.7). After excluding the same targets and non-Homo sapiens targets from the screened targets, the PDB IDs were transformed into the gene symbol and gene IDs via the UniProt database (http://www.uniprot.org/). The CRC-related targets were obtained from the GeneCards Human database (https://www.genecards.org) using “colorectal cancer” as a keyword. The crossover genes between AGS and CRC were screened by the R software using the Venn Diagram package after eliminating duplicates. A compound-disease-target network was constructed using the Cytoscape3.7.2 to further explore the therapeutic mechanism of AGS against CRC. The PPI network construction of the predicted targets of AGS in treating CRC was performed based on their interaction data by applying the Interacting Genes/Proteins (STRING) database  and visualized using Cytoscape v3.7.2 with the parameters of the minimum required interaction score = 0.9 and hide disconnected nodes in the network. The network can be used to predict the protein interactions , among which the nodes represent the intersected target proteins, and edges represent the predicted or validated interactions between proteins. All collective proteins/genes were subjected to Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using the database  for annotation, visualization, and integrated discovery. GO analysis consists of Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). KEGG analysis [http://www.bioinformatics.com.cn/).The potential active ingredients in AGS were determined by referring to the published literature by searching 3 databases, the CNKI database . Structures for the 6 bioactive ligands were prepared via the ‘LigPrep’ module for possible ionization and optical isomers with OPLS3e field forces. The 3D structures of the potential targets including SRC (PBD ID: 3G5D) [http://www.rcsb.org). All the proteins were processed by undergoing the following procedure before the docking calculations: preprocessing, reviewing, modification, and refinement . The binding sites were determined according to the coordinates of the protein ligands. All compounds were docked by docking with standard precision (SP). The docked conformers were evaluated using the docking score. PyMOL 2.4.0 software was used to study the interaction between the docked molecules.To explore the interactions and binding modes between the active ingredients of AGS and the predicted targets from the network analysis, a molecular docking simulation was carried out using Maestro Schrodinger software 12.5. Six characteristic active components which possess the most targets and the top 5 collective targets were selected for verifying molecular docking. The three-dimensional (3D) sdf format of the 6 active ingredients including 4-O-galloylbergenin, 11-O-galloylbergenin, 11-O-protocatechuoylbergenin, 11-O-syringylbergenin, bergenin, epicatechin-3-gallate was obtained from the PubChem database (D: 3G5D) , MAPK1 (D: 3G5D) , ESR1 (PD: 3G5D) , HSP90AAD: 3G5D) , MAPK8 (D: 3G5D)  were dowMolecular dynamics (MD) simulation analysis was performed to evaluate the stability of the protein-small molecule complex in the present study. MD can not only calculate the binding affinities of small molecules within the binding sites in the binding process but also exhibit dynamic conformational changes with the time scale , 29. TheMolecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) –41 was abinding) can be decomposed into five terms: internal energy , van der Waals (VDW), electrostatic interaction (elec), and free energy of solvation which consists of polar (GB) and non-polar solvation free energy (SA). Polar solvation free energy was calculated by the GB model (igb = 2) [SA = 0.0072 × In formula (1), the binding free energy (igb = 2)  and non-nt}ΔSASA . All theThe results were expressed as mean ± SEM analyzed with a one-way analysis of variance (ANOVA) using Prism 8.0.1 . P < 0.05 was considered to be significantly different.50 (50% inhibitory concentration) values of NBAGS, EAAGS, and PEAGS were 197.24, 264.85, 15.45 µg/mL on HCT-116 cells . According to the results, the core targets were predicted to be SRC, MAPK1, ESR1, HSP90AA1, MAPK8, which had more connections than other genes.Based on the crossover targets of AGS & CRC, the PPI network was built up by the String database. As shown in Fig. As a result, GO analysis showed that the numbers of BP, CC, and MF of AGS against CRC were 1079, 44, and 132, respectively  and the 6 characteristic active components were shown in Table ), which indicated that AGS had multiple ingredients and multiple targets against CRC.Altogether, the results showed that the target proteins SRC and ESR1 had stronger docking capability with AGS than the other targets. Four compounds  had a lower docking score than the ligand-protein of SRC, and two compounds  docked better than the ligand-protein of ESR1 (Table 1) were the most stable systems, with the lowest RMSD value fluctuating stably within the range of 2 Å, which were followed by SRC-11-O-protocatechuoylbergenin complex, SRC-epicatechin-3-gallate complex , and MAPK8-11-O-galloylbergenin complex  simulation was used to analyze the stability of compounds from AGS binding multiple targets including SRC, MAPK1, ESR1, HSP90AA1, and MAPK8. We analyzed the root mean square deviation (RMSD) plot, presenting the stability of protein, and the higher of RMSD value is, the more violent the fluctuation is. On the contrary, the protein binding is stable. As shown in Fig. To examine the structural-flexibility effect of small molecules on target proteins in the process of molecular dynamics simulation. We analyzed the root mean square fluctuation (RMSF) plot of the 10 complexes. In general, the flexibility of the target protein decreases after a compound binding to the protein, to stabilize and activate the protein . As showThe hydrogen bond is one of the strongest non-covalent interactions between small molecule compounds and proteins. During the molecular dynamic simulation, we analyzed the number of hydrogen bonds in all 100 ns of 10 complexes. As shown in Fig. In this study, MD trajectories were fully sampled to accurately calculate the binding energy between small molecules and proteins based on MM/GBSA calculation method. As shown in Table Colorectal cancer ranks third among the causes of cancer-related death and carries a huge global health burden . AlthougScutellaria baicalensis Georgi, Huangqin), curcumin , berberine  have been demonstrated to have good efficiencies in cancer treatment [Traditional Chinese herbal medicine (TCM) has been used for thousands of years in China, with the characteristics of definite therapeutic effects and low side effects, and has been used as an alternative therapy for cancer patients. Shi et al. found that TCM could significantly improve the disease-free survival of cancer patients, in particular with stage III patients , and Xu reatment –48. TherIn our study, we verified that the 3 parts of AGS extracts, PEAGS, EAAGS, and NBAGS showed a good anti-CRC effect against HCT-116 and SW620 cells proliferation determined by MTT assay. Literature searching indicated that the flavones  and terpenoids  were the major pharmacological active ingredients of AGS , 50. ChaNetwork pharmacology, an emerging discipline that is commonly used to predict the potential targets and mechanisms based on the accumulation of evidence from big data, would provide strong support and accelerate the process of new drug development. In our study, we utilized the network pharmacological methodology to explore the pharmacological mechanism of the active components of AGS against CRC, and molecular docking and molecular dynamic simulation were used to investigate the docking pattern and capability between major active compounds of AGS and potential target proteins. The results showed that 11 active ingredients in AGS would have effects on 170 overlapping genes that acting important roles in CRC treatment. PPI network study showed that SRC, MAPK1, ESR1, and HSP90AA1 were the most correlated proteins, followed by MAPK8. SRC family kinase is a key mediator of cellular tumor-promotion genic signals linked with tumor proliferation, migration, and invasion, and SRC could be activated in CRC through various mechanisms, including the regulations of the SRC-STAT3 signaling pathway, SRC-CTNNB1, and macro-autophagy/autophagy pathways , 57. TheGO analysis showed that the numbers of BP, CC, and MF of AGS against CRC were 1079, 44, and 132, respectively. It was concluded that AGS mainly participated in the biological process of steroid metabolic process. The major reaction site was the cytoplasm in CRC treatment, and the central molecular function mainly included steroid hormone receptor activity, nuclear receptor activity, transcription factor activity, steroid binding, and endopeptidase activity. KEGG pathway analysis showed that 96 pathways in all were included and the top 10 pathways were MAPK signaling pathway, lipid and atherosclerosis, proteoglycans in cancer, prostate cancer, adherens junction, endocrine resistance, progesterone-mediated oocyte maturation, relaxin signaling pathway, FoxO signaling pathway, apoptosis, among which the MAPK signaling pathway, lipid and atherosclerosis, proteoglycans in cancer, prostate cancer, FoxO signaling pathway, and apoptosis were commonly recognized to be cancer-related. It is reported that the MAPK signaling pathway plays an important role in cell proliferation, and is commonly activated by its upstream growth-factor receptors, such as the epidermal growth factor which is commonly over-expressed in colorectal cancer . The altAGS has a wide range of biological properties including anti-cancer activity. Gu found that the biotransformation product S1 from AGS had significant inhibition on 6 kinds of tumor cell lines and the potential mechanism may be related to cell cycle arrest . Cyclin In all, our study firstly investigates the anti-CRC of AGS, explores the potential mechanism, analyzes the docking patterns and binding capabilities between the active compounds and target proteins, as well as studies the stability of the complexes. The results suggest the potential application of AGS in colorectal cancer treatment or prevention for humans through integrating experimental evaluation and network study. The 11 potential active ingredients were showing a summary of the composition from AGS based on the existing database. Although the PEAGS showed higher cytotoxicity, the NBAGS and EAAGS also had good inhibitory potency against CRC cell growth. It was suspected that there probably were some unknown compounds with good anti-tumor activity, including the new compounds that have never been reported or excluded in the databases. Hence, it is needed to conduct a series of experiments to identify the constituent structures in PEAGS, which is our follow-up study.50 values of 197.24, 264.85, 15.45 µg/mL on HCT-116 cells, and 523.6, 323.59, 150.31 µg/mL on SW620 cells. Network pharmacological analysis suggested that eleven active ingredients were identified, including 4-O-galloylbergenin, 11-O-galloylbergenin, 11-O-protocatechuoylbergenin, 11-O-syringylbergenin, ardisiacrispin B, bergenin, epicatechin-3-gallate, gallic acid, quercetin, stigmasterol, stigmasterol-3-o-β-D-glucopyranoside. The PPI network showed that SRC, MAPK1, ESR1, HSP90AA1, and MAPK8 would probably be the core targets of AGS against CRC. GO analysis showed that the numbers of biological process, cellular component, and molecular function of AGS against CRC were 1079, 44, and 132, respectively, and KEGG enrichment suggested that 96 signaling pathways in all would probably be involved in AGS against CRC, among which MAPK signaling pathway, lipid and atherosclerosis, proteoglycans in cancer, prostate cancer, adherens junction would probably be the major pathways. The molecular docking study showed that the targets proteins SRC and ESR1 had strong docking capability with AGS than the other targets. The compounds of 11-O-galloylbergenin, 11-O-protocatechuoylbergenin, 11-O-syringylbergenin, epicatechin-3-gallate have higher docking scores than the ligand-protein of SRC, and the compounds of bergenin, epicatechin-3-gallate dock better than the ligand-protein of ESR1, suggesting that AGS has multiple ingredients, multiple targets, and multiple pathways against CRC. Our study for the first time investigates the anti-CRC potency of AGS, as well as uncovers the underlying mechanism. The results can probably provide valuable information for further study on the anti-CRC effect of AGS as well as its underlying mechanism.This study showed that all the three kinds of fractions from AGS, including the n-butanol extract (NBAGS), ethyl acetate fraction (EAAGS), and petroleum ether fraction (PEAGS), could significantly inhibit the proliferation of CRC cells, with the ICAdditional file 1. Targetsof the compounds from Ardisia gigantifolia Stapf. against colorectal cancerbased on network searching.Additional file 2. Thetherapeutic targets related to colorectal cancer obtained by searching theGenecards database.Additional file 3. Theoverlapped targets of Ardisia gigantifolia Stapf. and colorectal cancer.Additional file 4. GO Analysis.Additional file 5. KEGG pathway enrichment analysis.


Alzheimer’s disease (AD) as a neurodegenerative disease occupies 3/5–4/5 cases among patients with dementia, yet its pathogenetic mechanism remains unclear. Geraniol, on the other hand, is a well-known extract from essential oils of aromatic plants and has been proven that it has outstanding neuroprotective effects as well as ameliorating influence in memory impairment. Therefore, the present study aims to elucidate the potential of geraniol against AD by network pharmacology-based approach combined with molecular modeling study.Firstly, we evaluated the druggability of geraniol by ADME method. Then, we obtained the geraniol targets and AD-related targets from multiple open data sources. Afterward, we calculated the intersection through a Venn diagram to find common targets, and via Panther classification system to categorize them. In order to gain a macroscopic understanding of these common targets, we carried out GO terms and KEGG pathways enrichment analyses, according to which we constructed a compound–target–pathway–disease network. In addition, we built a preliminary PPI network which was further analyzed both functionally and topologically. Consequently, five hub targets were sorted out. Finally, we conducted molecular docking and molecular dynamic simulation to validate our findings.In the present study, the pharmacological properties of geraniol were assessed according to ADME and Lipinski’s rule, which demonstrate promising druggability. Then, from 10,972 AD-related targets and 33 geraniol targets, 29 common targets were identified, among which 38.1% of them are metabolite interconversion enzymes, 23.8% are protein modifying enzymes, 33.3% are transmembrane receptors, and the rest are transporters. Enrichment analyses hint that geraniol is involved in cholinergic synapse, serotonergic synapse, and neuroactive ligand–receptor interaction. We also built a preliminary PPI network to investigate the interplay between these targets and their extensive interactions. Then, by functionally clustering the preliminary PPI network, we gained a cluster of proteins which formed a subnetwork with score of 8.476, and 22 nodes. Its results of GO terms and KEGG pathways enrichment analyses once again suggests that geraniol actively participates in cholinergic synapse, serotonergic synapse, and neuroactive ligand–receptor interaction, which are believed to be strongly associated with AD pathogenesis. Besides, topological analyses of the preliminary PPI network helped find 5 hub targets . To verify their interaction with geraniol molecule, we conducted molecular docking, and found that CHRM3 possesses the highest affinity in binding, indicating that geraniol molecules are closely bound to each hub target, and CHRM3 may serve as a key target of geraniol against AD. It was then further confirmed by molecular dynamic simulation, the result of which supports our hypothesis.The present study shares a mechanistic insight of the potential of geraniol against AD, giving a reference to future experimental studies.The online version contains supplementary material available at 10.1186/s40001-022-00699-8. Alzheimer’s disease (AD) has been a common neurodegenerative disease that occupies 3/5–4/5 cases among patients with dementia, yet its pathogenetic mechanism of AD is very complicated and thus remains unclear , 2. The Figure https://pubchem.ncbi.nlm.nih.gov/) and was input into SwissADME (www.swissadme.ch) [The sdf file of geraniol’s structure was downloaded from PubChem (adme.ch)  that prowww.swisstargetprediction.ch) this time to find the putative targets of geraniol [https://www.genecards.org) to obtain the AD-related targets [http://jvenn.toulouse.inra.fr/app/example.html) to calculate the intersection of them which are their common targets [http://www.pantherdb.org/) [We used the sdf file again, but with Swiss Target Predictor  .www.webgestalt.org), as a robust online bioinformatic toolkit, was used for GO and KEGG pathway enrichment analyses of the common targets [WebGestalt  for better visualization . In addiWe used a Cytoscape plug-in, “MCODE” to functionally cluster the preliminary PPI network, which created a subnetwork . With thWe used another Cytoscape plug-in, “CytoHubba”, to extract a core PPI network that frames the preliminary PPI network via intersectional merge of the subnetworks created from degree, closeness, and betweenness methods .http://clab.labshare.cn/cb-dock/php/blinddock.php) based on AutoDock Vina [To predict the interaction between the targets and the compounds, we downloaded the crystal structures of the 5 hub targets from PDB library  and then docked them with geraniol molecule on CB-dock platform  is considered as potential druggable substance, geraniol possessing MW = 154.25 Da, Hdom = 1, Hacc = 1, RBN = 4, TPSA = 20.23 Å2, LogKp = − 4.71 cm/s, therefore is deemed to be one of such molecules that can be further optimized into orally administered drug.As usually a molecule fulfilling the parameters of RBN < 10, OB ≥ 20%, DL ≥ 0.1, TPSA < 60 ÅDetails are shown in Table Through searching, 10,972 AD-related targets and 33 geraniol targets were found from which 29 of them are common targets. They are demonstrated as an intersection in the Venn diagram in Fig. With the common targets, we conducted GO terms and KEGG pathways enrichment analyses to gain a macroscopic understanding of their functions, from which we found that GO:1901700 (response to oxygen-containing compound) is the most enriched GO term, followed by GO:0008015 (blood circulation), GO:0003013 (circulatory system process), and hsa04725 (cholinergic synapse), hsa04726 (serotonergic synapse), hsa00590 (arachidonic acid metabolism) are the most enriched KEGG pathways, as shown in Fig. Based on the enrichment results, we constructed the geraniol–target–pathway–AD network, as shown in Fig. K core value to 3 and keeping the rest parameters in default values, we functionally clustered the proteins involved in the preliminary PPI network as what Fig. By setting Based on these genes, we conducted the GO terms and KEGG pathways analyses to have a deeper insight of their functions. The results in Fig. By using the CytoHubba plug-in, we can make a further in-depth analysis of the topology of the network. We screen 3 subnetworks with top 10 ranking nodes in Fig. Molecular docking between geraniol and the hub targets was carried out to ascertain the binding mode between them. It was found that the best run came with CHRM3 with the lowest affinity energy of − 5.9 kcal/mol, followed by JAK1, JAK2 and PRKCA , and PRKCD , which suggests that CHRM3 may serve as the most important player of the underlying mechanism against AD. Detailed of the binding modes are available in the Additional file Although there has been numerous research on geraniol and AD separately, to the best of our knowledge, the present study is the very first one to integrate network pharmacology and molecular modeling to give a mechanistic insight of the potential mechanisms of geraniol in AD treatment.The network pharmacological analysis identified 10,972 AD-related targets, 33 geraniol targets, among which 29 of them are common targets. Among the 29 common targets, 38.1% of them are metabolite interconversion enzymes, 23.8% are protein modifying enzymes, 33.3% are transmembrane receptors, and the rest are transporters. They mainly participate in cholinergic synapse, serotonergic synapse, and neuroactive ligand–receptor interaction. Earlier progress in neuroscience revealed that high density of cholinergic synapse in the human central nervous system is critically important in terms of memory, learning, as well as attention and thus significantly associated with age-dependent neurological declines, such as AD . DysreguIn addition, we constructed a preliminary PPI network aiming to sort out the hub targets. After topological optimization of the network, 5 hub targets  were screened out. CHRM3 has been found to heterodimerize with CHRM2 which contributes to memory and cognition –13, 28. Furthermore, we conducted molecular modeling to study the robustness of the interaction between each hub target and geraniol molecule. Subsequently, it is found that CHRM3 possesses the highest affinity with geraniol molecule and thus may serve as the key target of geraniol molecule in AD treatment.In conclusion, the present study shares a mechanistic insight of the potential of geraniol against AD, giving a reference to future experimental studies.Additional file 1. S1_Rare data for target identification, S2_Rare data for GO terms and KEGG pathways analyses, S3_Rare data for GO terms and KEGG pathway analyses of functional clustering, S4_Rare data for molecular docking.


Based on the protein interaction network, the important targets of ÜS were significantly enriched in PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction pathway, Ras signaling pathway, etc. ÜS in ICVD has characteristics like multiple targets, multiple approaches, and multiple pathways. Results of molecular docking showed that the active components in ICVD had a good binding ability with the key targets. Its main biological mechanism may be related to the PI3K-Akt and Ras-MAPK centered signaling pathway. Our study demonstrated that ÜS exerted the effect of treating ICVD by regulating multiple targets and multiple channels with multiple components through the method of network pharmacology and molecular docking.This paper aims to study the potential biological mechanism of Üstikuddus Sherbiti (ÜS) in the treatment of ischemic cerebrovascular diseases (ICVD) by the network pharmacology method. Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to obtain effective constituents of ÜS by screening eligible oral utilization, drug similarity, and blood-brain barrier permeability threshold. By drug target prediction and stroke treatment target mining, 2 target data sets were analyzed to find intersection targets and the corresponding constituents were used as active constituents. An active constituent target network and an effective constituent target network were constructed by using Cytoscape 3.7.2 software. Degree parameters of the effective constituent target network were analyzed to find important effective constituents and targets. Through protein-protein interaction (PPI) analysis/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, potential signaling pathways of ÜS in ischemic stroke were found out. AutoDock was used for molecular docking verification. A total of 90 active constituents of ÜS were screened out. There were 10 active constituents against ICVD, including quercetin, luteolin, kaempferol, and naringenin, and 10 important targets for anticerebral ischemia, namely, PIK3CA, APP, PIK3R1, MAPK1, MAPK3, AKT1, PRKCD, Fyn, RAC1, and NF- Cerebral ischemia is one of the main causes of adult death and disability. Due to its high incidence, high disability, and high fatality rate, it has become one of the common problems faced by various countries in the field of public health , 2. AccoLavandula angustifolia Mill., [Adiantum flabellulatum L., [Paeonia lactiflora Pall., [Anchusa Italica Retz., [Liquiritiae radix [, Cordia dichotoma seeds [Althaea Rosea L. [Viola tianschanica Maxim. [Foeniculum vulgare Mill. [Thrombolysis and thrombectomy are conventional and effective treatments for ICVD. The treatment time window is short and is accompanied by reperfusion injury. Evidence-based medicine shows that intravenous thrombolysis and mechanical thrombolysis are aimed at the early pathological characteristics of ischemic stroke and are safe and effective treatment methods . Howevera Mill.,  Adiantumatum L., , 15 Paeoa Pall., , 17 and a Retz., –20 whichae radix , 22, Corma seeds , 24, rosma seeds , 26, andma seeds , to enhaRosea L.  and Viola Maxim.  adjust ta Maxim.  and Foenre Mill.  correct re Mill. . In recere Mill. , 33, 34.re Mill. , 35, butre Mill. , 37.With the help of the network pharmacology platform, this research explores the potential biology of ÜS in the treatment of ICVD from the perspective of systems biology by mining and analyzing the effective components and related targets of ÜS against the ICVD mechanism.The Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform is a network pharmacology database that integrates the information of traditional Chinese medicine ingredients and corresponding target information. The database contains key parameters for the absorption, distribution, metabolism, and excretion of traditional Chinese medicine components, including oral bioavailability (OB), drug-likeness (DL), blood-brain barrier permeability (BBB). In this study, we used the TCMSP platform and searched the PubChem database to collect all the components of ÜS and obtained the OB, DL, and BBB values of each component, with OB ≥ 30%, DL ≥ 0.18, and BBB ≥ -0.3 as candidate active ingredients basis.https://www.drugbank.ca/DrugBank database: https://www.omim.org/Online Mendelian Inheritance in Man (OMIM) database: https://geneticassociationdb.nih.gov/Genetic Association Database (GAD): https://database.idrb.cqu.edu.cn/TTD/Therapeutic Target Database (TTD): https://stitch.embl.de/STITCH database: https://www.swisstargetprediction.ch/SwissTargetPrediction database: https://www.disgenet.org/home/DisGeNET database: https://bioinfogp.cnb.csic.es/tools/venny/Venny 2.1 software: https://www.tcmip.cn/TCMIP/index.php/Home/Index/index.htmlTCMIP database: https://scholar.google.com.hk/?hl=zh-CN/Google Scholar database: https://www.uniprot.orgUniProt database: https://david.ncifcrf.gov/DAVID database: https://www.alzdata.org/index.htmlAlzData database: https://www.omicshare.com/tools/OmicShare online data analysis platform: https://cytoscape.org/Cytoscape software: https://www.funrich.org/FunRich software: https://metascape.org/gp/index.htmlMetascape online data analysis platform: We use the TCMSP database to obtain the potential target information corresponding to each active ingredient screened in the ÜS, and search the targets from different sources in the UniProt database to obtain all target gene names, used for subsequent network pharmacology analysis.We use the keywords “cerebral ischemia,” “ischemic stroke,” and “brain oxidative stress” to search DrugBank, OMIM, GAD, STITCH, and DisGeNET databases to obtain information on cerebral ischemia targets.We enter the obtained targets of ICVD and the targets of ÜS components into the UniProt database to search for and obtain the gene symbols of all targets for subsequent analysis. In addition, we use Venny 2.1 online tool to draw the Venn diagram of the active ingredient targets of ÜS and the related targets of ICVD, and get the intersection target.Cytoscape 3.7.2 software was used to construct a compound-target interaction network and an anticerebral ischemia drug effect component target interaction network. Traditional medicine components or targets are expressed as nodes in the network, and their connections are expressed as edges. Network analyzer in Cytoscape 3.7.2 software is used to calculate the degree of nodes (degree) parameters to evaluate the importance of medicinal ingredients and targets.The above-obtained intersection targets were input into the AlzData database  to analyTo understand the interaction of target proteins at the system level, we use the Metascape database to analyze and obtain a protein interaction network diagram.P < 0.01 was considered significant. We drew bubble charts by OmicShare tool, a free online platform for data analysis [We used the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to perform Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes  signaling pathways to gain insight into the biological processes (BP), molecular functions (MF), and KEGG signaling pathways involved . FunRichanalysis . Moreoveanalysis .μm 2.1 ∗ 100 mm). The column temperature was set at 55°C, and the sample injection volume was set at 5 μL. The flow rate was set at 0.5 mL/min. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multistep linear elution gradient program was as follows: 0–11 min, 85–25% A; 11–12 min, 25–2% A; 12–14 min, 2–2% A; 14–14.1 min, 2–85% A; 14.1–15 min, 85–85% A; 15–16 min, 85–85% A. A Q Exactive Focus mass spectrometer coupled with Xcalibur software was employed to obtain the MS and MS/MS data based on the IDA acquisition mode. During each acquisition cycle, the mass range was from 100 to 1500, the top three of every cycle were screened, and the corresponding MS/MS data were further acquired. Sheath gas flow rate was 45 arb, aux gas flow rate 15 arb, capillary temperature 400°C, full MS resolution 70000, MS/MS resolution 17500, collision energy 15/30/45 in NCE mode, and spray voltage 4.0 kV (positive) or −3.6 kV (negative).The samples of ÜS were analyzed by an Agilent 1290 ultrahigh performance liquid chromatography (UHPLC) system with a Waters UPLC BEH C18 column (1.7 https://www.rcsb.org/) was used to retrieve and download the 3D structure files of key target proteins. 3D structure files of compounds were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) [To validate the compound-target associations, the AutoDock software (version 4.2) was used to perform the molecular docking program . RCSB PDih.gov/) . Finallyih.gov/) . Ligand ih.gov/) .We screened out 90 active ingredients based on OB ≥ 30%, DL ≥ 0.18, and BBB ≥ −0.3, including quercetin, luteolin, kaempferol, and naringenin. According to the OB value, A total of 443 targets for the active ingredients of ÜS were obtained by searching the TCMSP database. Through keyword search of 5 online databases, DrugBank, OMIM, GAD, TTD, and DisGeNET, 1,903 related targets of ICVD were obtained. The 283 intersection targets obtained from the intersection of the above two data sets are used for subsequent network pharmacology analysis, as shown in Using Cytoscape 3.7.2 software to construct the active ingredient-target interaction network, a network with 343 points and 1994 edges was obtained. Among them, the top 10 targets ranked by degree are quercetin, luteolin, naringenin, jaranol, ZINC519174 (2R-7-Hydroxy-2-(4-hydroxyphenyl)-2,3-dihydrochromen-4-one), catechin, isoformononetin, kaempfride, isobavachin, and kaempferol as shown in κB1.The obtained intersection target was taken as the pharmacodynamic target, and Cytoscape 3.7.2 was used to construct the pharmacodynamic ingredient-target interaction network see . The topWe import ÜS and anti-ischemic stroke-related targets into the Metascape database for analysis and obtain a protein interaction network diagram (see Figures 2P value, the smallest 15 cell components (We used FunRich software to analyze the tissue distribution of ÜS targets . The resmponents  were selmponents  and molemponents . We haveThe Venn diagram of the component targets of ÜS and the disease targets of ICVD was drawn, and 283 overlapping targets were obtained. The 283 targets were entered into the AlzData database, and the expression of these targets in different cells was analyzed. Cytoscape 3.7.2 software was used to construct a component-anti-ICVD target-cell interaction network. The experimental results show Figures  and 10 tP value, the top 15 signaling pathways related to ICVD are visualized (We use DAVID to perform KEGG enrichment analysis on the core PPI network. Sorted by sualized , includisualized –4. The rTo identify the, ÜS samples were analyzed by UHPLC-MS/MS. The total positive and some main major chemical components , total aκB1 with quercetin, and IKKβ with quercetin.Compound-target interactions with binding energy less than −5.0 kcal/mol are shown in κB signaling pathway, and PI3K/AKT signaling pathway.The pathway map of ÜS in treating ICVD was obtained from KEGG PATHWAY Database, as shown in More and more evidence shows that the pathophysiological mechanism of ICVD is very complicated, and a variety of biological processes and multiple signal pathways are involved in the process of ischemia-reperfusion injury. Traditional herb medicine is believed to be able to target energy metabolism and oxidation, multiple targets, multiple links, and multiple pathways such as stress, inflammation, endoplasmic reticulum stress, and apoptosis play a therapeutic role, which has obvious characteristics compared with traditional single compounds . In addiTo systematically explore the anti-ischemic brain of ÜS, the pharmacodynamic material basis and potential biological mechanism of vascular diseases, we use network pharmacology platform technology, through active ingredient screening, pharmacodynamic target prediction and analysis, protein interaction analysis, and gene annotation enrichment analysis, we have obtained 10 important effective ingredients, namely, quercetin, luteolin, naringenin, jaranol, ZINC519174, catechin, isoformononetin, kaempfride, isobavachin, and kaempferol. 10 important signaling pathways, namely, PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction, Ras signaling pathway, cAMP signaling pathway, Rap1 signaling pathway, MAPK signaling pathway, sphingolipid signaling pathway, HIF-1 signaling pathway, and TNF signaling pathway.κB and MAPK signal pathways.We used FunRich software to perform tissue distribution enrichment analysis on all predicted targets of ÜS and found that these targets were widely enriched in the nervous system and circulatory system. The results of GO enrichment analysis showed that the target was significantly enriched in the items of biological processes or analytical functions related to cerebral ischemia, such as response to hypoxia, inflammatory response, calcium channel activity, negative regulation of the apoptotic process, and positive regulation of ERK1 and ERK2 cascade. The 283 targets obtained by the intersection of the Venn diagrams are not only the targets of the 90 components of ÜS but also the therapeutic targets of ICVD. Using AlzData, a database related to single-cell sequencing, we analyzed the high-expressing cell types of these 283 targets and found that the distribution of these targets in nerve cells is quite different. There are 112 targets highly expressed in neurons, 107 targets highly expressed in endothelial cells, 98 cells highly expressed in astrocytes, and 72 targets highly expressed in microglia. The above analysis shows that ÜS can act not only on neurons, but also on endothelial cells, astrocytes, and glial cells to resist ICVD. Because the brain is a steady-state organ integrating blood vessels, nerves, and immunity, and the nervous system is extremely sensitive to changes in cerebral blood flow, the pathophysiology of ICVD is extremely complicated. In the past two centuries, a large number of studies showed that the complexity of the interaction between the brain and its vascular system is unmatched by other organs . In thisP value.The pharmacological effects of ÜS may not only involve predicted targets and ischemic targets in the database. To expand the target network and to explore and screen important target networks, we used the PPI theory to construct the PPI network of the ÜS target and the PPI network of ICVD. Convergence of complex networks, based on the theory of molecular interaction, combined with the view of complex systems, the status of nodes in biological networks is closely related to their biological importance. Interventions on key parts of the biological network can change the entire system and are a manifestation of network vulnerability . We screWith research support, PI3K/Akt signaling pathway is one of the important therapeutic targets for ischemia-reperfusion injury. Upregulation of this signaling pathway helps to restore neurological function after ICVD, has antiapoptotic effects, inhibits autophagy and antioxidative stress, and promotes angiogenesis –56. Therαβ activation without changing its surface expression [With research support, the Ras signaling pathway is one of the important therapeutic targets for ischemia-reperfusion injury . Blockinpression . The MAPpression . Mitogenpression . HIF-1 apression .κB signaling pathway, preventing ischemic stroke damage via antiapoptotic and antioxidant effects [It is worth noting that among the top 10 important medicinal ingredients of ÜS against ICVD, quercetin , luteoli effects , 76. Kae effects . These fκB1 were the top ten targets with high degrees. Followed by cluster of the PPI network, the network could be divided into four modules, which were mainly related to angiogenesis, inflammation, coagulation, and blood-brain barrier [β-amyloid deposition, most likely through interference with amyloid clearance pathways [β neurotoxicity [PPI analysis showed that PIK3CA, APP, PIK3R1, MAPK1, MAPK3, AKT1, PRKCD, Fyn, RAC1, and NF- barrier . Accumul barrier , 80. Strpathways . PRKCD mpathways , and Fyntoxicity . RAC1 retoxicity .In our research, ÜS samples were examined by UHPLC-MS/MS. The main chemical components of ÜS, quercetin, luteolin, naringenin, kaempfride, and kaempferol, were examined. Compound-target interactions verified molecular docking, ; these rPI3K-Akt signaling pathway, distributed in neuronal cells, endothelial cells, glial cells, immune cells, etc., can interact with a variety of ligands. The complex biological activity of this pathway is also reflected in its ability to bind ligands, the different activation of different signal transduction pathways in the cell, and triggering different downstream effects , 86. TheIn summary, the anti-ICVD effect of ÜS has the characteristics of multiple targets and multiple pathways, and its main biological mechanism may be related to the PI3K-Akt signaling pathway as the central signaling pathway. There are still some limitations in this study, such as the need to further optimize the methods for finding and confirming relevant targets of active ingredients, interaction of ÜS and neuroactive ligand-receptor, MAPK signaling, apoptosis, autophagy, and Rap1 signaling, and the relationship between these pathways requires experimental evidence. The rationality of the PI3K-Akt signaling pathway as the underlying biological basis for the treatment of different diseases in ÜS requires further biological evidence.


As a type of regulated cell death (RCD) mode, pyroptosis plays an important role in several kinds of cancers. Pyroptosis is induced by different stimuli, whose pathways are divided into the canonical pathway and the noncanonical pathway depending on the formation of the inflammasomes. The canonical pathway is triggered by the assembly of inflammasomes, and the activation of caspase-1 and then the cleavage of effector protein gasdermin D (GSDMD) are promoted. While in the noncanonical pathway, the caspase-4/5/11 (caspase 4/5 in humans and caspase 11 in mice) directly cleave GSDMD without the assembly of inflammasomes. Pyroptosis is involved in various cancers, such as lung cancer, gastric cancer, hepatic carcinoma, breast cancer, and colorectal carcinoma. Pyroptosis in gastric cancer, hepatic carcinoma, breast cancer, and colorectal carcinoma is related to the canonical pathway, while both the canonical and noncanonical pathway participate in lung cancer. Moreover, simvastatin, metformin, and curcumin have effect on these cancers and simultaneously promote the pyroptosis of cancer cells. Accordingly, pyroptosis may be an important therapeutic target for cancer. Cancer seriously threatens human health worldwide. Based on the most recent data compiled by the International Agency for Research on Cancer (IARC), 19.3 million new cancer cases were diagnosed in 2020 , 5. Accoβ (IL-1β) and interleukin-18 (IL-18), and consequent death of cells [In normal mammalian cells, cells undergo death and renewal as a result of cell aging, infection, or damage, which present homeostasis of cells. Currently, several types of cell death are found, including apoptosis, autophagy, necrosis, necrotic apoptosis, and pyroptosis . ApoptosShigella flexneri was mistaken for apoptosis at that time, but this programmed cell death is actually pyroptosis [In 1992, a programmed cell death of host macrophages caused by roptosis . The Celroptosis , and in roptosis .β and IL-18 are released through those cell membrane pores; consequently, the pyroptosis continues. Accordingly, pyroptosis is different from apoptosis whose morphological characteristics are manifested in cell shrinkage, DNA degradation, nuclear membrane rupture, and cell membrane integrity [The morphological characteristics of pyroptosis include cell enlargement, small amount of DNA damage, and chromatin concentration, but the nucleus remains intact . In the ntegrity . It is gntegrity .β, and the pro-IL-18 [The main pathways of pyroptosis are divided into the canonical pathway and the noncanonical pathway according to the upstream signal transducing mechanism . In the ro-IL-18 , 29. Whiro-IL-18 .β and pro-IL-18 [The pattern-recognition receptor (PRR) is a vital part of our natural immune system . PRRs rero-IL-18 –42.β and pro-IL-18 [Activated caspase-1 also facilitates the cleavage of gasdermins besides the pro-IL-1ro-IL-18 . The gasro-IL-18 . Most ofro-IL-18 . When garo-IL-18 . Increasro-IL-18 , 47. Howro-IL-18 .β and pro-IL-18 [β and IL-18, to be released [κB) signaling pathway takes part in the activation of NLRP3 inflammasome [κB, which mainly regulates the NLRP3 inflammasome [The upstream signaling of canonical pyroptosis pathway stimulates the assembly of inflammasome whose PRR is generally NLRP3 . The NLRro-IL-18 , 37, 50.ro-IL-18 , 51. Thereleased , 53. Conammasome . ROS proammasome , 56  of Gram-negative bacteria and then promote the cleavage of GSDMD to form pores rather than assemble into inflammasomes [The downstream of noncanonical pyroptosis pathway is the same as that of canonical pyroptosis pathway and presents GSDMD as the effector protein which causes the formation of cell membrane pores , 47. In mmasomes , 57, 58.mmasomes   seriously threatens human health worldwide. The survival rate for a period of 5 years is less than 15% , 60. LunSCLC accounts for approximately 15% of all lung cancers and is classified as a high-grade neuroendocrine (NE) tumor which has a high death rate and poor prognosis . HoweverκB/NLRP3/GSDMD signal axis and inhibits the progression of NSCLC [X (Bax) and p53 expression, and arrests the cell cycle in G2/M; thus, apoptosis of NSCLC cells is triggered [Xinactive-specific transcript (lncRNA-XIST)/miR-let-7c/IL-6/signal transducer and activator of transcription 3 (STAT3) axis and suppressing the mitogen-activated protein kinases (MAPK) and PI3K pathways [G2/M phase and downregulates the level of Bcl-2, thus inducing apoptosis via the STAT3 pathway [In pyroptosis, it is clear that the assembly of NLRP3 inflammasome is closely related to the activation of caspase-1, which is involved in the cleavage and maturation of the GSDMD, and the GSDMD is the executor of pyroptosis. It indicates that NLRP3 inflammasome, caspase-1, and GSDMD are crucial factors in the process of pyroptosis. Accordingly, inducing pyroptosis of NSCLC cells through the NLRP3/caspase-1/GSDMD pathway may be potential targets for inhibiting the tumor progression of NSCLC . Wang etof NSCLC . In addiriggered , 74. Meariggered . The cucriggered . Also, Criggered . Moreovepathways , 78. Add pathway , 80. In  pathway , 82. It  pathway . DAS als pathway , 85. In  pathway .κB pathway is involved in the expression of NLRP3 inflammasome which indicates that both the ROS and the NF-κB act vital roles in pyroptosis [κB are targeted to induce pyroptosis in NSCLC therapy [κB/caspase-3/GSDME pathway [κB, activating the cleavage of GSDME by caspase-3 [κB [κB signaling pathway [The ROS/NF-roptosis , 87, 88. therapy . Chalcon therapy . Meanwhi therapy , 91. Met pathway . The mecaspase-3 . Moreoveaspase-3 , 94. Pipse-3 [κB . It is sse-3 [κB , 97. It  pathway , 99. Fur pathway , 101.Apurinic endonuclease 1 (APE1) acts as a key factor in base excision repair (BER) and exerts the function of apurinic sites excision . It is rG2/M phase cycle of lung adenocarcinoma (LUAD) cells by inhibiting MELK to trigger the pyroptosis [Maternal embryonic leucine zipper kinase (MELK) is a carcinogenic kinase and is essential in NSCLC mitotic progression, metastasis by regulating the process of cell death . It is rroptosis . The inhroptosis .Other member of the gasdermins such as GSDME serves as special targets to induce pyroptosis in NSCLC therapy . It is rIntracellular LPS induces the pyroptosis through the noncanonical pathway , 116. Inβ, and IL-18 genes in A549 cells [It is proposed that the pyroptosis is induced by inhibiting the lncRNA-XIST through the Mir-335/SOD2/ROS signaling pathway, and then the NSCLC is inhibited . In addi49 cells   is one of the most common cancers which is seriously harmful to human health , 126. AcGC cells , 138. IcGC cells . MeanwhiGC cells . It is cGC cells . FurtherGC cells  (Table 1Helicobacter pylori (H. pylori) [H. pylori-triggered inflammatory response and the depression of pyroptosis via the ROS/NLRP3/caspase-1/IL-1β pathway may suppress the progression of GC [However, the cytotoxin-related gene A (CagA) protein, an important pathogenic factor of  pylori) , promoteon of GC . These rSalvia miltiorrhiza Bunge, promotes the accumulation of intracellular ROS and induces the GSDME-dependent pyroptosis of HCC [G0/G1 phase in the cell cycle and induces mitochondrial-dependent apoptosis in HCC cell lines [β (GSK-3β) signaling pathway [β-estradiol (E2) is a kind of hormonally active compounds [G2/M phase [κB/p65 pathway [Hepatic carcinoma (HCC) is a common kind of cancers which seriously hazards human health . Howevers of HCC . Likewiss of HCC . Furthers of HCC . In addis of HCC . Moreovell lines . It is cll lines . Meanwhill lines –166. Furll lines . It is dll lines . In addill lines . Moreove pathway , 171. 17ompounds . It is sompounds . In addiompounds . Further/M phase . It is c pathway , 177. Ad pathway . These rG0/G1 cell cycle arrest of BC cells [κB pathway [Breast cancer (BC) does great harm to women health which ranks second among cancer-related death in women . It is cBC cells . It is d pathway . In addi pathway , 192. Te pathway . Likewis pathway . AccordiColorectal carcinoma (CRC) is the third most common form of cancer in adults which has a poor prognosis and significantly damages the patient's daily life and mental health . Effectiβ and IL-18, resulting in the morphological characteristics of pyroptosis. While in the noncanonical pathway, the caspase-4/5/11 directly cleave GSDMD, resulting in the pyroptosis. In addition, pyroptosis is affected by the ROS and NF-κB which influence the upstream pathway of pyroptosis.As a type of RCD mode, pyroptosis plays an important role in several kinds of cancers, whose pathways are divided into the canonical and noncanonical pathway depending on whether formation of the inflammasomes. The canonical pathway is triggered by the assembly of inflammasomes and mainly regulated by the activation of caspase-1. Activated caspase-1 not only promotes the cleavage of effector protein GSDMD but also promotes the proteolytic maturation of proinflammatory cytokines IL-1Pyroptosis in gastric cancer, hepatic carcinoma, breast cancer, and colorectal carcinoma is related to the canonical pathway, while both the canonical and noncanonical pathway participate in lung cancer. Moreover, simvastatin, metformin, and curcumin have effect on these cancers and simultaneously promote the pyroptosis of cancer cells. Accordingly, pyroptosis may be an important therapeutic target to cancer though the relationship between pyroptosis and a few cancers such as CRC and SCLC remain unclear, and more researches on pyroptosis in these cancers are needed in future.