The results indicated that TSD protected against VaD through multicomponent and multitarget modes. Though the detailed mechanism of action of various active ingredients needs to be further illustrated, TSD still showed a promising therapeutic agent for VaD due to its biological activity.Vascular dementia (VaD) is the second most prevalent dementia, which is attributable to neurovascular dysfunction. Currently, no approved pharmaceuticals are available. Taohong Siwu decoction (TSD) is a traditional Chinese medicine prescription with powerful antiapoptosis and anti-inflammatory properties. In this study, a network pharmacology approach together with molecular docking validation was used to explore the probable mechanism of action of TSD against VaD. A total of 44 active components, 202 potential targets of components, and 3,613 VaD-related targets including 161 intersecting were obtained. The potential chemical components including kaempferol, baicalein, beta-carotene, luteolin, quercetin, and beta-sitosterol involved in the inflammatory response, oxidative stress, and apoptosis might have potential therapeutic effects on the treatment of VaD. The potential core targets including AKT1, CASP3, IL1 Vascular dementia (VaD) is one of the most prevalent types of dementia involved in a series of symptoms that are mainly characterized by progressive neurocognitive impairment attributed to various cerebrovascular pathologies , 2. VaD Radix Salviae (Danshen in Chinese), Huperzia serrata (Qiancengta in Chinese), Ligusticum chuanxiong (Chuanxiong in Chinese), Ginkgo biloba (Yinxing in Chinese), and Panax ginseng (Renshen in Chinese), have been revealed the clinical pharmacological effects on dementia and dementia-like disorders [Traditional Chinese medicine (TCM), as a major component of alternative and complementary medical remedies, has attracted increasing attention worldwide due to its high clinical effectiveness and security . It was isorders , 6, 8.Paeoniae Radix Alba (Baishao in Chinese), Rehmanniae Radix Praeparata (Shudihuang in Chinese), Angelicae Sinensis Radix (Danggui in Chinese), Chuanxiong Rhizoma (Chuanxiong in Chinese), Persicae Semen (Taoren in Chinese), and Carthami Flos (Honghua in Chinese). As a Chinese medicine prescription, it has been used primarily to treat deficiency and stasis of qi and blood, which are the main causes of dementia [Taohong Siwu decoction (TSD), a multiherbal formula, was initially documented in Wu Qian's “Yi Zong Jin Jian” (Qing Dynasty of China). The formula is composed of six herbs including dementia . Increasdementia –13. AlthIn TCM, network pharmacology is a common research approach. Based on network analysis, it could reveal potential mechanisms of multi-ingredient action of Chinese herbal compounds as a whole in treating diseases. In the process of network pharmacological analysis, herbs and their targets are discovered by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), which has proven successful in drug discovery as one of the most prevalent system pharmacology platforms for Chinese herbal medicines . DespitePaeoniae Radix Alba (Baishao in Chinese), Rehmanniae Radix Praeparata (Shudihuang in Chinese), Angelicae Sinensis Radix (Danggui in Chinese), Chuanxiong Rhizoma (Chuanxiong in Chinese), Persicae Semen (Taoren in Chinese), and Carthami Flos (Honghua in Chinese) in TSD prescription , were retrieved from the TCMSP database [https://pubchem.ncbi.nlm.nih.gov/).Chemical ingredients of six herbs, including database , 16. SubHomo sapiens” was chosen for the prediction results. The indexes with high probability (above 0.65 for 2D or above 0.85 for 3D) were selected as potential targets of the ingredients within TSD according to the SwissTargetPrediction interpretation. Eventually, the UniProt Knowledgebase (https://www.uniprot.org/) was employed to convert protein names into corresponding gene symbols.The potential targets of bioactive ingredients in six herbal medicines were retrieved from the SwissTargetPrediction . The SMIHomo sapiens.” All disease targets were merged, duplicate items were excluded, and then VaD-related target genes were reserved for further research.The VaD-related gene targets were screened from four databases: GeneCards: the human gene database, DrugBank Online database, Online Mendelian Inheritance in Man (OMIM) database, and PharmGKB database with the keywords of “vascular dementia” and “The Venn R package was used to compile a Venn diagram following the acquisition of the TSD and VaD targets. The intersection targets were considered as a potential target gene set for TSD to exert therapeutic effects on VaD.https://david.ncifcrf.gov/) to annotate the function of target genes and disclose potential biological pathways of the active constituents of TSD. Terms with a threshold value of P ≤ 0.05 were conducted for further research, and then, the number of enriched genes was ranked within descending order. The clusterProfiler R package was conducted to demonstrate advanced bubble diagrams. Through enrichment analysis, the potential biological processes and pathways via which TSD had a therapeutic effect on VaD could be predicted.Enrichment analyses of GO and KEGG were conducted through the Database for Annotation, Visualization and Integrated Discovery (DAVID) v 6.8 (https://cytoscape.org/) was utilized in this research.To provide insight into the mechanisms of action of TSD in treating VaD, networks of compound-target and pathway-target were conducted. In these networks, nodes symbolize constituents, targets, or pathways, while edges refer to links between them. To visualize networks, the Cytoscape 3.7.1 software (https://cn.string-db.org/) to construct the PPI network. Target proteins restricted to the species of “Homo sapiens” and isolated proteins in the PPI network were concealed. For the purpose of understanding the relationship between proteins, the network topology was generated using the Cytoscape 3.7.1 software. The nodes in the network refer to the targets, while the edges refer to the PPI. A circle was formed based on these parameters for each protein. High centrality values and degrees implied a major role in the network. The probable core targets were then selected using the plugins MCODE and CytoHubba [The intersection targets of TSD and VaD were entered into the STRING database (ytoHubba .https://pymol.org/2/). Docking was performed via AutoDock Vina software according to the manufacturer's process. The molecule with energy-minimized conformation in the docking was selected, and the interaction between ingredients and targets was visualized via the PyMOL software [Molecular docking was conducted to predict the binding affinity between active ingredients and their hub targets through the AutoDock Vina software. Structures of candidate active ingredients and targets were obtained from TCMSP and PDB databases, respectively. Dehydration, hydrogenation, and ligand separation were accomplished using the PyMOL software  in this study. For visualization, we further selected the top 10 catalogs in each of the three categories: BP, CC, and MF . The main pathways of enrichment included lipid and atherosclerosis, Kaposi's sarcoma-associated herpesvirus infection, human cytomegalovirus infection, hepatitis B, fluid shear stress and atherosclerosis, pathways of neurodegeneration-multiple diseases, PI3K/Akt signaling pathway, MAPK signaling pathway, chemical carcinogenesis-receptor activation, and human T-cell leukemia virus 1 infection  items, 77 cell composition (CC) items, and 200 molecular function (MF) items were revealed  with six active monomers  of TSD. The results illustrated that most of the binding energies between two molecules were less than -7 kcal/mol, and the binding energy between beta-carotene and AKT1 was less than -12 kcal/mol (lowest) (β-sitosterol formed hydrogen bonds with the THR-62 residue. In IL1B (5R86), quercetin formed hydrogen bonds with MET-20, GLU-37, GLN-39, and VAL-41 residues, respectively. In JUN (4Y46), beta-sitosterol formed hydrogen bonds with the ALA-74 residue  . Molecul residue . The remRadix Salviae (Danshen in Chinese), Huperzia serrate (Qianchenta in Chinese), Chuanxiong Rhizoma (Chuanxiong in Chinese), Ginkgo Folium (Yinxingye in Chinese), and Panax ginseng (Renshen in Chinese), have been widely applied in clinical practice in the treatment of VaD [VaD arises after multiple risk factors due to primary injury in microhemodynamics and alteration in blood vessel thickening and dysfunction. As a consequence, a comparatively effective intervention for VaD should improve the cognitive function as well as reduce the production of vascular risk factors. Up to now, various herbs, including t of VaD . In the Carthami Flos and Paeoniae Radix Alba, plays an important role in improving the damage of striatal neurons [β-Sitosterol derived from Persicae Semen, Paeoniae Radix Alba, Carthami Flos, and Angelicae Sinensis Radix is regarded to have a crucial role in potentially reducing neuron demyelination and blood lipids [arthami Flos' major ingredients, is involved in the inflammatory response of VaD by inhibiting the expression of AP-1 and NF-κB [Carthami Flos and play an important role in antioxidation and anti-inflammation, which is closely related to relevant therapeutic strategies in VaD [In this study, it was revealed that kaempferol, baicalein, beta-carotene, luteolin, quercetin, and beta-sitosterol in TSD might have potential therapeutic effects on the treatment of VaD based on network analysis. Kaempferol, mainly derived from  neurons . β-Sitosd lipids . Luteolind NF-κB . Quercets in VaD . These fβ, JUN, and TP53, which are associated with cell apoptosis and inflammatory. Through Akt activation, the p-Akt protein was produced, which inhibited apoptosis and promoted hippocampal nerve cell survival [β is increasingly involved in the process of neuroinflammation, indicating that it may be strongly implicated in the progression of VaD [α and IL-1β release, further suggesting that IL-1β was a potential target of TSD [2+-induced neurotoxicity and neuronal cell death [β1–42 induced neurotoxicity [β in cerebral amyloid angiopathy or cerebral blood vessels were related to vascular cognitive impairment, indicating a novel role of TP53 in VaD [TCM therapeutics has the advantage of integrity and simultaneous capture of multiple targets and multiple pathways, which produces a stronger robust synergistic effect than any single compound. In this study, it was revealed that the therapeutic effects of TSD on VaD are mostly attributed to the potential core targets of AKT1, CASP3, IL1survival . The incsurvival . Proinfln of VaD . TSD hadt of TSD . It was ll death . TP53 hatoxicity . Intrigu3 in VaD . In addiin vitro cerebral ischemia-reperfusion injury model, TSD's protective effect, was significantly attenuated by inhibiting PI3K activity, which indicated that this pathway was critical to TSD protection [In this study, it's also revealed that the therapeutic effects of TSD on VaD are mostly related to PI3K/Akt and MAPK signaling pathways, which were closely associated with cell apoptosis and inflammation. PI3K is able to be induced by neurotrophic factors , and the phosphorylation of Akt is also enhanced after ischemia . There iotection . MAPK siotection . TherefoTCM has been extensively used and accumulated a wealth of experience in the treatment of dementia due to its reducing adverse effects, prolonging survival time, and improving quality of life in patients. Modern studies have also revealed that a growing number of Chinese medicines have been reported to have neuroprotective effects against VaD . For insNetwork pharmacology along with bioinformatics analyses revealed the compositions and targets of TSD, as well as VaD-related genes and targets. In the study, we predicted probable therapeutic targets of TSD on VaD, revealed its action mechanisms through core compounds and genes, and provided scientific evidence for TSD to VaD. However, it is worth noting that the results from network pharmacology together with bioinformatics are a certain false-positive rate as a result of relying only on existing databases and results for network modeling. Therefore, the absence of experimental validation is the major limitation of this study. According to the findings from this study, the validation of molecular levels is of great significance to conduct in future studies.β, JUN, and TP53), which might be through two main VaD-related signaling pathways  in this network study. This study revealed potential mechanisms of TSD with multiple components, multiple targets, and multiple pathways for treating VaD, providing a scientific foundation for studying the mechanism in vivo and in vitro by wet experiment in the future.In brief, to investigate the potential action mechanisms of TSD for treating VaD, compound and target prediction and map analysis were performed by using network pharmacology together with bioinformatics methods in this study. A total of 44 compounds and 202 target genes were identified in TSD and finally found 161 intersecting targets. TSD intervened VaD mainly by six active monomers  and five key target genes (AKT1, CASP3, IL1


Thermosulfidibacter takaii, which was recently demonstrated to take place via an unexpected reverse reaction of citrate synthase, was reproduced using a kinetic network model, and a competition between reductive and oxidative fluxes on rTCA due to an acetyl coenzyme A (ACOA) influx upon acetate uptake was revealed. Avoiding ACOA direct influx into rTCA from WL is, therefore, raised as a kinetically necessary condition to maintain a complete rTCA. This hypothesis was confirmed for deep-branching bacteria and archaea, and explains the kinetic factors governing elementary processes in carbon metabolism evolution from the last universal common ancestor.The origin of life is believed to be chemoautotrophic, deriving all biomass components from carbon dioxide, and all energy from inorganic redox couples in the environment. The reductive tricarboxylic acid cycle (rTCA) and the Wood–Ljungdahl pathway (WL) have been recognized as the most ancient carbon fixation pathways. The rTCA of the chemolithotrophic  The reductive tricarboxylic acid cycle and the reductive acetyl-CoA pathways are thought to be ancient carbon fixation pathways in deepbranching bacteria and archaea. Here kinetic modelling suggests a conflict between these pathways based on acetyl-CoA influx. Therefore, most of the organic carbon on early Earth was obtained by chemoautotrophic carbon fixation, thus sustaining life. Currently, there are seven known different autotrophic pathways responsible for carbon fixation, including the newly demonstrated reductive glycine (rGly) pathway2. These pathways are recognized to have partial overlaps, but the reasons for the redundancy and the forces of selection in their evolution remain unclear. The WL pathway, also called the reductive acetyl-CoA (coenzyme A) pathway, and the reductive tricarboxylic acid (rTCA) cycle are among the most ancient pathways responsible for the biosynthesis of the universal precursors of anabolism6. Even if we focus only on these ancient carbon metabolism pathways, diverse observations on the phylogenetic distribution of the network topology have not yet been given a universal and phylum-compatible interpretation.The concept of a last universal common ancestor (LUCA) has been attracting much interest among researchers on the early evolution and origin of life. Based on an extensive investigation of protein-coding genes in prokaryotic genomes, LUCA has been inferred to be an anaerobic chemoautotroph that uses the Wood–Ljungdahl (WL) pathway and exists in a hydrothermal setting8. Therefore, in organisms using the rTCA cycle, CS has been interpreted to be substituted either by an adenosine triphosphate (ATP)-dependent citrate lyase or other homologous enzymes that catalyze the same reaction in two steps11. In contrast, however, two research groups demonstrated that CS activity drove the rTCA cycle in the thermophilic bacteria Thermosulfidibacter takaii and Desulfurella acetivorans grown chemolithoautotrophically14.The rTCA cycle is a reversal of the oxidative tricarboxylic acid (oTCA) cycle, which provides energy, reducing agents, and precursors for certain amino acids. However, citrate synthesis from acetyl-CoA (ACOA) and oxaloacetate, catalyzed by citrate synthase (CS), is regarded as one of the irreversible reactions in the oTCA cycle14 is also simulated. Interestingly, it was observed in dynamical systems that a conflict between the reductive and oxidative fluxes in the TCA cycle was caused by an influx of ACOA upon uptake of acetate. This indicates that the coexistence of a WL pathway and a rTCA cycle, which yields ACOA influx into the rTCA cycle, results in a partial reduction of fluxes in the TCA cycle. Consequently, the less active enzymes of the rTCA cycle due to this coexistence would be lost to evolution or enzymes lacked in the rTCA cycle would be difficult to be newly incorporated into the less active part of that. In the pioneering work by Braakman and Smith5, a single connected redundant network consisting of a complete WL pathway and a full rTCA cycle was proposed as a more robust and plausible topology that LUCA or (proto) biological systems leading up to the LUCA possibly possess. However, our kinetic results do not support the coexistence of these two pathways in one organism, unless other predominant selective pressure that favors their combination exists as well. Remarkably, it was confirmed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) carbon metabolism database15 that our kinetic hypothesis, that a complete rTCA cycle does not coexist with a WL pathway, was held for deeply branching archaea and bacteria18. Based on the kinetic hypothesis and the fluxes simulated for these organisms, a fundamental model of carbon metabolism evolution for ancestral bacteria and archaea starting from LUCA is presented.In the present study, kinetic simulations of the autotrophic rTCA cycle for these bacteria are presented and the mechanism by which the CS reaction is reversed, which was thought to function only in the oxidative direction, is revealed in dynamical systems. In addition, bifurcation of the TCA cycle into reductive and oxidative reactions that has been demonstrated in the presence of succinate and acetateT. takaii and D. acetivorans, typical key enzymes involved in the rTCA cycle of autotrophs were identified, namely, the ferredoxin-dependent enzymes 2-oxoglutarate:ferredoxin oxidoreductase and pyruvate:ferredoxin oxidoreductase. On the other hand, genes for ATP citrate lyase and its two-step variant citryl-CoA synthetase/citryl-CoA lyase, which are regarded to be necessary for organisms with autotrophic rTCA cycle, were missing14: these organisms instead possessed genes of CS that were thought to be active only in oTCA cycle. In the present study, the kinetic reaction models for enzymes identified for carbon metabolism in T. takaii were developed (Eqs. B1–B18 in the Supplementary Information (SI)), and the kinetic network model in which these enzymatic reactions were incorporated was presented. To investigate the direction of carbon metabolic fluxes on the network, the steady-state fluxes were determined by the kinetic network model (Eqs. 1–16 in the SI) with the fixed concentrations of chemical species listed in the SI Table T. takaii grown chemolithoautotrophically, except for the directions between malate (MAL) and PYR and between OAA and PEP13. In addition, it was confirmed that the kinetic network simulations involving the gene knockouts of malic enzyme between MAL and PYR and/or of phosphoenolpyruvate carboxykinase between OAA and PEP reproduced a complete rTCA flux  and oxidized ferredoxin (Fdxox) , which converts ACOA to PYR, and pyruvate carboxylase (PC), which converts PYR to OAA, affects the rTCA cycle. It is assumed that the model of D. acetivorans gains a PYR influx into the rTCA cycle via serine (SER) metabolism, whereas that of T. takaii does not because of its lack of the necessary enzyme   Fig.  and acetUC) Fig. 13. Upon ved Fig. . The dirved Fig. . The dirlux Fig. . This islux Fig. . This is15. The results for deep-branching bacteria grown autotrophically, the Firmicutes, Cyanobacteria, Aquificae, and Proteobacteria18, are shown in the SI Fig. 20 and was simulated with a lower reduced to oxidized agent ratio but was later incorporated in subsequent discussions. These representative flux patterns are classified according to whether the WL pathway coexists with the TCA cycle and yields an ACOA influx into the rTCA cycle. In the type-B1 pathway, the WL pathway produces ACOA influx, resulting in MAL influx through PYR into the partial rTCA pathway. This is applicable for H2/CO2-utilizing acetogens, e.g., Sporomusa termitida, which grows both autotrophically and heterotrophically21. Next, as an alternative pathway where a WL pathway coexists with CS is the type-B2 pathway, where the ACOA influx yielded by a WL pathway causes the partial oTCA pathway to synthesize AKG. However, because of the absence of several enzymes acting in the rTCA pathway, the competition between oxidative and reductive fluxes does not occur. This is found for acetogenic anaerobic bacteria, e.g., Moorella thermoacetica, which also grows autotrophically and heterotrophically22. In the type-B3 pathway, neither the incomplete WL pathway nor the incomplete rGly pathway produces an ACOA influx; hence, there is no competition between reductive and oxidative flux, and the necessary kinetic condition with respect to PC is also satisfied, thus a complete rTCA flux is produced. This is observed for sulfur-reducing bacteria, T. takaii13 and D. acetivorans14, which grow chemolithoautotrophically and heterotrophically. In the type-B4 pathway, if the rGly pathway produces ACOA influx, the kinetic hypothesis does not allow a complete rTCA cycle, yielding OAA influx through PEP into the partial rTCA pathway. This is observed for anaerobic anoxygenic photoheterotrophs, e.g., Heliobacterium modesticaldum24. Interestingly, it was expected that, according to the KEGG genome database15, H. modesticaldum possessed an rGly pathway similar to the one recently identified for Desulfovibrio desulfuricans grown chemolithoautotrophically2. The absence of complete rTCA cycle in H. modesticaldum possessing no WL pathway but an rGly pathway supports the extended applicability of kinetic hypothesis. Taken together, the proposed kinetic hypothesis is basically satisfied because the network patterns observed for deep-branching bacteria do not involve impaired rTCA fluxes.To assess the validity of the kinetic hypothesis, the fluxes of carbon metabolism for deep-branching bacteria were examined using the kinetic network model simulations with enzymes identified by the KEGG genome database15. The results for the deep-branching archaea grown autotrophically, namely, Euryarchaeota, Candidatus Bathyarchaeota, and Crenarchaeota18, are shown in the SI Fig. Methanococcus maripaludis27. In the type-A2 pathway, the absence of WL pathway does not prevent a complete rTCA cycle under autotrophic growth conditions. Nevertheless, the rTCA cycle is kinetically inhibited because of the absence of PC in this pathway. This absence led to the loss of the OAA influx from PYR into the rTCA cycle, thus causing a decrease in the concentration of CIT, which is the product of rTCA cycle, and resulted in the impairment of the reversed CS reaction. Consequently, the right half of the rTCA flux was significantly reduced  to oxidized agent (Fdxox). This is found for the heterotrophic anaerobic archaeon Vulcanisaeta distributa28.Next, the fluxes of carbon metabolism for deep-branching archaea were examined using the kinetic network model simulations with enzymes identified by the KEGG genome databaseThermoproteus tenax and Acidianus hospitalis possess the dicarboxylate/4-hydroxybutyrate (DC/4HB)29 and 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) pathways30, respectively. These alternative carbon fixation pathways yield an ACOA influx under chemolithoautotrophic growth conditions and thus impairs the rTCA flux on the right-hand side biological systems leading up to the LUCA should possess on the basis of phylometabolic analysis. In contrast, the results obtained from our kinetic network model do not support the coexistence of those pathway in one organism, unless a more dominant and selective pressure that favors the combination of those pathways exists in parallel. The key difference between their work and ours is whether the kinetic factors are taken into consideration. If it is assumed that, as widely accepted, LUCA is an anaerobic hydrothermal chemoautotroph possessing at least a WL pathway1, the confirmed kinetic hypothesis raises a model of evolution processes on carbon metabolism for deep-branching archaea and bacteria  cycle  and is thus predicted to be a plausible candidate for the carbon metabolism phenotype of LUCA. Alternatively, if LUCA does not possess a partial rTCA pathway, such ancestral chemoautotrophs would gain enzymes necessary for a partial rTCA pathway that coexists with the WL pathway during evolution toward A1 or B1. On the other hand, in cases where ancestral chemoautotrophs gained CS during the early stages of type-I evolution, they would gain enzymes necessary for a partial oTCA pathway to synthesize AKG, as seen in B2. This corresponds to acetogenic anaerobic bacteria growing autotrophically and heterotrophically  were incorporated into the kinetic network model for carbon metabolism described by the ordinary differential Eqs. 1–16 listed in the SI  were also incorporated into the kinetic network model  for the kinetic network model comprised of 22 reaction equations and 29 chemical species were solved using COPASI biochemical system simulator (ver. 4.28)Peer Review FileSupplementary Information


Interleukin‐1β (IL‐1β) is a pro‐inflammatory cytokine mainly produced by monocytes and macrophages with a wide range of biological effects. Evidence has shown that IL‐1β plays a vital role in the process of apoptosis; however, the specific mechanisms, by which IL‐1β induces apoptosis, vary due to different cellular and experimental conditions. Therefore, this present reviewstudy aimed to systematically review the association between the molecular mechanisms of IL‐1β‐induced apoptosis in pathological processes and the role of signaling pathways. This article also sought to briefly investigate the potential of signaling pathway‐targeted therapy in the prevention and treatment of disease.This is a literature review article. The present discourse aim is first to scrutinize and assess the available literature on IL‐1β and apoptosis. The relevant studies using the keywords of “IL‐1β‐induced apoptosis” and “signaling pathways” were searched in the databases of PubMed, Scopus, Google Scholar, and Web of Science. Gathered relevant material, and extracted information was then assessed.kB signaling pathway, p38MAPK, and JNK signaling pathways mainly play a proapoptotic part, and the ERK1/2 pathway has a bidirectional role in regulating apoptosis, while activation of the PI3K‐Akt signaling pathway can inhibit apoptosis.IL‐1β can induce apoptosis in various types of cells under different external stimuli via the mitochondrial pathway, death receptor pathway and endoplasmic reticulum pathway, and that the different pathways are often interconnected. The NF‐This review indicates that IL‐1β‐induced apoptosis plays an important role in pathogenesis and development of pathology of many inflammatory diseases. Elucidating the role of the signaling pathways will aid the development of targeted therapeutic treatments. Evidence has shown that interleukin‐1β (IL‐1β) plays a vital role in the process of apoptosis. This paper reviews the molecular mechanisms of IL‐1β‐induced apoptosis in pathological processes, focusing on the role of signaling pathways. This article also briefly describes the potential of signal pathway‐targeted therapy in the prevention and treatment of disease, using the application of signal pathways in the treatment of disc degeneration and osteoarthritis as examples. The biological functions of IL‐1β mainly include mediating inflammatory responses and innate immunity,Apoptosis is the autonomous and orderly death of cells under certain physiological or pathological conditions, which is controlled by a series of activation, expression, and regulation of genes. Currently, the three well‐known pathways for apoptosis are mitochondria pathway, death receptor pathway, and endoplasmic reticulum stress pathway, whose ability to mediate IL‐1β‐induced apoptosis have been proven by studies. The difference between the mechanism of the three pathways in regulating the apoptosis process may result from the type, origin, growth environment of cells, and the external factors to initiate apoptosis, based on which the signal transduction pathways involved also differ.1.11.1.1The interleukin‐1 superfamily includes IL‐1α, ‐1Ra, ‐18, ‐33, ‐36Ra, ‐36α, ‐36β, ‐3 6γ, ‐37, and ‐38, of which IL‐1β is one of the earliest discovered and most important members. The IL‐1β gene is encoded by seven exons and six introns. The abundance of AU sequence on the 3′ untranslated zone of the seventh exon causes IL‐1β to be extremely unstable and easily degradable by enzymes,1.1.2d‐mediated secretory pathways.IL‐1β is not through the classical endoplasmic reticulum‐Golgi secretion pathway but sequential activation of vesicular or gasdermin In the cytoplasm, there is usually inactive pro‐interleukin‐1 (pro‐IL‐1β), which is the processed by the IL‐1β‐converting enzyme (ICE) and converted into active and mature IL‐1. The mature IL‐1βthen binds to the type 1 IL‐1 receptor (IL‐1R1) to exert biological functions.1.21.2.1Mitochondria are organelles critical to the process of apoptosis. In experiments related to IL‐1β‐induced apoptosis of nucleus pulposus cells1.2.2In recent years, Hui et al.1.2.32+ imbalance and upregulation of endoplasmic reticulum stress‐related proteins GRP78 and CHOP. Li et al.2+.Dong et al.For IL‐1β to induce apoptosis, a single pathway can work in a stand‐alone fashion or interact with other pathways to take effect collaboratively. Some scholars suggest that IL‐1β upregulates Bax, cyt‐c, caspase‐9, and caspase‐3 to induce apoptosis via the mitochondrial damage pathway.1.3IL‐1β‐induced apoptosis is the result of the interaction and joint regulation of multiple signaling pathways including nuclear factor‐κB (NF‐κB) signaling pathway, mitogen‐activated protein kinases (MAPKs) signaling pathway, and phosphoinositol‐3 kinase (PI3K)/Akt signaling pathway, etc. Among them, NF‐κB signaling pathway, p38 mitogen‐activated protein kinase (p38MAPK) signaling pathway, and c‐ Jun N‐ terminal kinase (JNK) signaling pathway mainly play a pro‐apoptotic role, and the extracellular signal‐regulated protein kinase (ERK) 1/2 pathway has a bidirectional role in regulating apoptosis, while activation of the PI3K‐Akt signaling pathway can inhibit apoptosis  NO promotes apoptosis by regulating Bax/Bcl‐2 proteins and upregulating caspase‐3 through a p38/p53‐dependent pathway,The antiapoptotic mechanisms of NO of low concentrations include inducing the formation of heat shock protein 70,IL‐1β induces JNK signaling,The ERK1/2 pathway has both antiapoptotic and proapoptotic effects, depending on the experimental conditions and/or cell types. One of the mechanisms involved in IL‐1β‐induced apoptosis may be through the MEK/ERK signaling pathway,In recent years, there has been increasing evidence that regulation of the cell cycle through the PI3K/Akt signaling pathway can inhibit IL‐1β‐induced apoptosis and inflammatory.1.4IL‐1β has been shown to play an integral role in the pathology of many inflammatory diseases, including the induction of apoptosis and the mediation of inflammatory responses. The clarification of the mechanisms of disease progression provides the molecular basis for research into new drugs and opens up new rationales. Signaling pathway‐targeted therapies have been studied more centrally in the treatment of disc degeneration and osteoarthritis, and as such are illustrated below as an example.A recent review revealed that the activation of the PI3K/Akt signaling pathway alleviated intervertebral disc degeneration and focused attention on the therapeutic effects of estrogen.In IL‐1β‐induced chondrocyte injury, NaHS partially inhibited IL‐1β‐induced phosphorylation of the MAPK cascade, significantly reversing apoptosis associated with mitochondrial dysfunctionApart from the currently reported drugs, numerous other biotherapeutic studies have been published. circular RNAs2This paper reviews various literature and studies to indicate that IL‐1β can induce apoptosis in various types of cells under different external stimuli via the mitochondrial pathway, death receptor pathway and endoplasmic reticulum pathway, and that the different pathways are often interconnected. The NF‐κB signaling pathway, p38MAPK, and JNK signaling pathways mainly play a proapoptotic part, and the ERK1/2 pathway has a bidirectional role in regulating apoptosis, while activation of the PI3K‐Akt signaling pathway can inhibit apoptosis. As apoptosis is a complex process, the molecules, substrates, mechanisms of action and regulation of the signal transduction pathway remain to be further investigated. The study of signal pathways has led to a clearer understanding of the pathophysiology of disease and has provided a theoretical basis and new ideas for the development of targeted therapeutics that interfere specifically with the expression of molecules in one or more signal pathways to stop disease progression accurately and effectively. We look forward to a future where the treatment of IL‐1β and its signal pathways may become a new target for the treatment of inflammatory diseases, and inhibitors of the caspase downstream pathway are promising new loci for treatment. At present, many of these targeted therapeutics remain in in vitro models with a long way to go before they can be used clinically. In addition, the research of normal physiological activity is no less important than the study of the onset of disease.In the future, it may be possible to explore the role of IL‐1β in inducing apoptosis during physiological processes, such as physiological root resorption in deciduous teeth, based on an understanding of the IL‐1β signal pathway, which has great potential for preventive applications.The authors declare no competing interests.


Duhuo Jisheng Decoction (DHJSD) is an ancient compound widely used in the treatment of ankylosing spondylitis (AS). However, its efficacy is controversial, and its mechanism of action is not clear enough. Using meta-analysis and network pharmacology, our study evaluated the clinical efficacy of DHJSD in the treatment of AS and explored its mechanisms of action.  We searched medical databases, including Embase, PubMed, the China National Knowledge Infrastructure databases, Wanfang, and the Chinese Scientific Journal Database, to identify studies that met the inclusion criteria. RevMan 5.3 software was used for the meta-analysis. The compounds and the potential protein targets of DHJSD were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform. AS was treated as a search query in the NCBI, PharmGKB, TTD, DrugBank, and OMIM databases to obtain disease-related genes. The overlapping targets of DHJSD and AS were identified, and then Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed. Cytoscape was employed to construct a drug-compound-target network and a protein-protein interaction (PPI) network. CytoHubba was utilized to select the hub genes.  A total of 10 studies involving 860 participants were included in the meta-analysis. Compared with the control, DHJSD treatment significantly improved clinical symptoms; reduced the erythrocyte sedimentation rate (ESR), the C-reactive protein (CPR), and interleukin 6 (IL-6) levels; increased the degree of motion of the chest; reduced the visual analog scale (VAS) pain score; reduced Schober's test values; reduced the finger-to-floor distance; reduced the duration of morning stiffness. However, the differences were not statistically significant in the Bath Ankylosing Spondylitis Functional Index scores, the Bath Ankylosing Spondylitis Disease Activity Index scores, the bone Gla-containing protein (BGP) levels, or the bone alkaline phosphatase (BALP) levels. In terms of adverse events, DHJSD treatment of AS reduced the incidence of gastrointestinal events, the incidence of skin events, and the incidence of abnormal liver function; however, there was no statistically significant reduction in the incidence of adverse renal function events. Subgroup analysis showed that in the treatment of AS, the clinical effect of DHJSD for AS was better than that of the controls for both treatment durations, ≤2 months and >2 months. A total of 178 active compounds and 47 related potential targets were identified for DHJSD in the treatment of AS, including four hub genes . The core active ingredients of DHJSD in the treatment of AS were mainly quercetin, kaempferol, licochalcone A, and isorhamnetin. DHJSD treatment of AS-related pathways mainly involved the IL-17 signaling pathway, the TNF signaling pathway, and the rheumatoid arthritis signaling pathway.  The above results suggest that DHJSD acts on AS through multiple targets, components, and pathways with significant clinical efficacy. Future studies may further explore the active components of DHJSD. Ankylosing spondylitis is a chronic inflammatory disease that mainly affects large joints, such as the sacroiliac joint and the spine, but it can damage the surrounding small joints as well. It can cause spinal stiffness and deformity. The disability rate is as high as 30%, which seriously affects the quality of life of patients , 2.At present, this disease still cannot be cured. Treatment is used mainly to control the clinical symptoms, slow down disease progression, and improve the patient's prognosis. Western medicines are mainly antirheumatic drugs, such as NSAIDs and glucocorticoids –5. HowevAnkylosing spondylitis falls into the categories of “nephroparalysis” and “bone impediment” in traditional Chinese medicine (TCM) –10. In tDuhuo Jisheng Decoction (DHJSD) is a classic prescription commonly used to treat liver and kidney deficiency, deficiency of essence and blood, and chronic arthralgia. It has the functions of nourishing the liver and kidneys, nourishing essence and blood, strengthening muscles and bones, removing dampness, and relieving arthralgia , 14.Eucommiae Ulmoidis), Ren Shen (Panax Ginseng), Gan Cao (Radix Glycyrrhizae), Dang Gui (Radix Angelicae Sinensis), Niu Xi (Radix Achyranthis Bidentatae), Shu Di Huang (Radix Rehmanniae Preparata), and Rou Gui (Cortex Cinnamomi) [DHJSD is mainly composed of Du Huo (Radix Angelicae Pubescentis), Sang Ji Sheng (Herba Taxilli), Qin Jiao (Radix Gentianae Macrophyllae), Fang Feng (Radix Saposhnikoviae), Xi Xin (Herba Asari), Fu Ling (Poria Cocos), Chuan Xiong (Rhizoma Chuanxiong), Bai Shao (Radix Paeoniae Alba), Du Zhong (Cortex nnamomi) . There aA systematic search of PubMed, Embase, the China National Knowledge Infrastructure databases, Wanfang, and the Chinese Scientific Journal Database was conducted from inception to June 30, 2021, with no language restriction. Randomized controlled trials (RCTs) that involved the use of DHJSD for the treatment of AS were collected. The keywords used were as follows: Duhuo Jisheng Decoction, Duhuo Jisheng Tang, DHJSD, Ankylosing Spondylitis, and Bechterew Disease. Furthermore, we manually searched the reference lists of the included studies for additional studies.Patients with a clear diagnosis of AS; no restrictions on race, sex, or age were imposedRCTsControl group for Western medicine treatment and experimental group for treatment with DHJSD based on the control groupOne or more of the following outcome indicators had to be present: (1) primary outcome: clinical efficacy; (2) secondary outcomes: Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Visual Analog Scale (VAS) pain score, Finger-to-floor distance, morning stiffness duration, degree of motion of chest, Schober's test values, Erythrocyte Sedimentation Rate (ESR), C-reactive protein (CPR), bone alkaline phosphatase (BALP), interleukin 6 (IL-6), bone Gla-containing protein (BGP), and adverse eventsThe inclusion criteria included the following:Republished studiesNon-RCTsExperiments on animalsStudies lacking literature dataReviews, conference reports, system reviews, protocols, or abstractsThe exclusion criteria included the following:All the retrieved search results were summarized and imported into Endnote, which was used to delete duplicate searches. By reading the title of the article and the abstract, the literature that did not meet the inclusion criteria was preliminarily excluded. The full text of the literature that met the inclusion criteria after the initial screening was downloaded, and two independent authors read the full text of each source carefully and cross-checked its content against the inclusion/exclusion criteria. For controversial literature, the two authors discussed their differences and either reached a resolution or asked the third author to evaluate the article and make a decision. The data extracted from the articles included the following: (1) general information: the name of the first author, research time, sample size, gender, and age; (2) research design method: random allocation of specific methods; blinding; loss to follow-up; withdrawal cases in the research analysis; (3) treatment status: the specific drug name, dose, frequency, course of treatment, and route of administration used in the treatment group and the control group; (4) outcome indicators: clinical effective rate and BASFI, BASDAI, and VAS scores. If some studies lacked data that affected the final statistical analysis, the author was contacted, as necessary, to supplement it.RCTs were evaluated by the researchers based on the Cochrane risk bias assessment tool. Assessments included random assignment methods, allocation concealment, blinding methods, missing outcome data, selecting result reports, and other biases.Meta-analysis was performed with RevMan 5.3 software. Dichotomous data were reported as risk ratios (RRs) with 95% confidence intervals. Continuous data were calculated as the standard mean difference (SMD) with associated 95% CIs. When the heterogeneity was too large or if we were unable to find the data source, the meta-analysis was abandoned, and only descriptive analysis was performed. A subgroup analysis was performed according to the course of treatment (≤2 months and >2 months). The sensitivity of each index was analyzed with the elimination method to test the stability of the results. If no fewer than ten articles were included, then Begg's funnel plot and Egger's regression analysis were used to test for publication bias.Saposhnikoviae, Herba Asari, Poria Cocos, Rhizoma Chuanxiong, Radix Paeoniae Alba, Cortex Eucommiae ulmoidis, Panax Ginseng, Radix Glycyrrhizae, Radix Angelicae Sinensis, Radix Achyranthis Biatae, Radix Rehmanniae Preax Ginsta, and Radix Glycyrrhizae, respectively.Du Huo, Qin Jiao, Sang Ji Sheng, Fang Feng, Fu Ling, Xi Xin, Chuan Xiong, Bai Shao, Du Zhong, Ren Shen, Gan Cao, Dang Gui, Niu Xi, Shu Di Huang, and Rou Gui are monarchs and minister herbs, which are regarded as the main active herbs in DHJSD that have the Latin names of Radix Angelicae Pubescentis, Herba Taxilli, Radix Gentianae Macrophyllae, Radix https://tcmspw.com/tcmsp.php database.The active chemical components of DHJSD's component drugs were searched using the TCMSP, Version: 2.3, The active ingredients screened in DHJSD were searched through the TCMSP platform to find their corresponding target proteins. The UniProt database was used to query the gene names corresponding to the target proteins, merge duplicate targets of action, and delete proteins without corresponding gene names.“Ankylosing Spondylitis” was used as the keyword search term in the National Center for Biotechnology Information (NCBI), Online Mendelian Inheritance in Man (OMIM), Pharmacogenomics Knowledgebase (PharmGKB), Therapeutic Target Database (TTD), and DrugBank.Cytoscape 3.8.0 software was used to build a “drug-compound-target” network and to analyze compounds and targets. Disease-drug names, active ingredients, and targets are represented by “nodes,” and the interrelationships between nodes are represented by “edges.”We imported the potential targets of DHJSD in treating AS into the STRING database, selected the species as “Homo sapiens,” selected the minimum required interaction score as medium confidence (0.400), removed the free nodes, obtained the protein interaction information, and saved it in “TSV” format. The “TSV” file was imported into Cytoscape 3.8.0 to draw a protein-protein interaction (PPI) network. We analyzed the nodes in the network through the plug-in CytoNCA. We calculated the betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigenvector centrality (EC), local average connectivity-based method centrality (LAC), and network centrality (NC) of each node in the network. Nodes with DC ≥ median, CC ≥ median, BC ≥ median, EC ≥ median, LAC ≥ median, and NC ≥ median were selected, and the first screening was completed. We established a subnetwork and once again used the plug-in CytoNCA to calculate the DC, BC, CC, EC, LAC, and NC of the nodes in the subnetwork and to filter out DC ≥ median, CC ≥ median, BC ≥ median, and EC ≥ median for nodes with LAC ≥ median and NC ≥ median; thus, we complete the second screening. We drew a flowchart to visualize the screening process.P < 0.05 was the screening condition.R 4.1.0 software and its “org.Hs.eg.db” package were used to obtain the ID of the intersection target. Then, program packages such as “clusterProfiler” were used to perform GO function enrichment analysis on the target and screen out the top 10 functional categories of biological process (BP), cellular component (CC), and molecular function (MF) and to draw them into a histogram. Then, KEGG enrichment analysis was performed on the target, the first 20 KEGG pathways were selected, and a histogram was drawn. A total of 396 studies were searched, and 249 studies were obtained after removing duplicates. After screening, ten papers were finally included. The literature screening process is shown in P=0.02, I2 = 53%). When the random effect model was used to merge RR values, the pooled RR was 1.30 . This indicated that the clinical effective rate was significantly higher in the experimental group than that in the control group . The random-effects model combined SMD, and the pooled SMD was −0.46 . This indicated that the difference in BASFI scores between the experimental and control groups after treatment was not statistically significant . The random-effects model combined SMD, and the pooled SMD was −0.36 . These results indicated that the difference in BASDAI scores between the experimental and control groups after treatment was not statistically significant . The random-effects model combined SMD, and the pooled SMD was -0.79 . Compared with the control groups, the DHJSD treatment groups were associated with reduced VAS pain scores . The random-effects model combined SMD, and the pooled SMD was −1.55 . Compared with the control groups, the DHJSD treatment groups were associated with reduced finger-to-floor distance . The random-effects model combined SMD, and the pooled SMD was−1.23 . Compared with the control groups, the DHJSD treatment groups were associated with reduced morning stiffness time . The fixed-effects model combined SMD, and the pooled SMD was 0.24 . Compared with the control groups, the DHJSD treatment groups were associated with an increased degree of motion of the chest . The fixed-effects model combined SMD, and the pooled SMD was 0.23 . Compared with the control groups, the DHJSD treatment groups were associated with increased Schober's test values . The random-effects model combined SMD, and the pooled SMD was −1.07 . Compared with the control groups, the DHJSD treatment groups were associated with reduced ESR levels . The random-effects model combined SMD, and the pooled SMD was −2.31 . Compared with the control groups, the DHJSD treatment groups were associated with reduced CPR levels . The random-effects model combined SMD, and the pooled SMD was 0.18 . This indicated that the difference in BALP levels between the experimental groups and the control groups after treatment was not statistically significant . The random-effects model combined SMD, and the pooled SMD was −1.72 . Compared with the control groups, the DHJSD treatment groups were associated with reduced IL-6 levels . The random-effects model combined SMD, and the pooled SMD was 3.14 . This indicated that the difference in BGP levels between the experimental groups and the control groups after treatment was not statistically significant . When the fixed effect model was used to merge RR values, the pooled RR was 0.27 . This indicated that the incidence of gastrointestinal events was significantly lower in the experimental groups than it was in the control groups . When the fixed-effects model was used to merge RR values, the pooled RR was 0.13 . This indicated that the incidence of skin events was significantly lower in the experimental groups than it was in the control groups . When the fixed-effects model was used to merge RR values, the pooled RR was 0.28 . This indicated that the incidence of abnormal liver function was significantly lower in the experimental groups than it was in the control groups . When the fixed effect model was used to merge RR values, the pooled RR was 0.29 . This indicated that the incidence of abnormal renal function was not significantly different between the experimental groups and the control groups  or >2 months , P=0.0006), and the difference was statistically significant  to analyze the clinical effective rates in AS patients. The results of subgroup analysis showed that the clinical effective rates of the experimental groups were higher than those of the control groups, regardless of whether the treatment course was ≤2 months , nificant .Sensitivity analysis of the clinical effective rate showed that the results of this meta-analysis were stable .P=0.012; Egger's test: P=0.023).Begg's funnel plot and Egger's test were performed to evaluate publication bias . The resEucommiae Ulmoides, 18 were from Radix Saposhnikoviae, six were from Poria Cocos, 88 were from Radix Glycyrrhizae, 16 were from Radix Achyranthis Bidentatae, two were from Radix Gentianae Macrophyllae, 17 were from Panax Ginseng, two were from Herba Taxilli, ten were from Cortex Cinnamomi, three were from Radix Rehmanniae Preparata, and seven were from Radix Herba Asari. In addition, based on these compounds, 262 predicted targets of DHJSD were found. A total of 178 active ingredients were extracted from the TCMSP database. Information on active compounds in DHJSD and the number of corresponding targets are shown in By querying the TCMSP database, after screening with OB ≥ 30% and DL ≥ 0.18, a total of 179 active compounds were obtained, of which seven compounds were from Radix Paeoniae Alba, six were from Rhizoma Chuanxiong, three compounds were from Radix Angelicae Sinensis, eight were from Radix Angelicae Pubescentis, 25 were from Cortex By searching the NCBI, OMIM, PharmGKB, TTD, and DrugBank databases, a total of 398 AS-related targets were obtained.The targets of DHJSD and AS-related targets were crossed, and 47 crossed targets were obtained; that is, there were 47 potential targets for DHJSD in the treatment of AS. The results are shown in Through the STRING database, we constructed a PPI network for DHJSD in the treatment AS. A node represents the potential target protein, and the connection between the two nodes represents the interactive relationship between the target proteins . The datP < 0.05, the top 10 different biological processes were selected for visual display  release, and alleviate inflammation [kB signaling pathway, inhibit the release of cytokines , and alleviate inflammation [We identified the active compounds and target genes of DHJSD through TCMSP. Then, 178 candidate target genes of DHJSD that are active in AS were identified, and the key compounds of DHJSD that act on AS were screened. The main key compounds are quercetin, kaempferol, licochalcone A, and isorhamnetin. Quercetin has pharmacological effects as an antioxidant and an anti-inflammatory, it protects bones and joints, it acts as an antiapoptosis agent, it inhibits osteoclast absorption, and it regulates immune function –30. Quersymptoms –30. Kaeme effect , 32. In ammation , 32. Licammation . As a diammation , 35. Expammation .PPI network analysis shows that DHJSD can affect AS through multiple targets. The main targets are CXCL8, PTGS2, VEGFA, and STAT3. The Entrez Gene summary of the CXCL8 gene shows that the protein encoded by this gene is a member of the CXC chemokine family and is the main mediator of the inflammatory response , 38. A pDuring the GO enrichment analysis in this study, it was discovered that the biological process of DHJSD in the treatment of AS includes responses to lipopolysaccharides, to molecules of bacterial origin, to fatty acid metabolic processes, to the external side of plasma membranes, to the nuclear envelope, to the nuclear membrane, to heme binding, to tetrapyrrole binding, to DNA-binding, to transcription factor binding, and to many other biological processes. These biological processes play a key role in the treatment of AS by DHJSD.KEGG enrichment pathway analysis showed that many pathways were closely related to the pathogenesis of AS. The main pathways included the IL-17 signaling pathway, the TNF signaling pathway, and the rheumatoid arthritis signaling pathway. The IL−17 signaling pathway and the TNF signaling pathway play a central role in the pathogenesis of AS . Mouse mMeta-analysis is a systematic, quantitative, and comprehensive statistical method based on previous research results. It is often used to evaluate interventional RCTs in evidence-based medicine to objectively evaluate clinical efficacy. Therefore, in this study, we compiled current evidence on the overall effect of DHJSD on AS in 860 patients from 10 studies. The results of the meta-analysis showed that DHJSD treatment of AS has clinical efficacy with a reduced occurrence of adverse reactions. Evidence-based medicine methods show the efficacy of DHJSD treatment on AS at the clinical level, which is more meaningful than experimental verification. Through the integration of meta-analysis and network pharmacology analysis, we successfully determined the clinical efficacy of DHJSD in the treatment of AS and the targets, components, and pathways of DHJSD in the treatment of AS.However, this study also includes some other limitations. First, due to the particularity of traditional Chinese medicine decoctions, it is difficult to implement allocation concealment and blinding. Second, pharmacological analysis is mainly used to demonstrate the synergistic effects of herbal chemical components obtained through existing databases, rather than experimental verification, which needs to be further verified in future studies.According to existing research data, DHJSD treatment of AS can improve clinical efficacy and reduce adverse reactions. Its mechanism of action works mainly through its active ingredients, such as quercetin, kaempferol, licochalcone A, and isorhamnetin, which act on key target genes such as CXCL8, PTGS2, VEGFA, and STAT3, thereby regulating the IL-17 signaling pathway, the TNF signaling pathway, the rheumatoid arthritis signaling pathway, and other related signaling pathways. DHJSD should eventually be given a formal role in the treatment of AS.


Despite the significant decline in mortality, cardiovascular diseases are still the leading cause of death worldwide. Among them, myocardial infarction (MI) seems to be the most important. A further decline in the death rate may be achieved by the introduction of molecularly targeted drugs. It seems that the components of the PI3K/Akt signaling pathway are good candidates for this. The PI3K/Akt pathway plays a key role in the regulation of the growth and survival of cells, such as cardiomyocytes. In addition, it has been shown that the activation of the PI3K/Akt pathway results in the alleviation of the negative post-infarct changes in the myocardium and is impaired in the state of diabetes. In this article, the role of this pathway was described in each step of ischemia and subsequent left ventricular remodeling. In addition, we point out the most promising substances which need more investigation before introduction into clinical practice. Moreover, we present the impact of diabetes and widely used cardiac and antidiabetic drugs on the PI3K/Akt pathway and discuss the molecular mechanism of its effects on myocardial ischemia and left ventricular remodeling. Cardiovascular diseases (CVD) are the leading cause of death globally, and myocardial infarction (MI) is of fundamental importance . MI is cThe 1-year mortality rate among patients after MI is still high . HoweverAt the same time, cardiovascular diseases are associated with growing public and private expenditure on healthcare worldwide . TherefoPost-infarction HF is mostly caused by adverse remodeling of the left ventricle (LV). This is a series of molecular, cellular, and interstitial changes following MI ,12,13. IThe phosphatidylinositol 3-kinase (PI3K)/protein kinase B(Akt) pathway is one of the most significant intracellular signal transduction pathways. Even though it was relatively recently discovered, it has caused a revolution mainly in personalized oncology. PI3K was identified by Lewis Cantley’s group in 1988 , while AThe proper activity of PI3K and Akt appears to be essential for the development, functioning, and survival of the organism. The lack of their expression leads to a blockade of cell division and embryonic lethality ,24. In rThe main molecules involved in the PI3K/Akt pathway are presented in the figure below a This paReceptor tyrosine kinases (RTKs) are surface receptors composed of three functional domains: an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain . After bDue to the importance of this pathway, its activity must be carefully regulated. Downregulation of the PI3K/Akt pathway may be achieved via two mechanisms. Firstly, phosphatase and tensin homolog (PTEN) may specifically dephosphorylate the 3-phosphate of the inositol ring in PIP3 to PIP2 and therefore reduce the concentration of PIP3 required for PI3K activation ,43,44. IThe activity of the described pathway has an impact on many processes associated with the peri-infarct period, such as myocardial fibrosis, and other components of post-infarct left ventricular remodeling b,c. All Both the physical obstruction of a coronary vessel and the redistribution of blood flow reduce the oxygen supply and are responsible for irreversible damage of the cardiomyocytes . Necrosi+, which arises from anaerobic glycolysis. In addition, the lack of ATP causes the dysfunction of the Na+/K+ ion pump. The increased level of H+ causes the need for their removal by the Na+/H+ exchanger. In response to elevated levels of Na+ (associated with the Na+/K+ ion pump failure and the Na+/H+ exchanger) inside of the cell, the Na+/Ca2+ exchanger operates in reverse mode and causes an increase in Ca2+ concentration. Elevated levels of all of these ions cause the swelling of the cell and promote the rupture of the cell membrane and necrosis. In one basic study, it was shown that pretreatment with HDL can protect the cardiomyocytes against oxygen and glucose deprivation-dependant necrosis. In addition, the authors brought about the inhibition of the PI3K/Akt pathway which abolished the protective effect of HDL  and cardiac death  during the one-year follow-up period [p < 0.0001) [MiR-145 can also play a cardioprotective role, attenuating both myocardial ischemia–reperfusion injury and post-infarct remodeling via targeting the PI3K/Akt pathway. It inhibits cardiac cells’ apoptosis, ROS activity and increases cells’ viability under hypoxic conditions in vitro. These effects are positively related to the activation of PI3K/Akt and expression of SGK1 (serum- and glucocorticoid-regulated kinase (1)—a downstream effector of PI3K associated with cell survival . Moreovep period . Moreove 0.0001) . MiRNAs may play and important role in the modulation of PI3K/Akt activity in the heart. They may serve as potential biomarkers and prognostic factors of the infarct size and adverse post-MI remodeling. Moreover, miRNAs can mediate the effect of DM on the impaired PI3K/Akt signaling in the heart.The PI3K/Akt pathway plays an important role in the survival and function of cardiomyocytes. It seems that this pathway may be an ideal target to protect the myocardium against most of the post-infarct changes described above. In addition, PI3K and other components of the described pathway are among the essential links in the inflammatory response after ischemia. Substances activating this pathway  are at an advanced stage of development in basic research, but still the results are not conclusive ,328,329.Besides the activators of this pathway, substances which inhibit  its activation are under investigation for oncology . Taking The PI3K/Akt pathway regulates the metabolism of cardiac cells on multiple levels, and plays an important role in the pathophysiology of myocardial infarction and the post-infarction remodeling of the left ventricle. The activation of this pathway is responsible for the cardioprotective effect of many drugs such as statins and ACEI, prescribed for patients with high cardiovascular risk or after MI. Moreover, altered activity of the PI3K/Akt pathway during diabetes may be responsible for the greater risk of death or post-MI HF development in this group of patients. The results of thebasic studies show that the modulation of this pathway by selective inhibitors or miRNA can influence both ischemic damage and post-infarction LV remodeling. Akt seems to be a key component of this signal transduction and is responsible for most of the positive effects of its activation in cardiomyocytes. There is still a long way to go before introducing drugs based on the activation or inhibition of this pathway, but this may bring significant clinical benefits.


Diabetic osteoporosis (DOP) is one of the chronic complications of diabetes mellitus, but without a standardized treatment plan till now. Liuwei Dihuang pill (LDP) has gradually exerted a remarkable effect on DOP in recent years; its specific mechanism is not clear yet.We adopted network pharmacology approaches, including multi-database search, pharmacokinetic screening, network construction analysis, gene ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis and molecular docking to elaborate the active components, signaling pathways and potential mechanisms of LDP in the treatment of DOP.Twenty-seven active ingredients and 55 related disease targets have been found through integrated network pharmacology. Functional enrichment analysis shows that five key active ingredients, including beta-sitosterol, stigmasterol, diosgenin, tetrahydroalstonine, and kadsurenone, may give full scope to insulin secretion estrogen-level raising and angiogenesis in biological process through the pivotal targets. In addition, the underlying effect of PI3K/AKT/FOXO and VEGF pathways is also suggested in the treatment.Based on systematic network pharmacology methods, we predicted the basic pharmacological effects and potential mechanisms of LDP in the treatment of DOP, revealing that LDP may treat DOP through multiple targets and multiple signaling pathways, which provide evidence for the further study of pharmacological mechanism and broader clinical thinking.The online version contains supplementary material available at 10.1186/s13018-022-03194-2. Along with the significant change in an individual’s lifestyle and diet habits, diabetes mellitus (DM) has gradually developed into a common endocrine and metabolic disease that accompanies a dramatically increasing prevalence worldwide. Osteoporosis (OS) is also a growing public health concern worldwide relating to the possibility of a loss of life, with more than 9 million osteoporotic fractures taking place every year . More anLiuwei Dihuang pill (DOP) is a classic prescription for nourishing kidney yin created by Qian Yi in the Song Dynasty. It consists of six traditional Chinese medicines: Rehmanniae Radix Preparata (SDH), Cornus Officinalis Sieb.et Zucc. (SZY), Poria cocos (Schw.) Wolf. (FL), Rhizoma Dioscoreae (SY), Alisma orientale (Sam.) Juz. (ZX), and Cortex Moutan (MDP). In traditional Chinese medicine, it is believed  that “kiNetwork pharmacology is an emerging discipline with scientific ideas and research strategies. It provides a new network model of “multiple targets, multiple effects, and complex diseases” to explain the mechanism of traditional Chinese medicine treatment . At preshttp://lsp.nwu.edu.cn/, Version 2.3) [Active components of the six LDP herbs were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  . In thision 2.3) , while cion 2.3) . Caco-2 ion 2.3) .http://www.uniprot.org/) [Subsequently, the TCMSP was further used to predict the potential targets of these bioactive compounds. We converted the obtained drug targets into gene names and restricted the species to “Homo sapiens” through The Universal Protein Resource  .https://www.genecards.org/) database [http://www.omim.org/) [In this study, human genes related to LDP came from two sources: (1) GeneCards (database . It is aim.org/)  is a comThe compound–D&L target interaction was established and visualized by Cytoscape (Version 3.7.1). By using the Cytoscape tool network analyzer to calculate three indicators: degree, intermediateness, and compactness, we evaluated the topological characteristics of the interactive network. We also adopted the MCC algorithm of another Cytoscape tool—cytoHubba —to perfohttps://string-db.org/), which contains 9,643,763 proteins and 1,380,838,440 protein interaction information [D&L's protein interaction (PPI) data are obtained from Search Tool for the Retrieval of Interacting Genes . We set the standard of p value to 0.05 and use RStudio 3.6.1 (GOplot) to visualize GO terms and KEGG pathways, and the false discovery rate (FDR) is < 0.05.Gene ontology (GO) functions are performed in three categories of GO terms: biological process (BP), molecular function (MF), and cellular component (CC). The “Kyoto Encyclopedia of Genes and Genomes” (KEGG) pathway enrichment analysis is an important method for characterizing candidate targets. Both of them were conducted to predict the specific mechanism through the Database for Annotation, Visualization, and Integrated Discovery  and PubChem (https://pubchem.ncbi.nlm.nih.gov/) was used to find the chemical and conformational information of the compounds. Then, before the docking experiments, we used AutoTools software to preprocess the crystal structure of the target protein, including removing excess protein chains, ligands, water molecules, and hydrogenation. Moreover, the ligand structure is needed to conform to a low-energy conformation. Furthermore, we used AutoDock Vina to simulate the docking state between proteins and small molecules [We chose the core compounds in LDP and the key proteins in core pathways for molecular docking. First, the 3D structure of the target protein was obtained from the RCSB PDB database  genes , Cornus Officinalis Sieb.et Zucc. (SZY), Poria cocos (Schw.) Wolf. (FL), Rhizoma Dioscoreae (SY), Alisma orientale (Sam.) Juz. (ZX), and Cortex Moutan (MDP). Among the D&L genes, 16, 31, 9, 45, 4, and 3 genes are relatively corresponding to 2, 13, 4, 10, and 2 compounds. Cornus Officinalis Sieb. Et Zucc and Rhizoma Dioscoreae are the two herbs that account for the highest proportion of collected compounds and targets  for protein–protein interaction analysis. As shown in Fig. To further clarify the core biological functions of LDP, GO analysis was performed on 17 potential targets in Module 1 from the aspects of biological process (BP), molecular function (MF), and cell composition (CC).P < 0.05, we obtained 133 GO entries, including 90 BP entries, 31 MF entries, and 12 CC entries  and the core target protein  in PI3k/Akt signaling pathway and VEGF signaling pathway. After searching AKTI, FOXO1, PI3K, and VEGFA from the PDB protein database, the three-dimensional structures of 6s9w, 6qvw, 3l54, and 3qtk proteins were obtained.The docking score suggests that most of them have excellent binding activity Table , which i–1, while there was no death in mice and rats after 14 days of administration of 15 g·kg–1 Liuwei Dihuang either [–1 in rats for 180 days and 0.5 g kg–1 in beagle dogs for 270 days, which is the equivalent of 25 and 30 times of the clinical dosage for humans, respectively [https://admetmesh.scbdd.com/) [In the long-term clinical application and clinical trials, no liver and kidney toxicity and adverse reactions to Liuwei Dihuang pill were observed , 20. Preg either . Resultsectively . Specifidd.com/) . ADMETladd.com/) . Accordi1/2α and t1/2β were 2.62/32.66, 0.46/4.71, and 1.30/23.51 h, and the climax times and concentrations were 0.56/683.75, 0.70/2826.11, and 0.62 h/ 4030.48 ng·mL−1 for HMFA, loganin, and paeonol, respectively [Although Liuwei Dihuang pill has a long history of clinical application, the scientific and systematic pharmacokinetic evaluation is far from comprehensive. There are few studies on the pharmacokinetic parameters and pharmacokinetic characteristics of Liuwei Dihuang pill, which mainly focus on its bioactive chemical components. The effects of Liuwei Dihuang pills on cytochrome P4502C19, cytochrome P4502D6, and cytochrome P4503A4 activities in 12 healthy Chinese subjects were evaluated in a single-center, controlled, open-label, two-way crossover clinical trial . The resNetwork pharmacology is a combination of clustering algorithms and network topology. It is good at expressing complex data interaction relationships. It is a huge project aimed at exploring the relationship between compounds and target genes of the disease through experiments. Therefore, we eliminate irrelevant interference factors through network pharmacology, aiming to screen the central compounds, the most potential gene targets, and pathways to provide a more precise direction for studying its mechanism.Cornus Officinalis Sieb. et Zucc. and Rhizoma Dioscoreae are the two main drugs that can adjust most compounds and targets in the LDP component analysis. After further research on herbal medicine, we found that five central compounds  play a critical regulatory role in the treatment of DOP. Molecular docking verification showed that these five compounds also have a good degree of binding with the core protein of the vital pathway we obtained (of which diosgenin is the most important). In vitro experiments have shown that diosgenin can reduce bone loss in ovariectomized rats by reducing the ratio of receptor activator of nuclear factor-κ B ligand (RANKL)/osteoprotegerin (OPG) , while iTo clarify the role of LDP in gene function, we performed GO function enrichment analysis on potential targets. The biological process shows that insulin secretion, increasing estrogen levels, and promoting angiogenesis are the three main aspects that LDP may play an important role in the treatment of DOP.The lack of insulin (INS) is directly related to the occurrence and development of DOP. INS sends signals to the insulin receptor (IR) on the surface of osteoblasts (OBs) to promote collagen synthesis and calcium deposition in bones . OsteocaSclerostin is secreted by bone cells and is a negative regulator of bone mass, which can effectively prevent the bone formation and promote osteoblast apoptosis. Experiments have shown that the expression of the sclerostin gene is increased in diabetic rats , while bBone is a connective tissue rich in blood vessels. As an influencing factor in bone development, regeneration, and remodeling, blood vessels are essential in reducing bone formation caused by impaired angiogenesis. Clinical studies have shown that decreased bone mineral density in women with Type 2 diabetes mellitus (T2DM) has a special relationship with diabetic microangiopathy . In the In addition, combined with the GO enrichment analysis and based on the enrichment results of KEGG, we speculate that the pathway of LDP on DOP may be mainly insulin-induced PI3K/AKT/FOXO signaling pathway and VEGF signaling pathway.The insulin signaling pathway is of great significance for maintaining average blood glucose levels and avoiding complications of hyperglycemia. In the insulin pathway, insulin can activate multiple intracellular signaling pathways, among which PI3K/AKT is the main downstream molecular pathway downstream and plays a central role in cell physiology. PI3K/AKT signaling pathways can effectively improve insulin sensitivity, relieve insulin resistance, and regulate glucose metabolism . It alsoThe PI3K/AKT signaling pathway is a typical pathway to regulate the transcriptional activity of FOXO . ThroughAs we all know, the VEGF signaling pathway is the fundamental way to regulate angiogenesis . HoweverIn conclusion, LDP may have an angiogenesis–osteogenesis coupling through the VEGF signal pathway and the VEGF/PI3K/AKT cascade, which will have a positive effect on DOP.The results of this network construction are in line with our initial bioinformatics expectations: LDP can achieve the potential features of multiple targets–multiple channels treatment through the LDP–DOP network. This study demonstrates the two most effective herbs, five central compounds, seven core target genes, three biological processes, and two core pathways. But at the same time, we only pointed out that diosgenin has excellent research potential and value, and there is still a lack of in-depth excavation and research. In addition, although we found that the PI3K/AKT/FOXO and VEGF pathways may be the potential mechanisms of treatment, and these compounds can bind core proteins well in both pathways, we cannot ignore that the inference of these two pathways is based on the existing research on DOP. KEGG analysis also found some other potential enrichment pathways worthy of our further study.In addition, this study is only used for the prediction and construction of network informatics, and its practicality and feasibility need to be further improved. It is necessary to carry out the clinical application and experimental implementation under conditions to judge the accuracy of this prediction. We sincerely hope that these findings can provide readers with some hints and new ideas.In conclusion, the use of network pharmacology and molecular docking in the study of LDP treatment in DOP reveals the potential pharmacological process and specific mechanisms that may be related to the several key active ingredients and signaling pathways Fig. . Beta-siAdditional file 1. Supplementary Table S1. The significant GO entries enriched by the potential target genes. Supplementary Table S2. The significant KEGG pathway enriched by the potential target genes.


Objective: Network pharmacology provides new methods and references for the research of traditional Chinese medicine, but some problems remain, such as single evaluation components and index methods, imperfect relevant databases, unscientific prediction results, and lack of verification of results. Herein, we used a modified network pharmacology research method to explore the potential network analysis mechanism of Huoxue Qingre decoction in the treatment of coronary heart disease and utilized clinical trials for assessment.Methods: Based on literature research, the targets corresponding to the drug were obtained with the assistance of the TCMSP database and Swiss Target Prediction, and the target proteins were corrected using the UniProt database. The targets related to coronary heart disease was obtained through the GeneCards database. A protein-protein interaction network diagram was constructed, and a “component-intersection target” network diagram was drawn based on Cytoscape 3.6.2 software. The mapped targets were imported into the DAVID bioinformatics platform, which underwent Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and the network pharmacology prediction results were evaluated through clinical trials.Results: We obtained 151 compounds related to Huoxue Qingre decoction, 286 genes after evaluation and deduplication, and 426 genes related to coronary heart disease. Finally, 81 common target genes were obtained with 32 pathways according to the KEGG pathway enrichment analysis. The validation results of the clinical trials showed that a total of 98 differential metabolites were found in the treatment of coronary heart disease with Huoxue Qingre decoction, involving a total of 16 metabolic pathways. Compared with the network pharmacology prediction results, it was found that only the pathways in cancer (hsa05200) were the common pathways in the top 32 signaling pathways predicted by network pharmacology. The expanded network pharmacology prediction results revealed that the sphingolipid signaling pathway (hsa04071) and prostate cancer pathway (hsa05215) matched the predicted metabolic pathways, with differential metabolites of N-oleoyl-D-sphingomyelin and 1-methyl-6-phenyl-1h-imidazolepyridine-2-amine.Conclusion: Through the network analysis and metabolomic evaluation, there may be three signaling pathways that involve the Huoxue Qingre decoction in the treatment of coronary heart disease: pathways in cancer (hsa05200), sphingolipid signaling pathway (hsa04071), and prostate cancer pathway (hsa05215). Network pharmacology is an emerging, cross and cutting-edge discipline in the systematic study of drugs in the era of artificial intelligence and big data. It emphasizes that the molecular associations between drugs and therapeutic objects can be obtained from the perspective of the system level and the whole of the biological network. It has been widely used in the interpretation of the overall mechanism of action of traditional Chinese medicine (TCM) compounds, and in the analysis of drug combinations and formula compatibility law, providing new ideas for the study of complex systems of TCM . In receCoronary heart disease (CHD), also known as coronary atherosclerotic heart disease, is caused by narrowing or obstruction of the vascular lumen due to atherosclerotic lesions in the coronary vessels, resulting in myocardial ischemia, hypoxia, or necrosis. Coronary atherosclerosis is a factor involved in the pathogenesis of CHD. It has been reported that the current number of patients with cardiovascular diseases in China is 290 million, of which the number of patients with CHD is as high as 11 million, and this has become a major public health problem in China .At present, there have been satisfactory results in the use of TCM for the treatment of CHD. The combination of western medicine disease identification and TCM syndrome differentiation has become an important form of clinical diagnosis, treatment, and rehabilitation of CHD. In TCM, blood stasis is considered to be a factor leading to the dystrophy of the heart vessels, which leads to pain when the heart vessels are abnormal, thus causing “chest obstruction” and “heartache.” Its characteristics are basically consistent with the symptoms of modern CHD. Qi is the commander of the blood, and the normal operation of blood depends on the normal promotion of Qi. If the vitality is deficient and unable to circulate the blood, the blood flow will be slow and there will be blood stasis. Therefore, the treatment of CHD should focus on promoting blood circulation and invigorating Qi .Salvia miltiorrhiza Bunge , Panax ginseng C.A.Mey. , Coptis chinensis Franch. , Pinellia ternata (Thunb.) Makino , Paeonia lactiflora Pall. , Panax notoginseng (Burk) F. H. Chen , Pueraria lobata (Willd.) Maesen & S.M. Almeida ex Sanjappa & Predeep , and Cistanche deserticola Ma . It has the effects of invigorating Qi and activating blood circulation, clearing heat, and removing phlegm. In this formula, Salvia miltiorrhiza is the monarch drug. Panax ginseng, Coptis chinensis, and Pinellia ternata are the minister drugs. Paeonia lactiflora, Panax notoginseng, Puerariae Lobatae, and Cistanche deserticola are adjuvant drugs. Previously, our group found that HXQR improves the mitochondrial structure and function of cardiomyocytes, inhibits inflammatory factors and myocardial apoptosis, and thus improves ventricular remodeling after myocardial infarction (Huoxue Qingre (HXQR) decoction is the clinical experience of Guang’anmen Hospital in the treatment of CHD with significant therapeutic effect. HXQR consists of eight herbs, including farction .Network pharmacology provides new methods and references for TCM research, but problems still remain such as single evaluation components and index methods, imperfect relevant databases, unscientific prediction results, and insufficient validation of results. Therefore, in the current study, we intend to adopt and improve the network pharmacology research method , exploreSalvia miltiorrhiza Bunge , Panax ginseng C.A.Mey. , Coptis chinensis Franch. , Pinellia ternata (Thunb.) Makino , Paeonia lactiflora Pall. , Panax notoginseng (Burk) F. H. Chen , Pueraria lobata (Willd.) Maesen & S.M. Almeida ex Sanjappa & Predeep , and Cistanche deserticola Ma   . All medThe medicinal herbs of HXQR were extracted by 10 times of water through heating reflux for twice, each time for 1.0 h. The extracts were mixed and concentrated under reduced pressure. The relative density was about 1.12 (measured at 20°C). Ethanol was added to bring the ethanol content to 70%. After 24 h, the extracts were concentrated (density 1.20 at 50°C), and dried in vacuum at 60°C. Finally, all the extracts were crushed and mixed to get an extract of HXQR.Coptis chinensis total alkaloids , 0.33% for ginsenoside Rb1, 1.60% for paeoniflorin, 1.95% for puerarin, 0.24% for echinacoside  ≥30% and drug-like (DL) ≥0.18, combined with the TCM pharmacopoeia and published literature to supplement active ingredients. According to the TCMSP database and Swiss Target Prediction, the targets corresponding to the drug were queried, and the obtained target proteins were evaluated and corrected using the UniProt database, converted into the corresponding standard gene names, and saved in the drug target database for HXQR.Based on the systematic database of TCM pharmacology and the analysis platform ), the components of the main chemicals in HXQR decoction were retrieved. In the GeneCards database, with “coronary heart disease” as the keyword, a search was performed for CHD-related genes. These genes were exported to the Excel program, and gene evaluation was performed with the median, and the median was taken for four times to evaluate the genes with a relevance score greater than the median, and a CHD target database was then established.Mapping targets of diseases and drugs were obtained. Cytoscape 3.6.2 software was used to associate each compound with the corresponding targets to construct an “active ingredients-common targets” network. The STRING database was used to select the “homo sapiens” identification, construct the PPI network, analyze its network topological parameters, and find the key targets with node degree as an important parameter.p value was used as the condition to analyze the pathway of target regulation biology. The main signaling pathways and biological processes of HXQR exerting pharmacological effects were analyzed to explore the possible mechanism of HXQR in the treatment of CHD.HXQR and CHD mapping targets were imported into the DAVID bioinformatics platform for Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The https://www.rcsb.org/) were used to find the PDB ID (Protein Data Bank Identification) of key targets, and obtain protein information, and utilized Pymol software to process molecules and saved them as pdb format. The active ingredient information of the drug corresponding to the target in the TCMSP database was downloaded and saved in mol2 format. Molecular docking was performed using SYBYL-X 2.0 software, and the results were analyzed by the obtained total score.The Uniprot database and RCSB PDB protein structure database . This trial has been approved by the ethics committee .There is a comprehensive understanding of the purpose, methods, benefits, and possible risks of this study, which has been registered in the TCM Clinical Trials Registry  was used for metabolomics analysis in the current study, and the metabolomics analysis method was performed according to the Fujisaka method . The anaChromatographic conditions: chromatographic column ACQUITY UPLC BEH C18 ; column temperature 45°C; mobile phase: (A) water (containing 0.1% formic acid), (B) acetonitrile/methanol (2/3) (v/v) (containing 0.1% formic acid); flow rate: 0.4 ml/min; injection volume: 5 μl; elution conditions: A% (95%–0) gradient elution. Mass spectrometric conditions: electrospray ionization (ESI) ion source, the positive and negative ion scanning modes were used for sample mass spectrometric signal acquisition.S. miltiorrhiza, 14 chemical constituents related to C. chinensis, 25 chemical constituents related to P. ginseng, 13 chemical constituents related to P. ternata, 27 chemical constituents related to P. veitchii, 11 chemical constituents related to P. notoginseng, five chemical constituents related to P. lobata, and eight chemical constituents related to C. deserticola. Combined with the results of the pharmacopoeia and literature analysis, nine components, salvianolic acid B, tanshinone I, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, notoginsenoside R1, puerarin, echinacoside, and acteoside, were added to obtain a total of 172 active ingredients, and 151 compounds were obtained after deleting the duplicates.The chemical constituents of each drug in HXQR were searched by TCMSP, including 67 chemical constituents related to P. ternata and P. veitchii; MOL002879, MOL005341, MOL005344, and MOL007476 are common components of P. ginseng and P. notoginseng; MOL005320 and MOL005384 are common components of P. ginseng and C. deserticola; MOL000098 is a common component of C. chinensis, P. notoginseng, and C. deserticola; MOL002776 is a common component of S. miltiorrhiza, P. ternata, and P. ternata; MOL000449 is a common component of P. ginseng, P. veitchii, P. veitchii, and P. notoginseng; and MOL000358 is a common component of P. ginseng, P. ternata, P. veitchii, P. notoginseng, P. lobata, and C. deserticola. The four components with the highest OB were: MOL007064 przewalskin b, MOL007132 (2R)-3--2-[(Z)-3-acryl]oxy-propionic acid, MOL002907 corchoroside aqt, and MOL005314 celabenzine. The four components with the highest DL were: MOL013352 obacunone, MOL012297 puerarin, MOL008875 echinacoside, and MOL008871 marckine. The results are shown in Among them, MOL002714 is a common component of https://www.uniprot.org/). Human genes corresponding to TCM drug targets were searched to remove duplicates, and finally, 286 genes were obtained. A network diagram of “HXQRs-active ingredient-drug targets” was drawn. The active ingredients in the figure are expressed according to the last five digits of the MOD ID. TCM drugs are represented by initials. There are a total of 445 nodes and 1734 edges, as shown in A total of 2166 constituent targets were retrieved through the TCMSP database and the UniProt website , β-2 adrenergic receptor (ADRB2), sodium channel protein type 5 subunit α (SCN5A), androgen receptor (AR), estrogen receptor (ESR1), inducible nitric oxide lyase (NOS2), sodium-dependent serotonin transporter (SLC6A4), endothelial nitric oxide synthase (NOS3), potassium voltage-gated channel subfamily H member 2 (KCNH2), peroxisome proliferator-activated receptor γ (PPARG), and other targets were the core nodes of the network, as shown in The drug and disease intersection targets from 81 mappings were analyzed by the STRING database to construct the potential target PPI network, and the results were shown in p < 0.01, 254 items were obtained for BP, 26 items for CC, and 41 items for MF. BPs mainly involve positive regulation of transcription by the RNA polymerase II promoter, positive regulation of transcription, DNA template, gene expression, signal transduction, inflammatory response, cell proliferation, aging, drug response, negative regulation of apoptotic process, and response to hypoxia; CCs mainly involve the plasma membrane, extracellular space, extracellular region, cytosol, and extracellular exosome; MFs mainly involve protein binding, identical protein binding, enzyme binding, protein homodimerization activity, and cytokine activity. Among them, three aspects are in the descending order of enrichment number, and the first 20 items were plotted. The p value is taken as the negative logarithm in the figure, and the significance is expressed by the depth of color. The number of enriched genes is expressed by the size of the bubbles, and the greater the number, the larger the bubble, as shown in The common targets of HXQR and CHD were subjected to GO enrichment analysis and included three aspects: biological process (BP), cellular component (CC), and molecular function (MF), and at p < 0.05 and number of genes greater than or equal to 10 resulted in 32 pathways. Among them, the enrichment number is arranged in descending order, and the bubble is plotted as shown in p value is taken as the negative logarithm, the color indicates the size, and the gene number is expressed as the bubble size.KEGG pathway enrichment analysis was performed on the common targets of HXQR and CHD, and it was determined that 105 pathways were associated with them. Evaluating with Five key targets with high relevance in the PPI network: IL6, AKT1, VEGFA, TNF, and CCL2 were selected to obtain PDB IDs and target structures, and the five targets were docked to their corresponding active ingredients, respectively, and when the target was combined with the compound structure, the higher the docking score, the more stable the binding of the target with the chemical . The avep = 0.033)  .The ratios were (38.42 ± 20.68)% and (52.00 ± 13.15)%, respectively, while there was no statistical difference between the average CT value of the target plaque and the calcification score of the target plaque , 10.p = 0.028) (The TCM symptom score of angina pectoris of CHD showed a significantly higher decrease of the treatment score than that of the control group (= 0.028) , and the= 0.028) .p-value of the t-test was <0.05 for differential metabolite identification, and a total of 98 differential metabolites were found  and prostate cancer pathway (hsa05215) matched the predicted metabolic pathway, indicating that HXQR may be involved in the treatment of CHD through three signaling pathways, including the cancer pathway (hsa05200), sphingolipid signaling pathway (hsa04071), and prostate cancer pathway (hsa05215). The differential metabolites may be N-oleoyl-D-sphingomyelin (d18:1/22:0) , 1-methyl-6-phenyl-1h-imidazolepyridine-2-amine and 1-phenyl-6-phenyl-1H-imidazopyridine-2-amine (downregulated).p < 0.01), and the level of S1P in serum was decreased (p = 0.08) , Cer (d18:2/23:0), and Cer (t18:0/20:0 (2OH)), SM (d18:1/22:0), and SM (d18:1/24:1 (15Z)), were significantly increased ( = 0.08) . 1-Methyp < 0.01), and the level of S1P in serum was decreased after treatment with HXQR (p = 0.08).The level of Cer (d18:2/23:0), Cer (t18:0/20:0 (2OH), SM (d18:1/22:0), and SM (d18:1/24:1 (15Z)), were significantly increased after treatment with HXQR , puerarin (MOL012297), luteolin (MOL000006), kaempferol (MOL000422), tanshinone iia (MOL007154), and baicalein (MOL002714) were associated with multiple targets and may be the main active components of HXQR decoction in the treatment of CHD. In vitro and in vivo experiments have also confirmed that quercetin has the effects of antihypertensive, regulating blood lipid, lowering blood pressure, anti-atherosclerosis and inhibiting cardiotoxicity , the sphingolipid signaling pathway (hsa04071), and the prostate cancer pathway (hsa05215). However, we found that these three signaling pathways only had cancer pathway (hsa05200) in the top 32 predicted pathways, and prostate cancer pathway (hsa05215) and sphingolipid signaling pathway (hsa04071) in the 36th and 60th of the KEGG prediction results of network pharmacology, respectively, which also indicated that the unvalidated network pharmacology results may be severely biased and must be subject to the large sample size and strict scientific validation before arriving at any scientific conclusions.Metabolomics of clinical trials of HXQR for the treatment of CHD showed that the differential metabolites were identified by VIP value of the first principal component of the OPLS-DA model >0.1 and SM is mainly located on the cell membrane, lipoproteins , and other lipid-rich tissue structures, which is very important for maintaining the micro-control function of the cell membrane structure so that it can regulate the activity of growth factor receptors and extracellular matrix proteins. Its degradation and anabolic intermediates are known as sphingomyelins, which have the effect of regulating cell biological functions . The metThrough network analysis and metabolomic evaluation, there may be three signaling pathways that involve the Huoxue Qingre decoction in the treatment of CHD: pathways in cancer (hsa05200), sphingolipid signaling pathway (hsa04071), and prostate cancer pathway (hsa05215). Network pharmacology provides a method reference for the study of modernization of TCM. However, it is necessary to recognize the existing problems of network pharmacology and adopt a reasonable method to solve these problems in order to draw possible and correct conclusions.