Radix Bupleuri and Rhizoma Cyperi are listed as herbs dispersing stagnated liver Qi in China. They have been used clinically to treat liver diseases for many years and recent pharmacological studies have shown that they inhibit the proliferation of hepatocellular carcinoma (HCC). However, the pharmacological mechanisms, potential targets, and clinical value of the Radix Bupleuri-Rhizoma Cyperi herb pair (CXP) for suppressing HCC growth have not been fully elucidated. We identified 44 CXP targets involved in the treatment of HCC using the GEO dataset and HERB database. An analysis of the Traditional Chinese Medicine System Pharmacology Database (TCMSP) showed that CXP exerts synergistic effects through 4 active ingredients, including quercetin, stigmasterol, isorhamnetin, and kaempferol. GO and KEGG analyses revealed that CXP mainly regulates HCC progression through metabolic pathways, the p53 signaling pathway, and the cell cycle. Additionally, we applied The Cancer Genome Atlas (TCGA)-liver hepatocellular carcinoma (LIHC) database to perform the expression patterns, clinical features, and prognosis of 6 genes  in cell cycle pathways to reveal that CXP suppresses HCC clinical therapeutic value. Moreover, based on molecular docking, we further verified that CXP exerts its anti-HCC activity through the interaction of multiple active components with cell cycle-related genes. We systematically revealed the potential pharmacological mechanisms and targets of CXP in HCC using multilevel data integration and molecular docking strategies.Traditional Chinese medicine (TCM) is a promising and effective treatment for cancer with minimal side effects through a multi-active ingredient multitarget network.  The liver is a key organ for glucose storage, detoxification and processing of exogenous substances, lipid and cholesterol homeostasis, metabolism, endocrine regulation of growth signals, and immune surveillance . HoweverRadix Bupleuri and Rhizoma Cyperi are considered the chief herbs for soothing the liver, and their combination is believed to produce a synergistic effect that increases the efficacy, but this synergistic effect still lacks support from modern pharmacological evidence. Nevertheless, Radix Bupleuri (Chai Hu)-Rhizoma Cyperi (Xiang Fu), a common herb pair, is widely used as an important ingredient in liver-soothing prescriptions in China. For example, Radix Bupleuri (Chai Hu)-Rhizoma Cyperi (Xiang Fu) herb pair (CXP) is an important component of the classic prescription Chaihu Shugan San (CSS), which was first recorded in the ‘Jing Yue Quan Shu’ in the Ming Dynasty [Radix Bupleuri or Rhizoma Cyperi administered as a single herb exhibits significant anticancer activity against HCC cells [in vivo and in vitro, including on HCC [Traditional Chinese medicine (TCM), one of the most popular complementary and alternative medicine models in China, has been used clinically in Asia for thousands of years. Meanwhile, TCM has been gradually accepted by non-Chinese people because of its efficacy, accessibility, and lower toxicity. Several studies have shown that TCM formulas used alone or as an adjunct to conventional chemotherapy have shown good efficacy in the clinical treatment of cancer, including HCC and lung cancer –13. In T Dynasty . Modern  Dynasty , 17. FurCC cells , 19. Addg on HCC , 21. HowDespite many technological advances, experimental elucidation of the interaction of a large number of compounds in CXP with proteins in HCC and the underlying pharmacological mechanisms is still difficult. With the rapid development of systems biology and polypharmacology, the emergence of network pharmacology has provided great opportunities for breakthroughs in TCM research . To dateThe GEO dataset GSE60502 was analyzed to identify DEGs in adjacent nontumor liver tissues and HCC tissues. First, an expression matrix was constructed with 18 pairs of samples, the nonlinear dimensionality reduction algorithm UMAP was used to determine two clusters, and each sample was assigned to the nearest cluster. The results showed a clear difference between the two groups . NotablyA GO enrichment analysis was performed to further investigate the biological functions of the 1110 DEGs. We constructed bubble plots to display the top 20 enriched GO terms. The top 20 enriched GO terms showed that these DEGs were mainly enriched in metabolic processes , includiAdditionally, in the KEGG pathway analysis, the top 20 KEGG pathways related to the 1110 DEGs were identified . KEGG paP values were retained as functional descriptions of the corresponding components, as shown in Next, the PPI network was constructed to reveal the interconnections and roles of these 1110 DEGs. These 1110 DEGs (|log2FC| >1) were input into the STRING database to construct a PPI network. The results were obtained according to an interaction score ≥0.9 and hidden disconnected nodes to improve the reliability of the PPI network . This PPRadix Bupleuri and Rhizoma Cyperi contained 17 and 18 active ingredients, respectively, and both contained the four active compounds quercetin, stigmasterol, isorhamnetin, and kaempferol. The structures of these compounds are shown in Radix Bupleuri were obtained from the HERB database. Similarly, 159 potential targets of 18 active compounds of Rhizoma Cyperi were obtained. Finally, 230 potential targets of 31 CXP active ingredients were identified (According to the two criteria of drug-likeness (DL) ≥0.18 and oral bioavailability (OB) ≥30% , 27, 31 entified .Radix Bupleuri and Rhizoma Cyperi exerted anti-HCC effects through 25 common potential targets. We speculated that these comment targets may explain why CXP exerts a synergistic anti-HCC pharmacological effect. We constructed a network of Herb-Compound-Targets (H-C-T) to further understand the interconnectedness of herbs, active compounds, and potential anti-HCC targets ”, “Cellular senescence (ko04218)”, “Bladder cancer (ko05219)”, “Chemical carcinogenesis (ko05204)”, “IL-17 signaling pathway (ko04657)”, “Pathways in cancer (ko05200)”, “Endocrine resistance (ko01522)”, “Hepatocellular carcinoma (ko05225)”, “Metabolism of xenobiotics by cytochrome P450 (ko00980)”, “Cell cycle (ko04110)”, “Fluid shear stress and atherosclerosis (ko05418)”, “TNF signaling pathway (ko04668)”, “Steroid hormone biosynthesis (ko00140)”, “Kaposi sarcoma-associated herpesvirus infection (ko05167)”, “Retinol metabolism (ko00830)”, “Drug metabolism-cytochrome P450 (ko00982)”, “Bile secretion (ko04976)”, “Platinum drug resistance (ko01524)”, “Chronic myeloid leukemia (ko05220)”, and “HLCV-I infection (ko05166)” . Six of P < 0.01, HR >1)  . In addi, HR >1) .P = 0.001). Subsequently, a time-dependent receiver operating characteristic (ROC) analysis was performed, and the results showed that the risk score performed well in predicting 1-, 3-, and 5-year OS, with areas under the curves (AUCs) of 0.751, 0.678, and 0.644, respectively  were analyzed using the least absolute shrinkage and selection operator (LASSO) Cox regression analysis to construct a cell cycle-related signature for predicting survival. Four cell cycle-related genes  were used to establish a risk score to predict the overall survival (OS) of patients with hepatocellular carcinoma (LIHC) in TCGA training set using LASSO Cox regression analysis  and 8E. ectively .P < 0.05) (P < 0.05) (P < 0.05) (P < 0.05)  . We then < 0.05) ; meanwhi < 0.05) . Moreove < 0.05)  and 9E.P < 0.05). These results speculate that the expression of the four cell cycle-related genes in LIHC was related to different degrees of immune cell infiltration through different pathways, further supporting that these four cell cycle-related genes may be effective factors affecting patient survival and prognosis.We next analyzed the correlation between the abundance of 6 immune cell markers and the expression of 4 cell cycle-related genes using RNA-seq data and the TIMER2.0 database. The correlation analysis showed that the levels of infiltrating B cells, CD8+ T cells, CD4+ T cells, macrophages, and myeloid dendritic cells were positively correlated with the expression level of 4 the cell cycle-related genes  in LIHC  bind these cell cycle-related targets  well with low binding energy . This efThe pathological mechanism of HCC is very complex and involves multiple targets and signaling pathways during its progression. We used the public GEO dataset to analyze the gene expression profiles of human liver cancer tissues relative to adjacent tissues as a method to understand the genes and mechanisms that regulate HCC progression. A total of 1110 DEGs were identified, including 491 upregulated genes and 619 downregulated genes, indicating that the progression of HCC is regulated by a complex and large gene network. Most of these DEGs are involved in regulating cellular metabolism or cellular processes. Published studies have shown that DEGs, including MEP1A , AKR1B10Radix Bupleuri and Rhizoma Cyperi, herbs dispersing stagnated liver Qi, have been suggested to possess hepatoprotective activity in modern pharmacological studies [Radix Bupleuri water extract reduced the viability of HepG2 cells and that Radix Bupleuri enhanced the pharmacological effects of 5-fluorouracil-induced HepG2 cell death. Furthermore, as shown in the study by Mannarreddy et al. [Rhizoma Cyperi displays significant anticancer activity against multiple cancer cell lines, including HepG2, without inhibiting noncancer cells. However, the pharmacological mechanisms involved in the suppressing HCC remain largely unknown. In addition, Radix Bupleuri and Rhizoma Cyperi are often used as compatible drug pairs in in classic TCM prescriptions, such as CSS [Radix Bupleuri-Rhizoma Cyperi drug pair in treating HCC are necessary.TCM, as a multicomponent and multitarget empirical therapy, has been used clinically in Asia for thousands of years. Because of this unique feature, TCM treatments are considered promising therapeutic strategies for complex diseases, including liver cancer . Radix B studies , 37, 38. studies  showed ty et al. , the meth as CSS . TherefoHowever, due to the limitation of the complex molecular network characteristics of TCM in treating diseases, modern pharmacological research on TCM has not achieved great progress, which undoubtedly widens the research gap between TCM and Western medicine. In recent years, the introduction of network pharmacology has provided a good research strategy for investigating the modern pharmacology of TCM. Therefore, we attempted to utilize this strategy to explore how CXP exerts its pharmacological anti-HCC effects.Radix Bupleuri and Rhizoma Cyperi, respectively. These key active ingredients were mainly sterols  and flavonoids . Notably, both herbs contain the following four active ingredients: quercetin, stigmasterol, isorhamnetin, and kaempferol. We hypothesized that these 4 active ingredients may be the important basis for the synergistic anti-HCC effect of the two herbs on CXP. Pan et al. [According to the TCMSP database, 17 and 18 key active ingredients were identified in n et al.  showed tn et al.  showed tn et al. . Accordin et al. , kaempfen et al. , and then et al. . OverallForty-four potential therapeutic targets of CXP against HCC were identified from the GEO dataset and the TCM target prediction database HERB. Subsequently, according to the H-C-T network diagram, CXP may exert its anti-HCC effect through the actions of multiple active components on multiple targets. Among them, the two herbs in CXP exert synergistic anti-HCC effects on multiple targets mainly through their shared active ingredients: quercetin, stigmasterol, isorhamnetin, and kaempferol. TCM prescriptions are a rational combination of a variety of herbal medicines based on TCM theoretical compatibility, but they still lack the support of modern pharmacology, which limits TCMs progress . Here, wWe performed enrichment analyses of 44 potential therapeutic targets to better elucidate the molecular mechanism of CXP against HCC. The results showed that CXP mainly regulates HCC progression through metabolic and cellular processes, including the p53 signaling pathway, cellular senescence, the cell cycle, and retinol metabolism. These enrichment results largely overlapped with our previous KEGG enrichment results for HCC progression, such as the involvement of the p53 signaling pathway and cell cycle. Moreover, the foregoing discussion has clarified that the progression of HCC is closely related to the p53 pathway and the cell cycle. Additionally, several cell cycle-related genes have recently been identified as potential therapeutic targets for the treatment of HCC, including CDK1 , CDK4 4, CCNB1 , and CHEAs the roles of cell cycle-related signatures in HCC are largely underexplored, a broader understanding of their expression profiles in cancer and prognostic analyses are important. In this study, we analyzed the expression patterns, prognostic value, and clinical characteristics of cell cycle-related genes in LIHC to determine the value of CXP clinical application in the treatment of HCC. We identified five cell cycle-related genes, CDK1, CDK4, CHEK1, CCNB1, and CDKN2A, which were significantly associated with the OS of patients with LIHC and were considered risk factors. Subsequently, the results of LASSO Cox regression model and analysis of the Kaplan-Meier survival curve revealed that patients with LIHC presenting low expression of cell cycle-related genes  had a better prognosis than those with high expression. Moreover, the clinical characteristics of these genes were analyzed in the LIHC dataset, and the results revealed that their expression levels were closely related to the pathological stage of LIHC and the progression of vascular invasion. These data strongly suggest that CDK4, CHEK1, CCNB1, and CDKN2A are potential prognostic biomarkers for HCC and key therapeutic targets for CXP to suppress HCC.Given the achievements of immunotherapy in the field of cancer treatment , revealiIn conclusion, we elucidate the underlying pharmacological mechanisms and targets of CXP action in HCC therapy by integrating multiple databases. CXP inhibits tumors through multiple metabolic pathways and cellular processes. An analysis of TCGA data showed that CXP may improve the prognosis and clinical outcomes of patients with HCC by regulating cell cycle-related genes, revealing its clinical application value. Furthermore, CDK4, CDKN2A, CCNB1, and CHEK1 genes were identified as key therapeutic targets for CXP treatment of HCC. In recent years, TCM has become a promising cancer treatment strategy because of its multiple active components and multiple targets. However, due to the complexity of TCM pharmacological mechanisms, TCM research has not been rapidly established, and the proposal of network pharmacology strategies is gradually changing this dilemma. To our knowledge, this study represents the first systematic pharmacological analysis of CXP-mediated HCC therapy. Therefore, although this study still has limitations, it provides innovative research methods and breakthroughs for TCM research.https://old.tcmsp-e.com/tcmsp.php), a database that provides the relationship between chemical components, targets, and diseases. Moreover, the database contains the pharmacokinetic properties of various chemical components, including their OB and DL. First, Chinese characters such as “Chai Hu” or “Xiang Fu” were entered into the database to determine their respective components and corresponding pharmacokinetic data. Then, the absorption, distribution, metabolism, and excretion (ADME) evaluation system was used to select potential active ingredients. Here, we selected two pharmacokinetic parameters as screening criteria to determine the active ingredients in Radix Bupleuri or Rhizoma Cyperi, OB ≥30% and DL ≥0.18. Active ingredients that met the criteria were considered candidates for subsequent analysis.We collected the chemical components of CXP from the TCMSP (http://herb.ac.cn/) database for active components of CXP for potential therapeutic target mining to identify targets that bind to CXP active compounds. HERB is a high-throughput experiment- and reference-guided database for TCM. We downloaded GSE60502 from the GEO (http://www.ncbi.nlm.nih.gov/geo/) database to screen for significant DEGs in HCC and identify HCC-related pathological genes. Wang et al. [https://www.r-project.org). The uniform manifold approximation and projection (UMAP) was analyzed with the ‘umap’ package and visualized with the ‘ggplot2’ package. The DEGs in HCC were screened and obtained using the ‘limma’ package of R language Bioconductor (http://www.bioconductor.org/packages/release/bioc/html/affy.html) with a false discovery rate (FDR) <0.05 and |log2FC| >1. Subsequently, these DEGs were processed using the ‘ggplot2’ package in R language and visualized by constructing a volcano plot. The ‘ComplexHeatmap’ package in R language was used to analyze the top 50 upregulated or downregulated DEGs in HCC. Finally, these genes were compared to obtain the potential overlapping CXP targets and HCC DEGs. These overlapping targets are those that CXP may modulate to treat HCC.We searched the HERB . Fragments per kilobase per million (FPKM) values for TCGA cohort were converted to transcripts per million (TPM) values before further analysis. Subsequently, differences in gene expression between adjacent nontumorous tissues (n = 50) and tumor tissues (n = 374) were statistically analyzed and visualized using the “ggplot” R package.We downloaded RNA-seq and clinical data from 374 patients with HCC in TCGA (Homo sapiens’) was submitted to the OmicShare tool for KEGG pathway and GO enrichment analyses. OmicShare is a free online platform for data analysis (https://www.omicshare.com/tools) that can be used for GO analyses, KEGG analyses, Venn diagrams, heatmaps, network construction, volcano plot analyses, etc.A list of DEGs in HCC or CXP anti-HCC-related targets (species limited to ‘Homo sapiens’ and hidden disconnected nodes (interaction score ≥0.90), to construct a PPI network. Images were exported, and a topological analysis of nodes was performed. A list of DEGs in HCC (|log2FC| >2) or CXP anti-HCC-related targets was submitted to the Metascape webtool (http://metascape.org), with the species limited to ‘Homo sapiens’, to construct the PPI network. For each specific gene list, the Metascape platform performed a PPI enrichment analysis using the following databases: STRING, BioGrid, OmniPath, and InWeb_IM. The MCODE algorithm was used to identify densely connected network components if the network contained 3 to 500 proteins. This novel graph-theoretical clustering algorithm, MCODE, may represent densely connected regions in large PPI networks of molecular complexes [A list of DEGs in HCC (|log2FC| >1) was input into the STRING database, with the species limited to ‘omplexes . We downPrognostic cell cycle-related genes were identified by performing a univariate Cox analysis of OS of patients included in TCGA-LIHC set using the “survival” package in the R language. The R packages “glmnet” and “survival” were used to perform the LASSO Cox regression analysis with random seeds to build the risk score model that best predicts survival in the training cohort. A risk score formula was then established based on the normalized expression level of each gene and its corresponding regression coefficient: risk score = (0.1159 × CDK4 expression level) + (0.0234 × CHEK1 expression level) + (0.1968 × CCNB1 expression level) + (0.0322 × CDKN2A expression level). The patients were then divided into the low-risk and high-risk groups according to the median risk score.http://timer.comp-genomics.org/), including the TIMER, EPIC, XCELL, QUANTISEQ, and MCPCOUNTER algorithms, we analyzed the relationship between the expression of cell cycle-related genes  and immune cell infiltration levels in patients with LIHC. Pearson’s correlation analysis was performed to elucidate the correlations between the expression of cell cycle-related genes expression and immune cell infiltration.We integrated several computational tools to estimate immune cell infiltration in TCGA RNA-seq cohorts. Based on the centralized algorithms in the online database TIMER2.0 (https://www.rcsb.org/). Then, the PDB file was opened with PyMOL 1.8 to remove water molecules and heteroatoms from the protein crystal structure, add hydrogen atoms, and calculate the charge. Meanwhile, the 3D chemical structures of the active components of CXP were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) with the format for SDF and converted to a.pdb format by PyMOL 1.8. Next, the proteins and active ingredients were converted into.pdb format files by AutoDockTools (version 1.5.6). Second, the grid box function of AutoDockTools was used to define specific docking pockets in selected proteins that potentially bind to the active ingredient. Finally, the molecular docking analysis was performed using the command prompt, and the docking results were visualized using PyMOL. The binding energy was calculated to assess the theoretical binding affinity of the active ingredient for the selected proteins.AutoDock Vina and PyMOL 1.8 software were utilized to perform molecular docking studies of CXP active components and selected proteins. X-ray crystal structures of selected proteins were obtained from the Protein Data Bank  and with the OmicShare tool. Comparisons between the two groups were performed using the unpaired Student’s Publicly available datasets were analyzed in this study. This data can be found here: TCMSP, HERB, TCGA, GEO, TIMER2.0, etc.Supplementary Tables 1 and 5Supplementary Tables 2-4 and 6-8


Patients with Hantavirus-caused epidemic hemorrhagic fever (EHF) are at risk of contracting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is currently no validated EHF/SARS-CoV-2 strategy. Several studies have recently shown Puerarin, a natural product, has potent antiviral properties. The goal of present study was to determine the mechanism of puerarin in patients with EHF/COVID-19. We use network pharmacology and bioinformatics to investigate the possible pharmacological targets, bioactivities, and molecular mechanisms of puerarin in the treatment of patients with EHF/SARS-CoV-2. The study investigated the pathogenesis of COVID-19 and EHF and the signaling pathway impacted by puerarin. 68 common genes linked to puerarin and EHF/SARS-CoV-2 were discovered during the investigation. By using protein-protein interaction (PPI) network, we identified RELA, JUN, NF-B1, NF-B2, and FOS as potential therapeutic targets. The bioactivity and signaling pathways of puerarin have also been demonstrated in the treatment of EHF and COVID-19. According to present study, puerarin could reduce excessive immune responses and inflammation through the NF-B, TNF, and HIF-1 signaling pathways. This study explored the potential therapeutic targets and mechanisms of Puerarin in the treatment of EHF/COVID-19. COVID-19 is a novel zoonotic disease that causes severe flu-like symptoms and respiratory distress . The novThe World Health Organization considers epidemic hemorrhagic fever, Ebola virus Disease, influenza, SARS-CoV-2, SARS, and other diseases with epidemic or epidemic potential impacting human society substantially –17. The Puerarin is the main biologically active ingredient extracted from Pueraria Lobata, widely used in the therapy of various diseases such as diabetes, Parkinson’s disease, Alzheimer’s disease, and cancer . Studieshttp://www.lilab-ecust.cn/pharmmapper/), Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform , comparative toxicogenomics database , batman (http://bionet.ncpsb.org.cn/batman-tcm/index.php), and SwissTargetPrediction (http://www.swisstargetprediction.ch/) database to search for potential therapeutic targets of puerarin. Only the top 100 genes in each database were filtered for further analysis to increase the study’s accuracy. As the targets in the CTD database had no index for ranking, they were all included in present study.We used PharmMapper (https://www.ncbi.nlm.nih.gov/geo/) to collect EHF/COVID-19-related genes. DEGs were identified by using the “limma” package for R with a false discovery rate (FDR) <0.05 and log fold change (FC) absolute value > 1 as filter requirements. In addition, CTD (http://ctdbase.org/), Kyoto Encyclopedia of Genes and Genomes , PubChem (https://pubchem.ncbi.nlm.nih.gov/), GeneCards (https://www.genecards.org/), OMIM (https://omim.org/), and DisGeNET (https://www.disgenet.org/) databases were also used to find genes associated with EHF and COVID-19.The GSE157103, GSE152075, GSE86282, and GSE133751 datasets were downloaded from the gene expression omnibus website (http://genemania.org/) to develop the PPI network. To determine the key genes of puerarin in the therapy of EHF/COVID-19, we used the Cytoscape software to import the gene interaction network obtained from the GeneMANIA database and the cytohubba plug-in to compute and visualize the degree value of each gene.The “Venn” package for R was employed to identify common genes for puerarin, COVID-19, and EHF. The GeneMANIA database, a widely used bioinformatics database, could predict gene functions and build protein interaction networks in nine species, including humans. The obtained common genes were uploaded to the GeneMANIA database  and KEGG pathway through R language and visualized the results . Next, by using the ClueGo plug-in of the Cytoscape software, we conducted the correlation analysis on the findings of the enrichment analysis . Finally, the “circlize” package for R was used to display the highly correlated pathways and corresponding common genes.The ability to distinguish the molecular origin of aberrant transcriptome regulation relied heavily on signaling pathway activity based on transcriptome data. By combining existing experimental data, the SPPED database could systematically obtain the consensus features of genes. It could predict differential expression patterns and upstream gene pathway activity more accurately than the usual collection of consensus features from a single data set. The SPEED2 database, which was utilized in this work, was based on SPEED and has been enhanced in several ways: (1) the number of anticipated pathways had been expanded to 16, (2) the gene dataset used to generate the database had been extended to 640, (3) the user was able to obtain upstream and downstream of the input gene list, and (4) statistical scores were provided to improve the accuracy of the prediction.To obtain the relevant targets of puerarin, we selected 100, 125, 100, 54, and 96 targets from batman, CTD, PharmMapper, TCMSP, and SwissTargetPrediction database, respectively  network , as shown in To further probe the probable molecular mechanism of puerarin in the therapy of EHF/COVID-19, the SPEED2 database was used to acquire the upstream pathway activities of common genes. The 16 upstream pathways were sorted according to adjusted p-values in COVID-19, a rapidly expanding disease, is still spreading over the world. However, specific treatments for COVID-19 are still in development. COVID-19 has caused economic, social, health, and personal lifestyle difficulties across the globe. According to the most recent figures issued by Johns Hopkins University in the United States on January 7, 2022, the number of confirmed cases of COVID-19 worldwide has surpassed 300 million. While it takes only five months for the total number of infected people to reach to 300 million from 200 millions. As a global emerging zoonotic pathogen, Hantavirus causes EHF in Asia and Europe. Each year, more than 20,000 hantavirus cases are reported worldwide, most of which come from Asia . The numin vivo and vitro  can be found in the article/CZ developed the concept of the project. The data was collected and evaluated by WL, XL, and HW with the help of KN, QM, and JH. All authors reviewed and discussed the results and contributed to the paper preparation. CZ, WL, XL, and HW wrote the manuscript. All authors have read and approved the manuscript.The study was supported by Shenzhen Key Laboratory Foundation (ZDSYS20200811143757022).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Atrial fibrillation (AF) is a common atrial arrhythmia for which there is no specific therapeutic drug. Quercetin (Que) has been used to treat cardiovascular diseases such as arrhythmias. In this study, we explored the mechanism of action of Que in AF using network pharmacology and molecular docking. The chemical structure of Que was obtained from Pubchem. TCMSP, Swiss Target Prediction, Drugbank, STITCH, Pharmmapper, CTD, GeneCards, DISGENET and TTD were used to obtain drug component targets and AF-related genes, and extract AF and normal tissue by GEO database differentially expressed genes by GEO database. The top targets were IL6, VEGFA, JUN, MMP9 and EGFR, and Que for AF treatment might involve the role of AGE-RAGE signaling pathway in diabetic complications, MAPK signaling pathway and IL-17 signaling pathway. Molecular docking showed that Que binds strongly to key targets and is differentially expressed in AF. In vivo results showed that Que significantly reduced the duration of AF fibrillation and improved atrial remodeling, reduced p-MAPK protein expression, and inhibited the progression of AF. Combining network pharmacology and molecular docking approaches with in vivo studies advance our understanding of the intensive mechanisms of Quercetin, and provide the targeted basis for clinical Atrial fibrillation treatment. The prevalence of AF increases with age, doubling every 10 years after age 50 years and reaching 10% in patients ≥ 80 years. Furthermore, the number of patients with AF is expected to increase significantly in the coming decades3. Oral anticoagulant drugs are currently one of the main treatment modalities for AF, such as rivaroxaban and dabigatran5. In addition, relevant studies have reported that AF patients will increase significantly in the next few decades. Therefore, exploring new drugs to reduce the danger of bleeding in patients with AF, improve the cardiac function of patients and the complications of AF requires further research.Atrial fibrillation (AF) is by far the most common type of atrial arrhythmia and is a risk factor for increased cardiovascular mortality7. Que has been reported to show significant therapeutic effects in animal models of heart failure, arrhythmias and hypertension in various cardiovascular diseases11. A randomized clinical trial showed that with the increase of Que dosage, the systolic and diastolic blood pressure in hypertensive patients were significantly reduced12. Recent studies have found that in a rat model of arrhythmia after myocardial ischemia and reperfusion injury, the administration of Que significantly reduced the time to arrhythmia as well as lactate dehydrogenase (LDH) levels in myocardial injury13. Based on these comprehensive data, we speculate that Que is one of the new therapeutic approaches for the prevention of cardiovascular diseases, but there is still lack of the related research in vitro and in vivo as well as clinical studies of Que for AF treatment, so we think it is necessary to do the systematic and in-depth studies on the molecular mechanisms of Que for AF treatment.Quercetin (Que) is widely found in vegetables and fruits, and Que is also found in several herbal medicines. The therapeutic potential of Que has been widely studied because of its ability to inhibit low-density lipoprotein (LDL) oxidation, reduce adhesion molecules and other inflammatory markers, prevent neuronal oxidation and inflammatory damage, and platelet anti-aggregation14. Therefore, by using a network pharmacological analysis method, we investigated the targets of action in Que for the treatment of AF and analyzed their relevant target biological pathways, with further experiment validation in rat AF model in vivo, which laid a good foundation for further in-depth study of the mechanism of action of Que for the treatment of AF. The detail flow chart of this study is shown in Fig. Network pharmacology is to further analyze the relationship between drugs, diseases and targets by collecting various database data, data analysis and simulationThe chemical structure of quercetin is shown in Fig. The Que and AF intersection targets were imported into String database, species limited to human, and the protein interactions network was obtained, which was further imported into Cytoscape for topological analysis, and the results suggested that there were 65 nodes and 575 edges in the network, and the key core targets were obtained using cytoHubba plugin Fig. e, rankinTo further clarify the mechanism of action of quercetin in the treatment of AF, we classified 65 core targets into proteins using the PANTHER database Fig. a, found The pathways of action were clarified by KEGG enrichment analysis of 65 intersecting genes, with 157 enrichment terms, of which the top 30 most significant pathways Fig. a, the coThe top 10 relevant pathways of Que for AF were screened Table  and impoThe five core targets with high Degree values were selected for molecular docking to verify the binding activity of Que active ingredients to potential anti-AF targets. The average docking affinity of the results was − 5.042 kcal/mol, as detailed in Table Using the difference data of control group and AF group in GEO database, the differential expression of the first five core targets in Que treatment of AF diseases Fig. . VEGFA wIn contrast with the visible of p-wave in CON group, in typical AF and Que + AF groups, f-wave was appeared of and of p-wave was disappeared by electrocardiogram measurement Fig. a. ComparNo significant changes in cardiac function were happened in the CON, AF and Que + AF groups according to the cardiac ultrasound results Fig. .Figure 9HE staining results showed that the atrial tissues in the CON group were densely and neatly arranged, disorganized arrangement, hypertrophy and widened cell gaps of the atrial myocytes were showed in the AF group. Compared with the AF group, the morphology and the degree of structural damage of atrial myocytes were improved significantly with the increasing concentrations of Que Fig. .Figure 1The masson staining results showed that in the atrial myocardial tissue of the CON group, no significant proliferation of blue collagen fibers was happened, and the numbers of collagen fibers in atrial tissues of AF was increased. Compared with the AF group, the numbers of collagen fibers in atrial tissues were decreased significantly with the increasing concentration of Que (P < 0.05) Fig. a.Figure Compared with the CON group, p-MAPK protein expression was increased in the AF group (P < 0.01). Compared with the AF group, p-MAPK protein expression was significantly decreased with the increasing concentration Que (P < 0.01). Compared with 75 mg/kg Que, 150 mg/kg Que in the AF group, p-MAPK protein expression was reduced (P < 0.05). The expression of MAPK protein in 75 mg/kg Que and 150 mg/kg Que groups were no significant difference (P > 0.05) Fig. b.15. It has been found that Que played the anti-ischemic and anti-arrhythmic effects in clinical studies11, it leads to great interest and concern of scientists, but the specific mechanisms of Que in the treatment of AF remains unclear. In this study, we investigated the mechanism of Que in the treatment of AF based on network pharmacology analysis. In this study, 449 Que potential targets and 2201 AF targets were obtained from each database respectively, 3096 AF differential genes were obtained from the GEO database. The intersection of the 3 was taken to obtain 65 core genes for Que treatment of AF. The above core genes were screened for the top 5 hub target genes of IL-6, VEGFA, JUN, MMP9 and EGFR by Degree value. GO analysis confirmed that Que had the potential to play an important role in the pathogenesis of AF through multiple biological pathways, and KEGG analysis obtained key pathways of Que for AF, mainly involving lipid and atherosclerosis pathways, the AGE-RAGE signaling pathwayin diabetic complications, MAPK signaling pathway and IL-17 signaling pathway, etc. Through further screening, we found that MAPK pathway plays a key role in the treatment of AF.One study found that the increased risk of cardiovascular disease in some populations was associated with low dietary intake of flavonoids, including Que16. Wu reported inhibiting IL-6 inflammatory factor release reduced early atrial fibrosis and its duration in SD rats with postoperative AF17. Studies based on clinical follow-up investigations have found that measurement of IL-6 concentrations could provide an independent prognostic signal as well as a predictor of recurrence for mortality in AF patients19. Therefore, IL-6 has the potential to be a therapeutic target for AF. The next key gene VEGFA is a key factor in the growth process of blood vessels and endothelial cells. Its main function is to induce endothelial cell proliferation and inhibit apoptosis20. It is well known that AF can lead to endothelial dysfunction21, and Wang reported that the plasma levels of VEGFA were significantly elevated in AF patients22, suggesting that endothelial dysfunction may be one of the etiologies of AF, VEGFA may be a potential therapeutic target for AF.IL-6 plays a key role in immune, tissue regeneration and metabolism. It was shown that IL-6 in the left atrium was found to promote early atrial fibrosis through the pSTAT3/STAT3 signaling pathway by establishing postoperative AF mice, and similarly higher IL-6 concentrations were found in the pericardial drainage fluid of patients with postoperative AF in a prospective clinical study23, activation of c-jun impaired cell–cell communication between atrial myocytes and ultimately promotes the development of atrial arrhythmias25. Therefore, JUN has the potential to be a therapeutic target for AF. The next gene MMP9 is a matrix metalloproteinase that plays an important role in local protein hydrolysis, cell migration, apoptosis and signaling in the extracellular matrix. During AF development, overproduction of reactive oxygen species activated MAPK signaling pathway further increasing MMP9 expression26. Moe GW reported inhibiting MMP9 expression in canine AF model improved cardiomyocyte hypertrophy and reduced myocardial fibrosis, as well as reduced AF duration27. Recent clinical studies have found that the plasma MMP9 level in AF patients were higher than that in controls, which suggested MMP9 could be as a target for the treatment of AF28. The last of the first five core genes is EGFR, whose main function is to regulate cell growth, proliferation and apoptosis, and is a key regulator of cardiac organogenesis and adult heart29. In AF fibrotic atrial tissues, EGFR expression was increased, probably due to the cleavage by pro-fibrotic metalloproteinases during atrial remodeling30. The plasma level of EGFR in clinical AF patients was significantly higher than that in the control group31, these results suggest that EGFR plays an important role in AF. Overall, the top five core genes were found to be supported by previous studies based on network pharmacology studies.The next gene is Transcription factor JUN, It has been shown that the transcription factor complex AP-1 plays a major role in cardiac hypertrophy, and knockout of c-jun pure mice develops in a process leading to cardiac malformations34. It is suggested that lipids and atherosclerosis may be involved in the pathogenesis of AF. The AGE-RAGE signaling pathway is an important link in the development of diabetic disease, and this signaling pathway directly mediates diabetic vascular calcification and vasoconstriction-diastolic dysfunction, and a meta-analysis showed that type 2 diabetes increased the risk of AF by 40%35. Recent studies had found that the AGE-RAGE signaling pathway was associated with the development of AF37, and that when AGE bind to RAGE, it produced inflammatory cytokines and cell adhesion molecules that further induced structural and electrical remodeling of the atria. Raposeiras-Roubín found significantly higher AGE and RAGE plasma levels in AF patients than in normal subjects, and several prospective studies have also shown that RAGE predicts future cardiovascular events and death in diabetic patients38. This suggesting that this pathway may mediate the progression of AF.The results of KEGG enrichment analysis suggested that the lipid and atherosclerosis pathway, AGE-RAGE signaling pathway in diabetic complications, MAPK signaling pathway and IL-17 signaling pathway played the major roles in the treatment of AF with Que. One of the main causative factors of atherosclerosis and cardiovascular disease is dyslipidemia, which is considered one of the major risk factors for AF, and several clinical studies had demonstrated that LDL and total cholesterol were negatively correlated with the occurrence of AF39. It has been demonstrated that activation of phosphorylated MAPK significantly increases susceptibility to AF40, in detail MAPK pathway 45, PharmMapper (http://www.lilab-ecust.cn/pharmmapper/)46, SwissTargetPrediction (http://www.swisstargetprediction.ch/)47, Drugbank (https://go.drugbank.com/)48, Binding database (http://www.bindingdb.org/bind/index.jsp)49, STITCH (http://stitch.embl.de/cgi/input.pl?UserId=MxcokIPgQVMy&sessionId=O32YjbzG5Gio) and other databases to obtain Que targets, default HumanProtein Targets Only (Retrieved on:2021.7.10).Acquisition of Que targets. Using the TCMSP (http://ctdbase.org)50, GeneCards (https://www.genecards.org/)51, DISGENET(https://www.disgenet.org/)52, Therapeutic Target Database (http://db.idrblab.net/ttd/)53, were used to “Atrial fibrillation” was used as a keyword to search for the relevant genes. The collected targets were combined and eliminated by the median algorithm to obtain disease targets. GSE31821 data were imported into the online analysis tool GEO2R for processing to obtain differentially expressed genes. The gene screening criteria were P < 0.05 and |logFC| ≥ 0.5 to obtain differentially expressed genes between normal tissues and AF, and further volcano maps of differentially expressed genes were drawn54.Acquisition of AF related targets. The CTD (http://www.ehbio.com/test/venn/#/), intersections of drug targets, disease targets and differential genes can be obtained.Cross-tabulation of drugs, diseases and GEO. By using the online mapping tool (55 (https://string-db.org/cgi/input.pl) to construct the protein–protein interaction network (PPI), and the species selectors, exported in tsv format. The tsv was imported into Cytoscape 3.7.2 software (http://www.cytoscape.org/) for adjustment as well as to obtain key targets using the cytoHubba plugin. Simultaneous protein functional classification of the intersecting targets using the Panther database.Construction of protein–protein interaction network (PPI). The intersecting genes were imported into the String database6, as well as the gene ontology (GO) of the above intersecting genes, GO analysis can further explain the role of the intersecting genes in molecular function (MF), biological process (BP) and cellular component (CC) of AF resistance. The Kyoto encyclopedia of genes and genomes (KEGG)56 enrichment analysis was used to investigate the major anti-AF signaling pathways involved in the crossover genes. The core target network of “Que-major pathway-AF” was also constructed.GO analysis and KEGG pathway and Que-primary pathway-AF network construction. To further understand the function and role in signaling pathways of the intersecting genes, the gene abbreviations (Symbol) were transformed into gene ID (EntrezID), and the results were analyzed visually by the R language package58. Changes in core targets were analyzed using differential expression between the normal and AF groups from the GEO database. GraphPad Prism software was used for graphical visualization. Values are expressed as mean ± SD.Molecular Docking and differential analysis of core targets. The known key targets were molecularly docked to Que to obtain docking affinities reflecting their stability. The PDB database was used to download the core protein molecular structures and PUBCHEM to download the structures of Que. The protein and Que structures were imported into Auto Dock Tools software for processing and molecular docking, and intermolecular docking affinities were calculated and PyMol 2.4.0 was used for visualization2 , BSA , p44/42 MAPK , Phospho-MAPK  , GAPDH .Quercetin , sodium pentobarbital , isoflurane , acetylcholine (ACh) and CaCl13 were divided into low-dose group  and high-dose group , which the rats were intragastric administrated with different concentrations of Que, once/day for 21 days, and the rats in CON and AF were intragastric administrated equal dose of salineion once/day for 21 days. At the end of 21 days, the rats in Que + AF and AF groups were injected with ACh-CaCl2 mixture (66 ug/ml ACh + 10 mg/ml CaCl2) in tail vein at a dose of 1 ml/kg once a day for 1 week, and equal amount of saline was injected in tail vein in CON group, and atrial tissue was taken after 7 days for next experiment.Twenty four male (Sprague-Dawley) SD grade rats (180–220 g) were obtained from Bengbu Medical College (SCXK-2022-0001). The animal protocol conformed to the guide for the care and use of laboratory animals published by the US National Academy of Sciences and the US National Institutes of Health  and the principles of laboratory animal care established by the National Institute for Medical Research. The study was conducted in accordance with the ARRIVE guidelines. The protocol was approved by the institutional animal care and use Committee of Bengbu Medical College. The rats were randomly divided into 3 groups, control group , AF group , and quercetin + AF group , where the rats in quercetin group44, and the time of induction and duration of AF were recorded.Before the experiment, rats were anesthetized using 2% pentobarbital sodium intraperitoneally, limb leads were performed using an electronic ECG machine, ECG was recorded in each group, and non-sinus rhythm was recorded and excluded. When the models of Que + AF and AF had been established, the appearance of f-wave and disappearance of p-wave were the markers for the occurrence of AF, and the disappearance of f-wave and the appearance of p-wave were the markers for the termination of AFAfter ECG recording was completed, echocardiographic testing was performed. Isoflurane anesthesia maintained with small animal ultrasound color Doppler ultrasonography , the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD) were measured in each group. The mean of 3 cardiac cycles were used for statistical analysis.The atrial tissues of each group were partially retained for protein extraction, and the rest were fixed in 4% paraformaldehyde for 24 h. After dehydration and embedding, paraffin sections were made, and the thickness of each section was 4 μm. HE staining and MASSON staining were performed in strict accordance with the instructions of the kit, and the morphology of atrial tissues and collagen deposition in the interstitial space of cardiomyocytes were observed under the light microscope.The proteins were extracted from the atrial tissue homogenates of each group, electrophoresed and then transferred to the PVDF membrane (cut the length of PVDF membrane according to the molecular weight of target protein), blocking at room temperature for 2 h in 5% non-fat milk powder, where Phospho-MAPK was blocked with 5% BSA for 2 h. GAPDH (1:3000), p44/42 MAPK (1:1000) and Phospho-MAPK (1:1000) antibodies were added sequentially and incubated on a shaker at 4 ℃ for 12 h. On the next day, the PVDF membranes were washed by TBS-T, and then given a secondary antibody incubation at room temperature for 2 h, the image was acquired by chemiluminescence system (VIVILBER machine), and the grayscale values of the strips were analyzed using Image J.Data were statistically processed using GraphPad Prism 9.0, and all data were expressed as mean ± standard deviation. One-way ANOVA was used between groups, and P < 0.05 was statistically significant.Supplementary Information.


Methanol, a relatively cheap and renewable single-carbon feedstock, has gained considerable attention as a substrate for the bio-production of commodity chemicals. Conventionally produced from syngas, along with emerging possibilities of generation from methane and CO2, this C1 substrate can serve as a pool for sequestering greenhouse gases while supporting a sustainable bio-economy. Methylotrophic organisms, with the inherent ability to use methanol as the sole carbon and energy source, are competent candidates as platform organisms. Accordingly, methanol bioconversion pathways have been an attractive target for biotechnological and bioengineering interventions in developing microbial cell factories. This review summarizes the recent advances in methanol-based production of various bulk and value-added chemicals exploiting the native and synthetic methylotrophic organisms. Finally, the current challenges and prospects of streamlining these methylotrophic platforms are discussed. Renewability and sustainability of carbon feedstock are one of the main aims and concerns in metabolic engineering toward producing value-added chemicals. Given the potential to be produced from methane and CO2 (two significant greenhouse gases) , methanoMethylorubrum extorquens, Bacillus methanolicus, and Pichia pastoris, where several chemicals have been targeted for production from methanol. Although various chemicals are produced, inefficiencies in carbon conversion, lack of product diversity, limitations in knowledge of regulatory networks, and updated genetic manipulation tools have been limiting factors for the practical applications of these organisms as an industrial production platform  or alcohol oxidase (Aox) catalyzes the oxidation of methanol to formaldehyde in prokaryotic and eukaryotic methylotrophs (yeast), respectively . Mdh utivia the oxidation of formaldehyde to formate and then CO2. Unlike the thiol-dependent formaldehyde dehydrogenases (Flds) of Escherichia coli  by hexulose-6-phosphate synthase (Hps) and then isomerized to form fructose 6-phosphate (F6P) by 6-phospho-3-hexuloisomerase (Phi)  to formaldehyde forming glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone (DHA) , xylulose monophosphate (XuMP), and serine pathway . In orgase (Phi) . In the ne (DHA) . Both pane (DHA) .M. extorquens, formaldehyde assimilation occurs via the serine pathway (via PPP pathway) regeneration of glyoxylate occurs through ethylmalonyl-CoA (EMC) pathway circulating the Serine cycle  and their route of carbon assimilation, various chemicals, intermediates, and value-added chemicals produced using methanol as a carbon source, will be discussed.The RuMP pathway is considered an efficient route for formaldehyde assimilation in terms of energy consumption and biomass yield since one molecule of ATP and three molecules of NAD(P)H are produced per molecule of pyruvate generation . MethyloB. methanolicus have been reported to produce up to 0.4 g/L L-lysine, 12 g/L L-alanine, 11 g/L L-aspartate, and 59 g/L L-glutamate , the chemical derivatives of lysine and glutamate, respectively. High cell density fed-batch fermentation using heterologous expression of E. coli L-lysine decarboxylase (cadA), which decarboxylates lysine to cadaverine, generated 6.5 g/L of cadaverine , dihydrodipicolinate synthase (DapA), and dihydrodipicolinate reductase (DapB), were characterized in M. methylotrophus and found to be allosterically inhibited by threonine and L-lysine  during the1970s, with production capacity reaching up to 50,000 tons per annum  to produce L-serine. This very step was utilized for the production of L-serine with glycine as a co-substrate. Using resting cells to prevent glycine toxicity and the freeze-thawing method for facilitating substrate permeability up to 54.5 g/L L-serine was produced  caused 11.7 mg/L of violacein production from M. extorquens AM1. Subsequently, through directed evolution of the host (for overcoming unidentified bottlenecks) and substrate co-utilization strategy , a final violacein production titer of 118 mg/L was achieved from methanol and acetate  production was attempted. 3HP, a precursor for acrylic acid, and 1,3-propanediol, which could be polymerized to form biodegradable polymers, was targeted for production in M. extorquens AM1 through the overexpression of malonyl-CoA reductase (mcr) gene of Chloroflexus aurantiacus DSM 635 encoding a bifunctional enzyme with alcohol dehydrogenase and aldehyde dehydrogenase activities. Using an adaptively evolved strain of M. extorquens AM1 for a faster growth rate -3-hydroxybutyryl-CoA to (R)-2-hydroxyisobutyryl-CoA were overexpressed in M. extorquens AM1. By utilizing the poly-3-hydroxybutyrate (PHB) overflow metabolism, the engineered strain was able to produce 2-HIBA to about 2.1 g/L along with PHB as a byproduct, with a maximal combined yield (2-HIBA and PHB) of 0.11 g/g methanol (2-Hydroxyisobutyric acid (2-HIBA), a branched C4-hydroxycarboxylic acid, is a precursor of methyl methacrylate, the key intermediate for poly-methyl methacrylate, also known as an alternative glass. An EMC pathway intermediate, 3-hydroxybutyryl-CoA, was employed for the production of 2-HIBA. The coenzyme B12-dependent (R)-3-hydroxybutyryl-CoA mutases (RcmAB) of methanol .M. extorquens AM1. Cis-aconitic acid an intermediate of the TCA cycle was decarboxylated with the overexpression of cis-aconitate decarboxylase (Cad) of (Aspergillus terreus) resulting in the production of 31.6 mg/L itaconic acid from methanol  gene from Blattella germanica, the HMG-CoA reductase (HmgR) gene from Trypanosoma cruzi, and the thiolase (PhaA) gene from Ralstonia eutropha. This engineered strain produced up to 2.2 g L−1 mevalonate with a yield and productivity of 28.4 mg/g and 7.16 mg/L/h under fed-batch fermentation conditions (fumC (encoding fumarase C), the highest yield of mevalonate (2.67 g/L) in engineered M. extorquens AM1 has been reported (M. extoquens AM1 by heterologous expression of α-humulene synthase (ZSS1) from Zingiber zerumbet and farnesyl pyrophosphate (FPP) synthase from Saccharomyces cerevisiae along with isoprenoid precursor supply from mevalonate pathway of Myxococcus xanthus . By using the crtN (diapophytoene dehydrogenase) knock-out pigment-less strain of M. extorquens AM1  host . Like the RuMP pathway, this pathway has also been exploited to produce various chemicals. Model methylotrophic yeast fe) host , P. pastrability . This orP. pastoris. D-Lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides was integrated into the rDNA locus via a multicopy integrative plasmid and through post transformational gene amplification strategy, leading to engineered strain successfully producing D-lactate (3.48 g/L) under fermentation condition (P. pastoris. With the multicopy integration of the pyruvate carboxylase gene (pc) and the cytoplasmic malate dehydrogenase gene (mdh1) in the genome of P. pastoris, Zhang et al., obtained production titers of 0.76, 42.3, and 9.42 g/L for fumaric acid, malic acid, and succinic acid, respectively  with high mechanical properties and hydrolysis resistance , was tarondition . Becauseectively  from a clic acid .P. pastoris has been used to produce nootkatone, a highly demanded and prized aromatic sesquiterpenoid naturally found in grapefruit, pummelo, or Nootka cypress trees. Initially, a whole-cell biocatalytic system was created by coexpressing the Arabidopsis thaliana cytochrome P450 reductase (CPR) and the Hyoscyamus muticus premnaspirodiene oxygenase (HPO), which hydroxylated extracellularly added (+)-valencene to (+)-nootkatone. Followed by the introduction of valencene synthase from Callitropsis nootkatensis for production of (+)-valencene  successfully produced 208 mg/L (+)-nootkatone from methanol using dodecane bilayer in a fed-batch bioreactor  production was demonstrated by expressing Aspergillus nidulans phosphopantetheinyl transferase (PPtase) gene npgA and A. terreus 6-methylsalicylic acid synthase (6-MSAS) gene atX driven by alcohol inducible AOX1 promoter. 6-MSAS, the first fungal polyketide synthase (PKS) gene to be cloned  and its precursor, monacolin J, have also been produced from methanol . The low terreus .E. coli require oxidative refolding for proper disulfide bond formation, resulting in low yield  (Margatoxin (MgTx) is a toxin naturally found in scorpion venom. Due to its high affinity for blocking voltage-gated potassium (Kv) channels, this toxin has been widely used for studying the physiological function of Kv ion channels in various cell types, including immune cells. MgTx maintain the biologically active tertiary structure with three disulfide bonds . The cheow yield . With th (rMgTx) .P. pastoris represents a promising microbial cell factory for methanol-based bio manufacturing. Compared to the methylotrophic bacteria, the eukaryotic nature of this organism enables higher tolerance to methanol and extreme environmental conditions (pH or temperature), in addition to the possession of protein assembly, folding and post-translational modification systems.Overall, via the corrinoid iron-sulfur protein (CoFeSP). 2) carbonyl branch, where CO or reduced CO2 is directly added to methyl-CoFeSP generated from the methyl-branch producing acetyl CoA. A diverse group of strictly anaerobic facultative autotrophic bacteria knowns as the acetogens utilizes this pathway for carbon assimilation  cycle, the reductive tricarboxylic acid cycle, the dicarboxylate–4-hydroxybutyrate cycle and reductive glycine pathway (rGLyP) also exist in natural organisms. Among these pathways, the reductive acetyl-CoA pathway is considered the most energetically efficient  and has milation . Some ofmilation . Accordimilation , and acemilation , acetatemilation , and butmilation  have beemilation . In addimilation . Consequ limosum  and acetm woodii . Introdumethanol . ConsideE. coli, C. glutamicum and S. cerevisiae have been reported   . In the revisiae .E. coli and S. cerevisiae  is a necessity (E. coli (P. pastoris) is a plausible strategy for eukaryotic hosts . Theoretically, only two enzymes are required to assimilate formaldehyde, i.e., 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). Increasing regeneration of formaldehyde acceptor, Ru5P, by the heterologous introduction of non-oxidative PPP enzymes and deletion of phosphoglucose isomerase (pgi) significantly increased methanolic carbon incorporation  for cell growth and biochemical production.Growth or cultivation of frmA) knock-out strain of E. coli was developed for the efficient uptake of formaldehyde, overexpressing Mdh of B. stearothermophilus and RuMP pathway enzymes Hps and Phi from B. methanolicus. The E. coli strain successfully produced the valuable compound naringenin using methanol and yeast extract. The introduction of 4-coumaroyl CoA ligase of A. thaliana and chalcone synthase of Petunia hybrida brought out 3.5 mg/L of naringenin production, where 18% of its carbon was derived from methanol  knock-out, equipped with Mdh from B. methanolicus, and Hps and Phi from B. subtilis, has been tested for cadaverine production. Combining the methanol-dependent complementation of this metabolic cutoff strain with ALE (Adaptive Laboratory Evolution) for increasing methanol dependency, the final strain produced 163 mg/L cadaverine with 43% labeled carbon using 13C-methanol and ribose as carbon substrates  deficient mutant, with Mdh of B. stearothermophilus and RuMP enzymes (Hps and Phi) of B. methanolicus has been engineered for production of glutamate. In a minimal medium supplemented with methanol and xylose (4 g/L each), the strain produced 90 mg/ml glutamate  route was avoided, where native Hpr exhibit higher catalytic activity for the irreversible conversion of intermediate glyoxylate to glycolate rather than converting hydroxypyruvate to glycerate. Instead of employing serine as an amino group donor, glyoxylate is transaminated with alanine to generate glycine in a reaction catalyzed by S. cerevisiae alanine-glyoxylate transaminase (Agt). Glycine accepts MTHF to generate serine which is finally deaminated to pyruvate through serine dehydratase (Sdh) from Cupriavidus necator. PEP is regenerated by the action of endogenous phosphoenolpyruvate synthetase (Pps), thus avoiding the formation of the intermediate hydroxypyruvate  catalyzes the direct condensation of glycine with formaldehyde to generate serine. Serine is directly converted to pyruvate, bypassing formaldehyde condensation to the CH2-THF route of the serine cycle, which follows the condensation of pyruvate with formaldehyde generating 4-hydroxy-2-oxobutanoate (HOB) via the promiscuous activity of E. coli 2-keto-3-deoxy-L-rhamnonate aldolase (RhmA). HOB is subsequently aminated to homoserine by HOB aminotransferase (Hat). Homoserine is later metabolized by homoserine kinase (Hsk) and threonine synthase (Ts) to produce threonine. Finally, the activity of LtaE cleaves threonine producing acetaldehyde and regenerating glycine  is a RuMP pathway derived carbon conserved and ATP-independent synthetic biocatalytic pathway that uses enzymatic reactions of the non-oxidative glycolysis (NOG) to convert formaldehyde to acetyl-CoA and water . Similar release . Using tin silico combinatorial algorithm called comb–flux balance analysis (In vitro demonstration of this pathway showed the production of 462.6 mg/L acetate after pathway optimization (Another novel C1 assimilation pathway based on phosphoketolase (Pkt) was developed through the analysis . In thisanalysis . In vitrmization .in vitro carbon yield was further improved (94%) compared to the GAA (88%)  pathway condenses the formaldehyde-derived glycoaldehyde with E4P generating 2R, 3R-stereo allose 6-phosphate (A6P), catalyzed by the novel aldolase DeoC. A6P is then isomerized to d-allulose 6-phosphate (Au6P) by allose 6-phosphate isomerase/ribose 5-phosphate isomerase B (RpiB) and subsequently epimerized to F6P by D-allulose-6-phosphate 3-epimerase (AlsE). Finally, F6P is hydrolyzed by Pkt to produce acetylphosphate regenerating E4P . With thAA (88%) .de novo engineered enzyme formolase (FLS), catalyzing the carboligation of three molecules of formate to a DHA molecule (B. methanolicus with formolase and native dihydroxyacetone kinase (Dhak), a linear pathway for methanol assimilation was demonstrated for the first time in E. coli  is a synthetic pathway, which theoretically provides the most energy-efficient formate assimilation route. This pathway starts with the ligation of formate with H4F and further reduces to CH2-THF. Then glycine cleavage/synthase system (GCS) reacts CH2-THF with CO2, ammonia, and NADH, generating glycine. Following this, another CH2-THF condenses with glycine to form serine. The pathway starting from formate up to serine is fully reversible, and the direction of metabolic flux is determined by the concentration of substrates and products . This pan source . Additioreported . The engrevisiae .via the action of Pta  pathway is a linear C1 assimilation pathway where two formaldehyde molecules are condensed to glycolaldehyde catalyzed by a glycolaldehyde synthase . Subsequently, an Actinobacteria-derived Pkt with acetylphosphate synthase activity converts the glycolaldehyde to acetylphosphate. Finally, acetyl-CoA is generated n of Pta . Althougd in PEP .via an acyl-CoA reductase, and the HACL catalyzes the ligation of formaldehyde with formyl-CoA to generate glycolyl-CoA, which can be converted to glycolate  pathway is another C1 assimilation pathway based on the activity of 2-hydroxyacyl-CoA-lyase (HACL). This enzyme can act reversibly and catalyze the ligation of carbonyl-containing molecules with formyl-CoA to produce C1-elongated 2-hydroxyacyl-CoAs . In thislycolate . Engineealdehyde .via alpha-keto acid decarboxylase to 3-hydroxypropionaldehyde (3-HPA) and finally reduced to a value-added product 1, 3-propanediol  using the enzyme 1, 3-PDO oxidoreductase. Engineered E. coli expressing this pathway successfully demonstrated the production of 32.7 mg/L 1, 3-propanediol from methanol with glucose supplement (Ketohydroxybutyrate (KHB) pathway is a pyruvate-based formaldehyde assimilatory pathway utilizing the aldol condensation activity of the natural enzyme 2-keto-4-hydroxyglutarate (KHG) aldolase. In this pathway, formaldehyde condenses with pyruvate to form HOB, which can be further decarboxylated pplement .in vitro and/or in vivo for the assimilation of C1 substrates, these novel engineered pathways are still far from optimum. The difficulties of increasing enzyme efficiencies, efficient metabolic rerouting, and regulation of host metabolism are still hurdles to overcome for transforming synthetic methylotrophs into a methylotrophic platform organism.All the aforementioned novel pathways have been designed to make effective use of endogenous cellular resources during methanol metabolism. Although functionally illustrated as proof of concept M. extorquens AM1, along with the essential regulatory role of the phosphoribulokinase gene  and methanol tolerance (up to 1.2 M) comparable to native methylotrophs have been reported (As for the synthetic methylotrophs, even with the vast knowledge and available tools, tolerance and efficient utilization of methanol with sufficient regeneration of pathway intermediates for incorporation of methanol-derived carbon remain a challenge. Techniques like improvement of substrate uptake and reduction of methanol toxicity , charactnic acid , ethanolnic acid  from metreported .Irrespective of their present status, both the methylotrophy approaches follow a parallel track in the pursuit of bioprocess from a renewable alternative carbon feedstock.


In recent years, the incidence of coronary heart disease and rheumatoid arthritis has been increasing, which has become a common public health problem worldwide. YiYiFuZi (YYFZ ) powder is a classical traditional Chinese prescription, which is commonly used to treat metabolic diseases such as rheumatoid arthritis, with an ideal curative effect, but the therapeutic mechanism is still unclear. In this study, from the perspective of clinical metabolomics, combined with network pharmacology, we sought the comorbidity mechanism and key targets of coronary heart disease and rheumatoid arthritis and the mechanism by which YYFZ powder exerts therapeutic effects, combined with molecular docking and atomic force microscopy to determine the effective components, and found that the higenamine and steroid components in YYFZ powder can bind acid sphingomyelinase enzymes to affect the sphingolipid pathway to produce therapeutic effects, which can bind to sugars existing as a glycoside. Coronary heart disease (CHD), a common cardiovascular disease, has become a major health problem in the general population for nearly half a century . RheumatAs an essential part of complementary and alternative medicine, traditional Chinese medicine (TCM) has long been used to treat various metabolic diseases including CHD. From the perspective of TCM, CHD and RA belong to the category of paralysis in TCM. Cold-damp paralysis is an important pathogenic mechanism in the early stage of RA and CHD, which is also an important pathological product in the development of the disease . YiYiFuZThis study aims to investigate the direct and indirect mechanisms of TCM YYFZ powder on the cotreatment of CHD and RA through clinical metabolomics combined with network pharmacology. First, the mechanism of metabolic disorders of the two diseases is explored through clinical metabolomics. Second, through network pharmacology, the common targets and signaling pathways of CHD and RA are collected, analyzed, and integrated. In addition, the common mechanisms of CHD and RA are explored, and the indirect mechanism of how YYFZ powder regulates the disease process of CHD and RA. At last, the main components of YYFZ powder were docked with the joint targets through molecular docking technology to discover the components of YYFZ powder that act directly on metabolic enzymes to exert disease therapeutic effects, and the results were then validated by atomic force. The results were then verified via atomic force microscopic imaging to establish the binding ability of the screened active ingredients to the protein.Formic acid was obtained from ROE Co., Ltd. (United States). Acetonitrile was supplied by Oceanpak Co., Ltd. (Sweden). Pure water was purchased from Watsons (China). Bruker Dimension ICO atomic force microscope (AFM) was obtained from Bruker (Germany). DK-98-II electric-heated thermostatic water bath was provided by Tianjin Taist Instrument Co., Ltd. (China). BT125D 1/100000 electronic analytical balance was purchased from Satorius (Germany). Recombinant human acid sphingomyelinase (ASM) protein was obtained from Shanghai Yubo Biotechnology Co., Ltd. (China). β-sitosterol, higenamine, and stigmasterol reference substances were obtained from Sichuan Vicki Biotechnology Co., Ltd. (China). Dimethyl sulfoxide was supplied by Beijing Solarbio Technology Co., Ltd. (China).This study included 150 subjects, aged 35–65 years, including 50 cases each in the RA group, CHD group, and normal saline (NS) group. All subjects were collected in the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine from April 2018 to July 2019. After being approved by the Ethics Committee of the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, data statistics have been performed on the age and clinical performance of each subject, and all subjects signed a patient informed consent.Healthy people who met the following conditions were included in the NS group: 1) The result of the physical examination was healthy. 2) There was no metabolic disease or inflammatory disease. 3) The four indicators of rheumatism , C-reactive protein (CRP), rheumatoid factor, and erythrocyte sedimentation rate) were normal.The inclusion criteria of the RA group are as follows: had more than four joint pains, morning stiffness not less than 30 min, increased platelet count, and increased four indicators of rheumatism; judged that the condition is consistent with the active stage of RA; and enrolled in the RA group as a candidate.The exclusion criteria of the RA group are as follows: 1) patients with severe joint deformities; 2) patients with other rheumatic diseases such as systemic lupus erythematosus, Sjogren’s syndrome, and knee osteoarthritis; 3) patients with metabolic diseases of the heart, brain, liver, kidney, and other systems; and 4) patients who have taken drugs for the treatment of RA before the study.The inclusion criteria of the CHD group are as follows: The CRP index of patients in serum biochemical indicators was significantly increased and accompanied by angina pectoris, dizziness, chills, and even syncope, and other clinical symptoms were included in the CHD group as candidates.The exclusion criteria of the CHD group are as follows: 1) patients with persistent atrial fibrillation, dilated cardiomyopathy, and cardiac insufficiency; 2) patients with severe electrolyte disorders, malignancies, and severe valvular disease; 3) patients with autoimmune diseases; 4) patients with other organic pathologies or other metabolic diseases; and 5) patients who took CHD-improving drugs before the study.The serum samples of the subjects were collected and centrifuged at 3,000 * g for 15 min at 4°C. In addition, the supernatant was transferred and centrifuged again at 3,500 * g for 4 min at 4°C, and then the supernatant was stored in a −80°C refrigerator for further serum metabolomics research.After thawing each group of samples frozen in a −80°C refrigerator, the samples were centrifuged at 4°C. A total of 100 μL of supernatant was mixed with acetonitrile in a volume ratio of 1:3, and the mixture was sonicated in an ice-water bath for 10 min and vortexed for 1 min and then centrifuged at 13,200 * g at 4°C. A total of 200 μL of supernatant was transferred into the sample vial for metabolomics analysis.In this experiment, UPLC/Q-TOF-MS  was used to characterize metabolic substances. UPLC separation was performed on the ACQUITY UPLC BEH C18 column , with a column temperature of 45°C. The mobile phase consisted of water (A) and acetonitrile (B) (both containing 0.1% formic acid). The gradient elution program was as follows: 0–0.5 min, 1% B; 0.5–2 min, 1%–50% B; 2–9 min, 50%–99% B; 9–10 min, 99% B; 10–10.5 min, 99%–1% B; and 10.5–12 min, 1% B. The flow rate was 0.3 ml/min. The injection volume was 5 μL.After separation, an electrospray ion source was used to detect and analyze mass spectrometry in positive and negative ionization modes. The ion source parameters were set as follows: a capillary voltage of 3.0 kV, drying gas temperature of 325°C, atomizing gas pressure of 310 kPa, drying gas flow of 0.26 ml/min, desolvation gas flow of 600 L/h, source temperature of 120°C, desolvation temperature of 350°C, and cone gas flow of 50 L/h.p < 0.05) in the statistical analysis. The metabolites screened by the above analysis are input into the MetaboAnalyst platform  for cluster analysis and Mate-Pa analysis.Raw data were exported by MarkerLynx 4.1  software, after which the data were imported into SIMCA 14.1 statistical software  for multivariate statistical analysis. Afterward, SPSS 26.0 was applied for statistical tests and the appropriate test was selected to determine whether metabolites changed significantly (http://ctdbase.org/), TTD (http://bidd.nus.edu.sg/group/cjttd/), durgbank (https://www.drugbank.ca/), and OMIM (http://www.omim.org/) database for retrieval.In this study, “Coronary heart disease” and “Rheumatoid arthritis” were used as search terms to search for CHD- and RA-related targets in the CTD (http://bioinfogp.cnb.csic.es/tools/venny/) website to obtain the common targets of the two diseases and draw a Venn diagram.The disease targets of the above CHD and RA target libraries were imported into the Venny2.1 (https://string-db.org/DAVID), and obtain the PPI network graph of the interaction between RA-CHD shared targets. Import the PPI map into Cytoscape 3.6.0 to visualize the PPI data between the potential treatment targets shared by the disease on the network.Import disease common target into STRING  websites. The information on the targets of the ingredients from the two websites was aggregated, and the common targets were removed to obtain the active ingredient target library.The TCMSP search of “Coix seed” and “Fuzi” was used to screen the main active ingredients in coix seed and Fuzi for the active ingredients. The screening of the main active ingredients in the two herbs of YYFZ powder was performed . Target via protein interaction analysis through the String website, and PPI maps were imported into Cytoscape 3.6.0 for in-network visualization of PPI data between potential therapeutic targets shared by diseases, and active ingredient–target–disease networks were mapped.PPI maps were created The KEGG pathway enrichment analysis was conducted on the nodes in the network using David’s website to obtain the functional pathways involved in the component action targets of YYFZ powder to modulate diseases, and the enriched pathways were visualized using the advanced bubble map function in OmicShare platform. The results will be compared with the functional pathways involved in the common targets of the two diseases, and a basis for the elaboration of the heterogeneous treatment mechanism of CHD and RA by YYFZ powder will be provided.The 3D structural formulas of the active compounds screened above were saved in .sdf format. The prepared major compounds were bonded into the CHARMM force field with partial charges calculated via the Momany–Rone option . The obtIn the PDB protein database, the eutectic structure of ASM and inhibitor (ABPA) (PDB ID: 5fi9) was selected as the receptor protein. Import the receptor protein into discovery studio 2017 R2 client software, and prepare the receptor protein in macromolecules. In automatic preparation, click prepare protein. The force field used for calculation was the CHARMM force field.First, the active site was defined for the location of the self-ligand of the 5FI9 protein complex. Second, the self-ligand of the 5FI9 protein complex was extracted for energy optimization, and then, the optimized ligand was docked with the active site of the 5FI9 protein, and the RMSD values of the docked conformation and the conformation in the crystal were calculated if the docked ligand could overlap well with the original crystal ligand structure and the RMSD value was <2. If the docked ligand is in good agreement with the original crystal ligand structure and the RMSD value is <2, then the docking procedure and the selected parameters are suitable for the screening of this study.The Docking Ligands (CDOCKER) semiflexible docking mode in the receptor–ligand interaction section was selected for molecular docking. The binding activity of the compound to the target is evaluated based on the score and the reasonableness of the bond between the ligand and the amino acid residues of the receptor protein.A buffer solution with pH 8.0 was prepared with 20 mM Tris and 150 mM NaCl, and ASM protein was prepared to 25 μg/ml of ASM protein solution by adding buffer. Higenamine, β-sitosterol, and stigmasterol were dissolved in DMSO, and the solution was configured to a final concentration of 25 μg/ml by adding buffer (DMSO ≤ 0.1%).The 25 μg/ml ASM protein solution and the pure water solvent were mixed 1:1 and added to the newly peeled silicon wafers for crystallization for 15 min, rinsed with ultrapure water, dried, and put into the AFM sample table for imaging. Mix 25 μg/ml of ASM protein solution and 25 μg/ml of standard solution 1:1, incubate for 30 min at room temperature, then add the solution to the silicon wafer for crystallization, rinse with ultrapure water to dry, and then put it into the AFM sample stage for imaging. The AFM was imaged in tap mode under gas phase room temperature conditions.First, unsupervised PCA models were established for the data in positive and negative ion modes for NS, CHD, and RA groups. As shown in p < 0.05) between CHD and RA groups and then screened 21 biomarkers common to CHD and RA. Among them, 10 metabolites showed upregulation in both disease groups, 11 metabolites showed downregulation in both diseases, and bilirubin showed opposite trends in both diseases, as shown in In this study, we used the PLS-DA results to screen the information of substances with the same significant difference . The enriched pathways were visualized by the advanced bubble map function of the OmicShare cloud platform  and the targets of YYFZ powder (543) are shown in The results of target pathway correspondence and pathway importance visualization are shown in In terms of biological processes, 103 enrichment results were obtained, and the regulatory targets of YYFZ powder that exerted allopathic treatment mainly involved collagen catabolic process, peptidyl serine phosphorylation, and positive regulation of RNA polymerase II promoter transcription. In terms of molecular function, a total of 19 enrichment results were obtained, with a total of 35 of the shared targets enriched in ATP binding function and nontransmembrane protein tyrosine kinase activity. The part of cellular composition involved 14 enrichment results, and target proteins were mainly present in the cytoplasm, nucleus, and cytoplasmic lysate. The top 10 analysis results of GO analysis are shown in Through the integration of network pharmacology and metabolomics results and the search of related literature, the following conclusions were drawn: Disorders of sphingolipid metabolism in CHD and RA cause activation of downstream signaling pathways such as apoptosis and inflammation, and inhibition of ceramide synthesis may be a key therapeutic target for the cotreatment of CHD and RA. The results of network enrichment analysis of the two disease comorbidities show that the sphingolipid signaling pathway occupies an important position in the two disease comorbidities . In addiThe literature research revealed that the ASM enzyme occupies an important role in ceramide synthesis, and the association of sphingolipase with the two diseases has been reported, which provides a basis for the metabolomics and network pharmacology experiments derived from “The importance of abnormalities in sphingolipid metabolism and sphingolipid signaling pathways in the comorbidity of the two diseases” and the possibility that ASM enzymes are key targets for the comorbidity of the two diseases provide theoretical support . As a clAs shown in According to the analysis of the docking scoring  results, the main components of YYFZ powder with drug-forming properties can bind well to ASM proteins and enter the protein cavity for reasonable interactions, among which the top three are higenamine (73.2564), β-sitosterol (47.8923), and stigmasterol (46.6821).As shown in 2+ ion in the ASM protein structure forms a salt bridge with the oxygen atom in the isoquinoline parent nucleus structure of desmethylaconitine, and the two key amino acids, His317 and His280, in the structure both form electron-absorbing interactions with the oxygen atom; on the other hand, His280 also forms a Pi-Pi T-sharp interaction with the benzene ring in the structure, and the amino acid ASP276 and the hydroxyl group in the structure form hydrogen bonding interactions to provide the stability of the structure , immunity , and regulation of vascular endothelial function .Ceramides are key signaling lipids that regulate the biophysical properties of membranes and are involved in many cellular processes as well as disease states. In a study on the regulation of sphingolipid metabolism in RA rats by Chinese herbal medicine, the Chinese herbal medicine compound IDM exerted antiarthritic effects by inhibiting Cer-mediated COX-2 activation and leading to PEG2 release, suggesting that ceramide plays an important role in RA and coronary atherosclerosis . Acid spThe binding strength of small molecules of YYFZ powder cochinchinensis epithelium to target proteins was evaluated using the molecular docking technique. In addition, the compounds were screened based on the scoring results, and the changes in the molecular morphology of target proteins induced by the selected active molecules were revealed via atomic force microscopy. The results showed that the components higenamine, β-sitosterol, and stigmasterol in YYFZ powder had strong interactions with ASM, and the results of AFM showed that higenamine, β-sitosterol, and stigmasterol were able to bind ASM protein at the protein level, affecting its structure and function. The results of this study confirmed the interaction between the active ingredients of the virtual screened drugs and the target proteins by the results of AFM experiments and may be useful for the screening and establishment of small molecule ligand drugs targeting receptor proteins.Based on clinical metabolomics combined with bioinformatics technology, this study attempted to characterize the intrinsic material basis of YYFZ powder in the treatment of CHD and RA. In addition, we attempted to explore the pharmacodynamic components of YYFZ powder in the treatment of different diseases from both direct and indirect perspectives. On the one hand, through the untargeted metabolomic analysis of serum samples from the two diseases and the NS group, the differential metabolic markers of CHD and RA comorbidity and the mechanism of comorbid metabolic disorders were obtained. This analysis revealed that the metabolic disorders of the two diseases mainly focused on sphingolipid metabolism, inflammation, oxidative stress metabolic pathways, and the abnormalities of ceramide played an important role in the two diseases. On the other hand, YYFZ powder is enriched in its component regulatory targets for the cotreatment of the two diseases through network pharmacology to elaborate its indirect mechanism for the treatment of the disease. The codisease targets of the two diseases mainly focus on inflammatory immunity, hormones, and lipid signaling pathways.Metabolomics and the analysis results of CHD and RA disease network modules were integrated and analyzed, and the abnormality of ceramide was judged to play an important role in the two diseases using the results of metabolomics. Through network pharmacology, the disease was enriched in common targets and enriched in ASM enzymes. At present, there are relevant reports on the research of ASM enzyme-amitriptyline in RA and atherosclerosis. In summary, it is inferred that ASM protein occupies an important position in the two diseases, and screening and discovering direct inhibitors of ASM enzyme may be a breakthrough in the treatment of the two diseases. The results of molecular docking and atomic force microscopy showed that the binding strength of higenamine, β-sitosterol, and stigmasterol to the ASM enzyme was high, and the binding conformation was reasonable, which might be the direct pharmacodynamic components of YYFZ powder to exert therapeutic effects.