In vivo murine studies revealed that KJK exposure increases the body weight and colon length, while reducing colonic epithelial injury, and the expression of inflammatory factors in colitis tissues such as TNF-α, IL-6, and IL-1β. Furthermore, KJK treatment downregulates the expression of pi3k and akt genes, as well as p-PI3K/PI3K and p-AKT/AKT proteins. Collectively, these findings describe the therapeutic effects and mechanisms of KJK in UC and highlight KJK as a potentially valuable therapeutic option for UC via modulation of the PI3K/AKT signaling pathway, thus providing a theoretical reference for the broader application of KJK in the clinical management of UC.Kui Jie Kang (KJK)—a traditional Chinese medicine—has demonstrated clinical therapeutic efficacy against ulcerative colitis (UC). However, the active compounds and their underlying mechanisms have not yet been fully characterized. Therefore, the current study sought to identify the volatile compounds in KJK responsible for eliciting the therapeutic effect against UC, while also analyzing key targets and potential mechanisms. To this end, systematic network pharmacology analysis was employed to obtain UC targets by using GeneCards, DisGeNET, OMIM, among others. A total of 145 candidate ingredients, 412 potential targets of KJK (12 herbs), and 1605 UC targets were identified. Of these KJK and UC targets, 205 intersected and further identified AKT1, JUN, MAPK, ESR, and TNF as the core targets and the PI3K/AKT signaling pathway as the top enriched pathway. Moreover, molecular docking and ultra-performance liquid chromatography Q Exactive-mass spectrometry analysis identified quercetin, kaempferol, luteolin, wogonin, and nobiletin as the core effective compounds of KJK.  Inflammatory bowel disease is a chronic, recurrent, and autoimmune disease that primarily occurs in the colon and bowel and includes Crohn's disease and ulcerative colitis (UC) , 2. UC iPoria cocos (Fu Ling), Paeoniae radix (Bai Shao), Citri Reticulatae Pericarpium (Chen Pi), Codonopsis pilosula (Dang Shen), Atractylodes macrocephala (Bai Zhu), Saposhnikovia divaricata (Fang Feng), Bletilla striata (Bai Ji), Sanguisorba officinalis (Di Yu), Dioscorea rhizoma (Shan Yao), Panax notoginseng (San Qi), Patrinia villosa (Bai Jiang Cao), and Platycodon grandiflorus (Jie Geng). It is a medication, based on an ancient Chinese “Tongxie” prescription, that has demonstrated clinical therapeutic efficacy. We have previously explored the preventive and therapeutic effects of this prescription in UC from various perspectives and found that KJK can downregulate NF-κB expression, inhibit the intestinal inflammatory response, restore intestinal mucosal barrier function, reduce the oxidative stress response, inhibit NLRP3 inflammasome activation, and regulate intestinal flora imbalance in a mouse model of UC. However, the effective components, key targets, and additional regulatory pathways for intervention remain unclear.Kui Jie Kang (KJK) comprise in vivo UC mouse model. These results provide new insights regarding the mechanism by which KJK contributes to the treatment of UC and provides a reference for its broader clinical application.Multi-pharmaceutical research is the basic strategy for the clinical application of traditional Chinese medicine (TCM). Although it is generally believed that the effective ingredients of TCMs are chemicals in drugs or metabolites, TCMs usually contain thousands of compounds, whose components may be altered by environmental factors or preparation methods . In addiβ (cat. no. MB-2906A), IL-6 (cat. no. MB-2899A), TNF-α (cat. no. MB-2868A), and enzyme-linked immunosorbent kits were purchased from Jiangsu Enzyme Labeling Biotechnology Co. .Chinese herbs of KJK were purchased from the Department of Pharmacy, the First Affiliated Hospital of Yunnan University of Traditional Chinese Medicine . Mesalazine enteric coated tablets  were purchased from Sunflower Pharmaceutical Group Jiamusi Luling Pharmaceutical Co., Ltd. . The SDS-PAGE Gel Matching Kit (cat. no. P0012A) and BCA Protein Determination Kit (cat. no. P0012) were purchased from Beyotime Biotech Inc. . Additionally, 4% paraformaldehyde was obtained from Sinopharm . Primers were designed by Generay Biotech Co. Ltd. . A Total RNA Extraction Kit (cat. no. DP419) was obtained from the Tiangen Biochemical Department, Technology Co., Ltd. . TaKaRa PrimeScript RT Master Mix (cat. no. RR036A) and TaKaRa TB Green Premix Ex Taq II (cat. no. RR820A) were purchased from Bao Biological Engineering  Co. . The anti-MPO antibody (ab9535) was purchased from Abcam, Inc. , and PI3K rabbit polyclonal antibody (cat. no. 4257S), p-PI3K rabbit polyclonal antibody (cat. no. D0615), AKT rabbit polyclonal antibody (cat. no. 4685S), and p-AKT rabbit polyclonal antibody (cat. no. 4060S) were purchased from Cell Signaling Technology . IRDye®680RD Goat anti-Rabbit IgG Secondary Antibody (cat. no. 926–68071) was purchased from LI-COR Biotechnology . The GAPDH rabbit polyclonal antibody (cat. no. 10494-1-AP) was purchased from Proteintech . DSS was purchased from MP Biomedicals . IL-1https://54.223.75.62:3838/), correlations between KJK and its components and the molecular basis of UC were evaluated. The specific operations were as follows: input UC (ULC004) and KJK , select hit, TCMID, stitch, and TCMSP databases, parameter property index QED set to 0.2, drug relevance threshold set to 400, compound target significance set to P < 0.05 [Through TCM Network Pharmacology Analysis System , a platform for the systematic pharmacological analysis of TCM, searches were performed for KJK active components and targets. Each component of KJK was used as an input. Oral availability (OB) was set to ≥30%, and drug similarity (DL) was set to ≥0.18 [https://www.swisstargetprediction.ch/) database. Using the PubChem database (https://www.lilab-ecust.cn/pharmmapper/), the active ingredients were imported in mol2 format, setting the species to human; 10 targets with the highest fit score were obtained by default. The UniProt database (https://www.uniprot.org/) defines the species as human, corrects the target corresponding to each active ingredient, and converts it into the corresponding gene name for further mechanistic research. With human genes as the background, “ulcerative colitis” was used as the search term for the identification of related genes in GeneCards (https://www.genecards.org/), DisGeNET (https://www.disgenet.org/), OMIM (https://omim.org/), and TTD (http://db.idrblab.net/ttd/). An information table including disease targets was exported, and redundant data were deleted.Using TCMSP (to ≥0.18 . The unvhttps://bioinfogp.cnb.csic.es/tools/venny/), a Venn diagram of the KJK targets and UC targets was generated. The active components of KJK and UC-related genes were introduced to the STRING database (https://string-db.org/) to evaluate core genes by the construction of a protein-protein interaction network, with “Homo sapiens” as the organism, a minimum required interaction score of 0.70 (high confidence), and deletion of highly disconnected nodes. Thereafter, a molecule network analysis was conducted using Cytoscape v3.8.2 and the CytoNCA plug-in. Pathway annotation of the core targets was performed using DAVID (https://david.ncifcrf.gov/). GO-BP, GO-CC, and GO-MF enrichment, and KEGG enrichment analyses were carried out . The “drug-target-pathway” table was imported into Cytoscape to generate network pharmacology figures.Using Venny 2.1 (https://plip-tool.biotec.tu-dresden.de/).AutoDockTools 1.5.6 was used to dock the KJK core active ingredients and the target proteins based on the UC target pathway. Autogrid calculations were performed, selecting AutoDock Vina as the docking algorithm, and AutoDock was used for molecular docking. A binding energy of less than −5 kcal/mol was selected as the reference, and the binding energy results were visualized using PyMOL 2.4.0. Moreover, hydrophobic force analysis was conducted via the website analysis , vortexed for 30 s, homogenized at 45 Hz for 4 min, and added to an ultrasonic  ice water bath for 1 h. After standing at −40°C for 1 h, samples were centrifuged at 4°C and 12004 × g for 15 min. The upper layer was carefully removed and subjected to 0.22 μM microporous membrane filtration. Samples were stored at −80°C until use. LC-MS/MS was performed using a Agilent ultra-high-performance liquid chromatography 1290 UPLC system with a Waters UPLC BEH C18 column . The flow rate was set to 0.4 mL/min, and the sample injection volume was set to 5 μL. The mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multi-step linear elution gradient program was carried out, and MS and MS/MS data were obtained as previously described [The sample (100 mg) was supplemented with 500 mg/All animal protocols were reviewed and approved the Intramural Committee on the Ethical and Humane Treatment of Experimental Animals of Yunnan Traditional Chinese Medicine Hospital/Medical Ethics Committee of the First Affiliated Hospital of Yunnan University of Traditional Chinese Medicine, and were in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals (Application No: SD2021-001).ad libitum for one week. They were then randomly divided into six groups, with 10 mice per group, as follows: normal diet control group (CON), 2.5% DSS group (MOD), 2.5% DSS + mesalazine group (MC), 2.5% DSS+10.08 mg/kg KJK group (KJK-H), 5.04 mg/kg KJK group (KJK-M), 2.5% DSS + 2.52 mg/kg KJK (KJK-L). Excluding the CON group, all groups had ad libitum access to 2.5% DSS (MW: 36000–50000 Da) solution for 7 days, and fresh DSS solution was added each day. Next, the CON and MOD groups were given normal saline, the MC group was administered mesalazine, and the remaining groups were administered corresponding doses of KJK for one week. The weight, diarrhea, and rectal bleeding of mice were recorded every 2 days, and the disease activity index (DAI) score was calculated as described in C57BL/6 female mice (18 ± 2 g) were purchased from Dashuo Biotech Co. . All mice were housed under standard conditions  with free access to food and water. Mice were acclimated by placing them on a diet administered μm sections, which were stained with hematoxylin-eosin (H & E) and examined under a light microscope  at 200× magnification. Five animals in each group were observed under the microscope in four fields of view each for changes in colonic tissue.The colon tissue was fixed with 4% paraformaldehyde for 24 hours and then sectioned by dehydration, embedded in paraffin, and cut into 5–7 μl of phosphate-buffered saline buffer (pH 7.4) and homogenize. The tissue was centrifuged at 12004 × g for 15 min at 4°C. The supernatant was then collected, and BCA quantification was performed. IL-1β, IL-6, and TNF-α were quantified by ELISA kits according to the manufacturer's protocol.Precisely weigh 80 mg of mice colon tissue into 720 μg) was equalized and converted to cDNA using the HiScript II Reverse Transcriptase Kit. The reaction system was as follows: PrimeScript RT Master Mix 4 μL, 1000 ng of total RNA, and ddH2O to 20 μL volume. The reaction conditions were as follows: 37°C for 15 min and 85°C for 5 s. Gene expression was measured by RT- qPCR  using SYBR green. A total of 1 μL of cDNA template was used for PCR amplification using the following primers: pi3k F, 5′-TCTACCCAGTGTCCAAATACCA-3´; R, 5′-AAATGCTTCGATAGCCGTTCT-3′; akt F, 5′-CGACCGCCTCTGCTTTGT-3′; R, 5′-AAGTCCGTTATCTTGATGTGCC-3′. The total reaction volume was 20 μL, including SYBR Premix Ex Taq II (10 μL), Primer F (0.3 μL), Primer R (0.3 μL), cDNA (1 μL), ddH2O (8.4 μL). The PCR conditions were as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing and extension for 20 s at 62°C, and amplification for 40 cycles. The ratio of target products to the internal reference GAPDH gray value  was calculated to determine the relative mRNA expression levels of pik3 and akt.Total RNA was prepared from the colon using TRIzol reagent according to the manufacturer's instructions. Total RNA  Protein Assay Kit. 50ug protein was separated by electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1 h at room temperature and then incubated with primary antibodies against p-PI3K/PI3K and p-AKT/AKT (dilute with primary antibody diluent in a dilution ratio of 1 : 1000) overnight at 4°C. The membranes were then washed and incubated with a horseradish peroxidase-labeled secondary antibody for 1 h at room temperature, and the signal was detected using an enhanced fluorescent luminescent substrate. Immunoreactive bands were quantified using ImageJ, and the density ratio of the target protein to GAPDH was used as the relative content of the target protein for statistical analyses.P < 0.05.Statistical analyses were performed using SPSS version 21.0 . Measurement data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) was conducted for group comparisons; differences were considered statistically significant at P=0.000191). In addition, the similarity of co-associated GO terms between KJK and UC was 0.74; the AUC for the top 30 co-associated GO terms between KJK and UC was 6.61  network of targets . AccordiP < 0.0001 with a count >5 are displayed in A total of 205 intersecting target genes were evaluated by GO and KEGG enrichment analyses using the DAVID database. The 15 most significant terms in the GO-BP, GO-MF, and GO-CC categories at P < 0.0001 and count >5, 29 disease-related pathways were screened (n = 55), cAMP (n = 42), MAPK (n = 39), calcium (n = 38), and Ras (n = 34) signaling pathways. Moreover, to elucidate the relationships among ingredients, targets, and pathways, 29 pathways identified in the KEGG enrichment analysis and intersecting genes were used to construct a target pathway network using Cytoscape 3.8.2, resulting in a “KJK active ingredient-UC target pathway” interaction network , JUN (PDB ID: 3U86), MAPK1 (PDB ID: 6G54), ESR1 (PDB ID: 5GTR), and TNF (PDB ID: 1FT4) protein receptors. The 2D structure of the small molecule compound was obtained using the PubChem database and was converted to a 3D structure using ChemOffice. The water molecules and ligands of these protein receptors were removed using PyMOL 2.4.0. We visualized the 3D binding of quercetin, kaempferol, luteolin, wogonin, and nobiletin to AKT1, JUN, MAPK1, ESR1, and TNF protein receptors using AutoDockTools and AutoDock Vina. A lower binding energy indicated a more stable structure . However, compared to levels in the MOD group, TNF-α, IL-6, IL-1β, and TNF-α expression levels were significantly lower in the colons of the KJK-M and KJK-H groups .The bioinformatics analysis showed that AKT1 was the most important target, and the PI3K-AKT signaling pathway was the most important pathway for the treatment of UC. Therefore, to further elucidate the mechanism underlying the anti-UC activity of KJK, we verified the expression of proteins in the PI3K-AKT signaling pathway using RT-qPCR and Western blotting . Comparepi3k mRNA expression in the KJK-L and KJK-M groups exceeded 1 , kaempferol (m/z 287.0544552), luteolin-4-O-glucoside (m/z 447.0960827), wogonin (m/z 283.0606575), and nobiletin (m/z 425.119079).The development of network pharmacology has provided new research ideas and technical means for studying diseases and the mechanisms underlying the effects of TCM compounds . Based oα, lipocalin-2, and other genes, regulate the structure of the intestinal flora, and inhibit the expression of proinflammatory factors in UC [κB pathway [Quercetin is a polyphenolic flavonoid that functions as an antioxidant, radical scavenger, protein kinase inhibitor, as well as an anti-inflammatory, antiviral, and antibacterial agent. As such, it has been used to treat or prevent diverse conditions, including hypercholesterolemia, rheumatic diseases, and infections –23. Querrs in UC , which frs in UC . Kaempfers in UC . Indeed,rs in UC . Luteoli pathway , 28. The pathway , 30.Based on the above research results, we constructed a PPI network, which facilitated the indentation of 15 potential core targets. Combined with the molecular docking results, AKT1, JUN, MAPK1, ESR1, and TNF were ultimately characterized as key targets of KJK in the treatment of UC. However, AKT is at the core of the key target network relationship of KJK in the treatment of UC. Interestingly, the main active components quercetin, kaempferol, luteolin, wogonin, and nobiletin of KJK have good binding activity with AKT1. AKT1 is one of three closely related serine/threonine-protein kinases  called the AKT kinase and is pivotal as a downstream effector of phosphatidylinositol kinase PI3K . Moreoveβ, and TNF-α [The KEGG pathway enrichment results showed that KJK treatment of UC primarily involved the PI3K-AKT, cAMP, MAPK, calcium, Ras, TNF, Foxo, and toll-like receptor signaling pathways. Furthermore, the PI3K/AKT signaling pathway was the most enriched. In recent years, the role of this pathway in intestinal inflammation and tumors has garnered increasing attention . In factnd TNF-α –39. Meannd TNF-α , 36.α, IL-6, and IL-1β abundance in UC mice colons and downregulate the expression of pi3k and akt, as well as the protein abundance of p-PI3K/PI3K and p-AKT/AKT. In summary, KJK can reduce the expression of genes in the PI3K/AKT signaling pathway and inhibit the activation of PI3K/AKT signaling in the colon, thus alleviating DSS-induced UC in mice.In line with the above findings, we found that KJK could effectively reverse the significant changes in body weight, colon weight index, colon weight, and colon length in mice with colitis. Moreover, colonic epithelial injury and inflammatory cell infiltration were reduced by treatment with KJK resulting in improved crypt inflammation, tissue edema, and necrosis. At the same time, KJK was found to significantly reduce TNF-in vitro analyses. Moreover, a rescue experiment will serve to further clarify the mediating effect of KJK and its key components on the PI3K/AKT pathway in the prevention and treatment of UC. Second, it is necessary to carry out more in-depth experimental research and verification from different dimensions in combination with the results of molecular docking and pathway enrichment analysis to further reveal the modern scientific connotation of “dysentery” TCM in the prevention and treatment of UC.Certain limitations were noted in the current study. First, it is necessary to verify which relevant genes are enriched in the PI3K/AKT signaling pathway using The aim of this study was to explore the molecular mechanisms underlying the therapeutic effect of KJK against UC via a comprehensive bioinformatics analysis. More specifically, we aimed to identify the main active components, key targets, and associated pathways involved in treatment of UC with KJK. Collectively, the results show that KJK primarily elicits its therapeutic effect on UC via modulation of the PI3K/AKT signaling pathway. Hence, KJK may represent a valuable therapeutic agent for the treatment of patients with UC. Nevertheless, additional molecular experiments are needed for further validation of the findings described herein.


In conclusion, the current study results demonstrated that Dahuang could treat hepatoma through a variety of active ingredients, targets, and multiantitumor pathways. Moreover, it effectively improved the prognosis of hepatoma patients. ESR1 is the potential key gene that is beneficial for the survival of hepatoma patients. Also, aloe-emodin and beta-sitosterol are the two main active crucial ingredients for hepatoma treatment. The study also provided some functional bases and references for the development of new drugs, target mining, and experimental animal research of hepatoma in the future.Hepatoma is one of the most common malignant tumors. The incidence rate is high in developing countries, and China has the most significant number of cases. Dahuang is a classic traditional antitumor drug commonly used in China and has also been applied to treat hepatoma. However, the potential mechanism of Dahuang in treating hepatoma is not clear. Therefore, this study is aimed at elucidating the possible molecular mechanism and key targets of Dahuang using methods of network pharmacology, molecular docking, and survival analysis. Firstly, the active ingredients and key targets of Dahuang were analyzed through public databases, and then the drug-ingredient-target-disease network diagram of Dahuang against hepatoma was constructed. Five main active components and five core targets were determined according to the enrichment degree. Enrichment analysis demonstrated that Dahuang treated hepatoma through the multiple pathways in cancer. Additionally, molecular docking predicted that aloe-emodin and PIK3CG depicted the best binding energy. Survival analysis indicated that a high/ESR1 gene expression had a relatively good prognosis for patients with hepatoma ( Hepatoma has become one of the major diseases threatening human health and hindering social development , 2. AccoRheum palmatum plant, Tanggu giant Dahuang, or medicinal Dahuang [Dahuang, also known as rhubarb, is the rhizome of the  Dahuang . Dahuang Dahuang . However Dahuang . Modern  Dahuang –19. In r Dahuang –22. Cert Dahuang –26. HoweNetwork pharmacology is based on multidisciplinary systems biology and multinomial pharmacology. The molecular mechanism of drug intervention in diseases is explored in a multidimensional form. It provides biological network construction and network visualization analysis for the complex interactions between drug ingredients and disease targets. This method is very suitable for the characteristics of multi-ingredient, multitarget, and multipathway action of TCM. It is widely used in studying ingredients and the mechanism of action of TCM . Moleculhttps://tcmsp-e.com/) were utilized to seek the active ingredients, chemical structure, and potential targets of Dahuang. According to the scheduling screening conditions, the oral bioavailability (OB) and druglikeness (DL) parameters were utilized to obtain the needed active ingredients, chemical structure, and potential targets. OB stands for the percentage of absorption of traditional Chinese medicine in the human circulatory system, and DL represents the similarity between the ingredients and known drugs [The traditional Chinese medicine systems pharmacology (TCMSP) database and analysis platform . Hence, the structures were redrawn using the ChemBioDraw Ultra software and then saved in the TIFF formats for better clarity.https://www.genecards.org/) and Online Mendelian Inheritance in Man (OMIM database) (https://omim.org/) were used to seek the potential related targets of hepatoma. The keyword “Hepatoma” was used in these two databases. The relevant downloaded targets from both databases were merged and then deduplicated to get the potential targets. Further, the R software was employed to draw a Venn diagram for achieving the core targets between “Dahuang” and “Hepatoma.”The GeneCards database (https://string-db.org/) website. PPI can help us understand the functions and relationships of proteins [The construction of the protein-protein interaction (PPI) network was completed on the STRING  functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis helped understand the function of the core targets and the critical pathways of Dahuang against hepatoma. The https://www.rcsb.org/), and the chemical structures of the main active ingredients were obtained from the TCMSP platform. The screening criteria for PDB ID are based on the best score ranking of each structure in the PDB official website and the latest updated structure form. Furthermore, Maestro, PyMoL, and ChemBioDraw Ultra software were utilized to process the abovementioned protein receptors and ligands, and a molecular docking process was conducted to obtain the affinity among them. The results of molecular docking are presented in two-dimensional and three-dimensional graphics [R software to draw a heat map based on the molecular docking results to analyze which active ingredients were more relevant to the target.The Maestro software was employed to achieve molecular docking between the five key targets in constructing the drug-ingredient-target-disease network and the main active ingredients of Dahuang . The strgraphics . The pregraphics . In addihttps://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga). The core target gene expression was ranked from high to low, and high (50%) and low (50%) cut-off values were utilized as the expression thresholds to divide the two groups, namely, the high expression group and the low expression group. The low expression group had a better prognosis, and it relatively reflected that the specific gene might inhibit tumor development and progress; the opposite was considered a risk factor. Data analysis was performed using the R software. The log-rank test and Kaplan-Meier method analyzed 371 samples from the TCGA database to acquire survival maps; p < 0.05 was considered a statistically significant difference.The impact of the main targets on the prognosis of hepatoma was analyzed through The Cancer Genome Atlas (TCGA) database -catechin) were docked with ESR1, JUN, PIK3CG, PTGS2, and TGFB1 target proteins. Aloe-emodin, beta-sitosterol, eupatin, toralactone, and (-)-catechin exhibited good binding and high matching degree with the five target proteins. Beta-sitosterol performed the worst, mainly because it is fat-soluble and exhibits strong hydrophobicity. Moreover, the molecule is large and not conducive to matching the protein pocket. The complex formed between the docking compound and the protein was visualized with PyMoL 2.1 software (the combination with the most negative docking score was selected for each target), and the binding mode of the compound and the protein was obtained. According to the binding mode, the amino acid residues of the compound and the protein pocket combination were clearly seen.For example, the amino acid residues of JUN interacting with the active sites of aloe-emodin included LEU-855, VAL-863, ALA-880, MET-929, GLU-930, VAL-911, GLY-993, LEU-983, SER-936, LEU-932, ASP-939, and TYR-931. Aloe-emodin is a strongly hydrophobic anthraquinone compound. It forms a strong hydrophobic interaction with active site amino acids  and plays an essential role in stabilizing the small molecules in the protein cavity. Additionally, the compound forms multiple hydrogen bonds with ASP-939, LEU-932, and GLU-930 amino acids; this bonding contributes considerably to anchor small molecules in the protein cavity. Several other compounds also exhibit hydrogen bonding and hydrophobic interactions with ESR1, PIK3CG, PTGS2, and TGFB1 targets; such interactions effectively promote the formation of stable complexes between small molecules and proteins. In conclusion, these compounds -catechin) and the target proteins  performed well in the docking scoring and binding mode. These compounds formed stable complexes with proteins and correlated strongly with the five targets. The detailed results of molecular docking is seen in p = 7.19e–5), indicating that it may inhibit the development and progress of hepatoma. The overall survival analysis of the remaining four core genes with high and low expression displayed no statistical significance (p < 0.05)  .R software. The top five active Dahuang ingredients involved in degree analysis were aloe-emodin, beta-sitosterol, eupatin, toralactone, and (-)-catechin, while the top five core targets were ESR1, JUN, PIK3CG, PTGS2, and TGFB1. The combination of aloe-emodin and PIK3CG exhibited the best binding affinity (-9.07 kJ·mol–1). Besides, ESR1 was found to inhibit the development and progress of hepatoma.In recent years, with advancements in traditional Chinese medicine and the continuous improvement of the technical field, traditional Chinese medicine has reached new heights. Traditional Chinese medicine exhibits various unique properties like varied active substances, multiple key targets, and low toxicity . More imst century proved the antihepatoma effect of aloe-emodin [50) values were 6.85 ± 0.61 μg/mL and 8.71 ± 0.21 μg/mL, respectively (p < 0.01). Moreover, beta-sitosterol exhibited cytotoxic activity by inducing apoptosis and activating caspase-3 and caspase-9 in these cells. These findings suggested that beta-sitosterol may be developed into a promising therapeutic agent for treating hepatoma. Eupatilin, a lipophilic flavonoid, is an agonist of PPARα and has antiapoptotic, antioxidant, and anti-inflammatory effects [Cassia obtusifolia. It mediates hepatoprotection via an Nrf2-dependent antioxidative mechanism. However, its anticancer action is not known [Dahuang contains five main active ingredients: aloe-emodin, beta-sitosterol, eupatin, toralactone, and (-)-catechin. Aloe-emodin  is an anthraquinone found in traditional medicinal plants such as Dahuang. The early studies of the 21e-emodin . Aloe-eme-emodin . These fe-emodin . Beta-si effects . Presentot known . Catechiot known –52. The ot known , 54.The drug-ingredient-target-disease network and degree ranking identified five core genes that included ESR1, JUN, PIK3CG, PTGS2, and TGFB1. These genes play an essential role in tumor development, particularly in the process of proliferation, migration, and apoptosis. The network analysis and survival analysis results identified ESR1 as an essential protective factor in hepatoma treatment. There was no significant difference in the survival analysis of other proteins in hepatoma patients. ESR1 has also been reported as a candidate tumor suppressor gene for hepatoma . The redThe GO functional enrichment analysis inferred some significant biological processes, cellular compositions, and intermolecular functions in the core targets . The KEGThe present study was based on public databases, which had restricted information, and needed continuous improvement. Besides, the study also ignored the clinical use of Dahuang, such as drug dose, medication frequency, and administration method. However, this study is also worthy of further exploration and verification to extract the intricate molecular mechanisms governing the therapeutic targets for hepatoma therapy.Based on the network pharmacology, molecular docking, and survival analysis, our study results predicted the critical active ingredients and core targets of Dahuang. Further, it explored the potential targets of hepatoma to design effective drug therapies for the future. The GO and KEGG enrichment analysis revealed that the possible working mechanism of Dahuang against hepatoma mainly focused on positive regulation of protein phosphorylation, regulation of kinase activity, rhythmic process, and cellular response to external stimulus. Moreover, a few important observations were drawn. Pathways in cancer and its related pathways are the main pathways of Dahuang against hepatoma. ESR1 is the potential key gene that is beneficial for the survival of hepatoma patients. Also, aloe-emodin and beta-sitosterol are the two main active ingredients crucial for hepatoma treatment. Nevertheless, this study needs further experimental verification to determine the exact situation of Dahuang and its active ingredients in the treatment of hepatoma. Later, the current team might conduct a pharmacological study on critical signaling pathways and targets of Dahuang against hepatoma to verify it.


The programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) signaling pathway is significantly upregulated in influenza virus infection, which impairs the antiviral response. Blocking this signaling pathway may reduce the damage, lower the virus titer in lung tissue, and alleviate the symptoms of infection to promote recovery. In addition to the enhanced viral immune response, using of immune checkpoint inhibitors in influenza virus infection is controversial, the aim of this study was to identify the key factors and regulatory mechanisms in the PD-1 checkpoint blockade response microenvironment in influenza infection.A BALB/c mouse model of influenza A/PR8(H1N1) infection was established then constructed, and whole-transcriptome sequencing including mRNAs, miRNAs (microRNAs), lncRNAs (long noncoding RNAs), and circRNAs (circular RNAs) of mice treated with PD-1 checkpoint blockade by antibody treatment and IgG2a isotype control before infection with A/PR8(H1N1) were performed. Subsequently, the differential expression of transcripts between these two groups was analyzed, followed by functional interaction prediction analysis to investigate gene-regulatory circuits.In total, 84 differentially expressed dif-mRNAs, 36 dif-miRNAs, 90 dif-lncRNAs and 22 dif-circRNAs were found in PD-1 antagonist treated A/PR8(H1N1) influenza-infected lungs compared with the controls (IgG2a isotype control treated before infection). In spleens between the above two groups, 45 dif-mRNAs, 36 dif-miRNAs, 57 dif-lncRNAs, and 24 dif-circRNAs were identified. Direct function enrichment analysis of dif-mRNAs and dif-miRNAs showed that these genes were mainly involved in myocardial damage related to viral infection, mitogen activated protein kinase (MAPK) signaling pathways, RAP1 (Ras-related protein 1) signaling pathway, and Axon guidance. Finally, 595 interaction pairs were obtained for the lungs and 462 interaction pairs for the spleens were obtained in the competing endogenous RNA (ceRNA) complex network, in which the downregulated mmu-miR-7043-3p and Vps39–204 were enriched significantly in PD-1 checkpoint blockade treated A/PR8(H1N1) infection group.The present study provided a basis for the identification of potential pathways and hub genes that might be involved in the PD-1 checkpoint blockade response microenvironment in influenza infection.The online version contains supplementary material available at 10.1186/s12863-022-01081-7. Programmed cell death 1 (PD-1) is a negative checkpoint molecule that downregulates T cell activity after binding with its ligand, PD-1 ligand 1 (PD-L1). In chronic infections or tumors, PD-1 overexpression after lasting antigen-exposure will impair clearance of the pathogens or degenerate cells , 3. The + T cells [Influenza virus, especially influenza A virus (IAV) infection, is a huge challenge to global public health, which, because of its high morbidity and mortality, and extremely high antigen mutation rate, has the possibility of causing epidemic outbreaks and even human-to-human transmission . Severe  T cells .+ cell migration to the lung, which plays an important role in maintaining immune homeostasis [The airway epithelium is the first barrier against influenza infection, which participates in host defense by producing cytokines and chemokines, and by regulating expression of surfactant proteins and adapter molecules. Experiments have confirmed that influenza virus infection can induce PD-1/PD-L1 signal overexpression and PD-1eostasis , 10. Theeostasis  and upreUsing of immune checkpoint inhibitors in IAV infection is controversial, in addition to the enhanced viral immune response, it is not the whole picture, some researchers concern role of PD-1/PD-L1 pathway in developing autoimmune dilated cardiomyopathy with production of high-titer autoantibodies against cardiac troponin I after infection , some wo6 median tissue culture infectious dose (TCID50) infective dose). 2. PD-1 antagonist followed with A/PR8(H1N1) infection group. The PD-1 antagonist comprised an antibody against PD-1 , which was administered via tail vein injection in 200 μg doses on days 1, 4, and 7 before infection. An antibody against IgG2a  was used as the isotype control. Mice were chemically restrained with 2,2,2-tribromoethanol (avertin) before intranasal challenge with 50 μL of 106 TCID50 virus diluted in phosphate-buffered saline (PBS) [BALB/c mice (6 to 7 weeks old) were purchased from Joint Ventures SIPPER-BK Experimental Animal Co. . All animals were bred and maintained in specific pathogen-free conditions in accordance with the Care and Use of Laboratory Animals of Zhejiang Province and were approved by the local Ethics Committee. Six mice were divided into two groups: 1. The isotype control followed by A/PR8(H1N1) infection group  . Mice we40–60 mg of lungs and spleens were homogenized by grinding in liquid nitrogen, and filled with TRIzol® reagent. After adding chloroform, the tubes were shaked vigorously for 15 s then incubated for 2–3 min. After centrifugation, the upper layer was transferred and added with isopropanol, and then centrifuged precipitate was washed with 75% alcohol. The RNA was dissolved in RNase-free water.A total of 3 μg RNA per sample was used as input material, and sequencing libraries were generated using an NEB Next®Multiplex Small RNA Library Prep Set . Briefly, the NEB 3′ SR Adaptor was ligated to the 3′ end of microRNAs (miRNA), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs), then the SR RT Primer hybridized to the excess of 3′ SR Adaptor and transformed the single-stranded DNA adaptor into a double-stranded DNA molecule. PCR amplification was performed, and then the amplicons were purified. DNA fragments corresponding to 140 ~ 160 bp were recovered and dissolved. Finally, library quality was assessed on an Agilent Bioanalyzer 2100 system  using DNA High Sensitivity Chips.The clustering of samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS . After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500/2000 platform and 50 bp single-end reads were generated.http://www.mirbase.org/ftp.shtml) to look for families; novel miRNA precursors were submitted to Rfam (http://rfam.sanger.ac.uk/search/) to look for Rfam families. Predicting the target genes of the miRNAs was performed using miRanda v1.0b. Differential expression analysis was performed using the DESeq R package v1.8.3 with a P-value of 0.05 set as the threshold. The P-values was adjusted using the Benjamini & Hochberg method.As described before , mapped Gene Ontology (GO) enrichment analysis was used on the target gene candidates of the differentially expressed miRNAs. GOseq based Wallenius non-central hyper-geometric distribution which could adjust for gene length bias. GO enrichment analysis was implemented by the clusterProfiler R package v4.0 . We usedA total of 3 μg RNA per sample was used as input material to construct sequencing libraries, which were generated using the rRNA-depleted RNA by NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina®. The clustering of samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina), the libraries were sequenced on an Illumina Hiseq 4000 platform and 150 bp paired-end reads were generated.As described before , For lncAll the transcripts were merged using Cuffmerge software. lncRNA and mRNAs were then identified from the assembled transcripts following four steps: (1) Removal of transcripts with uncertain chain directions; (2) Filtering the transcripts ≥200 bp and ≥ 2 exons; (3) Known mRNA and known lncRNA were identified by comparing the assembled transcripts with the reference genome GTF. (4) Filtering the transcripts with protein-coding capability using CNCI, Pfam and CPC2 database as Novel mRNA, filtering the transcripts without protein-coding capability using CNCI, Pfam and CPC2 database as Novel lncRNA.(FPKM + 1) values and clustered. Transcripts with P < 0.05 were assigned as differentially expressed. GO enrichment analysis and KEGG pathway enrichment analysis were performed as above.Quantification of the transcripts was performed using StringTie software and Fragments Per Kilobase of transcript per Million mapped reads (FPKM) was obtained. EdgeR was used for differential expression analysis. All the transcripts were merged using Cuff merge software. Using hierarchical clustering method, lncRNA and mRNA are converted to log10 Approximately 10 differentially expressed transcripts identified among the two groups of samples were verified by qRT-PCR using three biological replicates. Maize tubulin was used as an internal reference gene. The relative expression levels of each gene were calculated using the 2-∆∆CT method.P = 0.006). The viral load and pathological damage of lung tissue 6 day post infection are depicted in Supplementary Figs. We observed lethargy, ruffled fur, and loss of appetite, but no death in any IAV-challenged mice, the degree of weight loss of these two groups with 8 mice per group were shown in Fig. In the differential expression analysis of lungs Fig. , 85 diffIn the spleen data, 45 dif-mRNAs were obtained, of which 18 were upregulated and 27 were downregulated, functional genes in PD-1 antagonist treatment followed by A/PR8(H1N1) infection group included CAMP, Ltf, Ly6g, Bola1  et al.; 36 dif-miRNAs were identified, of which 19 were upregulated and 17 were downregulated; 57 dif-lncRNAs were obtained, including 22 upregulated and 35 downregulated; and 24 dif-circRNAs were found, of which 18 were upregulated and 6 were downregulated.P-value was set < 0.05, the dif-mRNAs were enriched in 11 pathways in lungs and 6 pathways in spleens. Dif-miRNAs were enriched in 11 pathways in lungs and 26 pathways in spleens. There was little degree of overlap of dif-mRNAs and dif-miRNAs in lungs between the most enriched clusters. The most enriched clusters of dif-mRNAs of lungs were related to muscle and heart biological behavior. More than 85% of the dif-miRNAs enriched clusters in lungs and spleens overlapped with each other, including localization, metabolic process, positive regulation of metabolic process, and regulation of molecular function in the biological process category; intracellular part, cytoplasm, intracellular, and membrane-bounded organelle in the cellular component category; and protein binding, enzyme binding, and molecular function regulator in the molecular function category.KEGG and GO analyses were used to investigate the functional associations of gene expression changes. Targeted genes of dif-mRNA and dif-miRNAs of lungs and spleens of the two groups: PD-1 antagonist followed with A/PR8(H1N1) infection group vs. Isotype control followed with A/PR8(H1N1) infection were predicted Figs.  and 4. TFunctional Enrichment analysis of mRNAs and miRNAs in lungs and spleens obtained from IAV infection mice treated with anti-PD-1 antibody clearly highlighted myocardial damage related to viral infection, mitogen-associated protein kinase (MAPK) signaling pathways, RAP1 (Ras-related protein 1) signaling pathway, and Axon guidance.The top 20 pathways and GO terms , CC (cellular component), and MF (molecular function)) enriched by 45 dif-mRNAs and 36 dif-miRNAs of spleens of the following groups: PD-1 antagonist treatment followed by A/PR8(H1N1) infection group vs. isotype control followed by A/PR8(H1N1) infection group. (A) Top 20 pathways enriched by dif-mRNAs (B) Top 20 pathways enriched by dif-miRNAs (C) Top 20 GO terms enriched by dif-mRNAs (D) Top 20 GO terms enriched by dif-miRNAs.P-value was set < 0.05, the dif- lncRNA target genes were enriched in 9 pathways in lungs and 12 pathways in spleens. The dif- circRNA target genes were enriched in 7 pathways in lungs and 4 pathways in spleens. There was a little degree of overlap of lncRNAs and circRNAs in lungs and spleens between the most enriched clusters except for Hypertrophic cardiomyopathy, MAPK signaling pathway, and the AMP-activated protein kinase (AMPK) signaling pathway.KEGG and GO analysis was performed for dif-lncRNA and dif-circRNA-related target genes Figs.  and 6. PTop 20 pathways and GO terms , CC (cellular component), and MF (molecular function)) enriched by 90 dif- lncRNAs and 22 dif-circRNAs of the lungs (A) Top 20 pathways enriched by dif-lncRNAs. (B) Top 20 pathways enriched by dif-circRNAs (C) Top 20 GO terms enriched by dif-lncRNAs (D) Top 20 GO terms enriched by dif-circRNAs.Top 20 pathways and GO terms , CC (cellular component), and MF (molecular function)) enriched by 57 dif- lncRNAs and 24 dif-circRNAs of spleens (A) Top 20 pathways enriched by dif-lncRNAs. (B) Top 20 pathways enriched by dif-circRNAs. (C) Top 20 GO terms enriched by dif-lncRNAs (D) Top 20 GO terms enriched by dif-circRNAs.According to the dif-lncRNA–dif-miRNA pairs and dif-miRNA–dif-mRNA pairs, differentially expressed lncRNAs and mRNAs regulated by the same miRNA were screened. In total, 77 lncRNA-miRNA-mRNA interactions in lungs were finally obtained  , 22, hepIn this study, by applying whole-transcriptome sequencing, we identified 84 dif-mRNAs, 36 dif-miRNAs, 90 dif-lncRNAs, and 22 dif-circRNAs in PD-1 antagonist treated A/PR8(H1N1) influenza infected lungs compared with those in the controls (IgG2a isotype control treated before infection). In the comparison between the spleen samples from the above two groups, 45 dif-mRNAs, 36 dif-miRNAs, 57 dif-lncRNAs, and 24 dif-circRNAs were identified. Direct functional enrichment analysis on the dif-mRNAs and dif-miRNAs showed that these genes were mainly involved in myocardial damage related to viral infection, MAPK signaling pathways, the RAP1 signaling pathway, and Axon guidance.Functional Enrichment analysis of mRNA and miRNA in lung and spleen clearly highlighted myocardial damage related to viral infection. Influenza virus is an etiological agent of myocarditis, and the relationship between acute respiratory virus infection, especially influenza, and associated viral myocardial damage is greatly underestimated. Many studies have reported that influenza virus infection, especially severe infection, causes fatal myocarditis in humans and experimental animals. Acute cardiovascular events even death triggered by influenza was first noted as early as the 1930s. Several studies have confirmed that acute respiratory infections or influenza-like illnesses were closely related to subsequent acute cardiovascular events , 27, virThe MAPK signaling pathway plays an important role in regulating cell proliferation, differentiation, invasion, metastasis, and death through phosphorylation activation. The relationship between MAPK signaling pathways and anti-PD-1 antibody in infectious disease has been discussed elsewhere, especially in chronic infection. MAPK activation is an important initiating event in the upregulation of PD-1 in HIV-1-infected cells, and inhibition of this signaling pathway can reduce infection . The HA Additionally, we identified the significant role of downregulated mmu-miR-7043-3p and Vps39–204 in the ceRNA network. Decreased expression of mmu-miR-7043-3p was proven to be one of remarkable miRNA signatures of myocardial reductive stress, which is associated with cardiac hypertrophy . Future In conclusion, this study explored the molecular mechanism of the PD-1 checkpoint blockade response microenvironment during influenza infection. Upregulated PD-1/PD-L1 expression-induced by IAV infection is an important component of the immunosuppressive microenvironment, and blocking this signaling pathway will regulate the following signal pathways: Myocardial damage related to viral infection, MAPK signaling pathways, Rap1 signaling pathway, and Axon guidance. Downregulated mmu-miR-7043-3p and Vps39–204 were most significantly enriched by PD-1 blockade. However, this study was limited by a small sample size and limited time points to provide a comprehensive overview of the PD-1 checkpoint response microenvironment. Further in vivo validation using a larger scale animal experiment and dynamic functional characterization are needed to delineate the exact mechanistic details.Additional file 1: Supplementary Figure 1. Viral titers of the lungs 6 day post infection. Viral titers of the lungs 6 days after wildtype IAV challenge (each group had 8 mice). Viral replication in the lungs of IAV-challenged BALB/c mice was determined using the TCID50 method in MDCK cells. Viral titers were expressed as the means ± SE of the log10 TCID50 per gram of tissue. ***P < 0.0001.Additional file 2: Supplementary Figure 2. The lung histopathology of mice after virus infection. The lung histopathology of mice treated with or without a PD-1 antagonist and subsequently intranasally inoculated with 106 TCID50 A/PR8 at 6 day post infection. Multiple 4-μm-thick sections were stained with H&E. Original magnification: 200×. Three independent pathologists calculated the inflammatory pathology score of lung tissues according to the standard of Underwood, which including perivascular and peribronchiolar eosinophilia, edema and epithelial damage. Comparing A/PR8(H1N1) infection group with PD-1 antagonists treated A/PR8(H1N1) infection group, pathology scores were 10 ± 0.6 to 7.8 ± 0.4.Additional file 3: Supplementary Figure 3. The lncRNA-miRNA-mRNA network of the lungs. Circles represent upregulation and rectangles represent downregulation. mRNAs, miRNAs, and lncRNAs in the network are presented in yellow, orange, and green, respectively.Additional file 4: Supplementary Figure 4. The lncRNA-miRNA-mRNA network of the spleens. Circles represent upregulation and rectangles represent downregulation. mRNAs, miRNAs, and lncRNAs in the network are presented in yellow, orange, and green, respectively.Additional file 5: Supplementary Figure 5. The circRNA-miRNA-mRNA network of the lungs. Circles represent upregulation and rectangles represent downregulation. mRNAs, miRNAs, and circRNAs in the network are presented in yellow, orange, and green, respectively.Additional file 6: Supplementary Figure 6. The circRNA-miRNA-mRNA network of the spleens. Circles represent upregulation and rectangles represent downregulation. mRNAs, miRNAs, and circRNAs in the network are presented in yellow, orange, and green, respectively.


Purpose: Diabetic retinopathy (DR) is a serious complication of diabetes mellitus, which nearly happens to all the diabetic sufferers. This study aims to identify the preliminary molecular regulation involved in the therapeutic efficacy of astragaloside IV (AS- IV) for DR.Methods: Diabetic rat models were established and treated with AS-IV. Optical coherence tomography (OCT) and Hematoxylin-eosin (HE) staining was employed to demonstrate the histopathological changes. The main targets of AS-IV were identified by searching from public databases of traditional Chinese medicine . Besides, disease targets of DR were also obtained by integrated data from GEO datasets and predicted from public databases. Protein-protein interaction (PPI) network was constructed by Cytoscape with overlapping genes and 10 core targets were selected, on which Gene Ontology (GO) along with Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted. The interaction between AS-IV and these crucial genes were analyzed using molecular docking. RT-qPCR and western blot were used to verify the expression variation of core targets.Results: OCT imaging and HE staining demonstrated that AS-IV administration significantly increased retinal thickness in diabetic rats, obviously alleviating DR induced histopathological changes as well as elevated blood glucose levels. 107 common targets of AS-IV and DR were determined after intersection. PPI network analysis filtered 10 hub genes potentially targeted by AS-IV, including VEGFA, CASP3, HIF1α, STAT3, CTNNB1, SRC, AKT1, EGFR, IL1β and IL6. Enrichment analysis indicated that these genes were mainly enriched in biological processes like T cell activation, epithelial cell proliferation and protein kinase B signaling, and involved in oxidative stress, apoptosis and inflammation-related pathways. The molecular docking prediction suggested that AS-IV exhibited stable binding to these core targets. In addition, mRNA levels of core targets in diabetic rats were differentially expressed before and after AS-IV treatment. Western blot further revealed that AS-IV treatment elevated DR-depressed protein levels of PI3K and AKT.Conclusion: Our study elucidated the effect of AS-IV in attenuating retinopathy induced by diabetes in rats and preliminarily unveiled the therapeutic efficacy of AS-IV in the treatment of DR might be attributed to activation of PI3K-AKT signaling pathway. Diabetic retinopathy (DR) represents as the most common and serious ocular complication of diabetes, with approximately 103 million DR patients worldwide as of 2020 and an estimated number of 160 million DR patients in 2045 . DR causqi and yin. On the basis, people may develop the types of yin deficiency and fire, yin deficiency and stomach heat, yin deficiency in lung and kidney, yin deficiency in liver and kidney, yin and yang loss, etc.  has been used in the prevention and treatment of chronic diseases for nearly 2,000 years and has made indelible contributions. TCM believes that the pathogenesis of DR can be derived from deficiency of both ss, etc. . Althouguropathy . Astragaoperties . In addioperties , inhibitoperties , and alsoperties . Despitein vivo experiments were applied to verify the reliability of these crucial targets  rats (180–220 g) were purchased from Experimental Animal Center of Kunming Medical University. The rats were kept in a controlled room at 22 ± 2.0°C and 50% ± 5% humidity, with light exposure for about 12 h a day and free access to food and water. One week after adaptive feeding, rats were injected intraperitoneally with streptozotocin  dissolved in a freshly prepared sodiumcitrate buffer  so as to induce diabetes mellitus . Age-matRats were anesthetized with intraperitoneal injection of 3% phenobarbital (50 mg/kg). Compound topicamide eye drops were applied externally to dilate the pupil, and flufloxacin protected the cornea during and after surgery. Rats in the control group, diabetic group, and AS-IV treatment group were recorded using the Micron IV Retinal imaging camera system . Finally, the retinal thickness of OCT images was measured by ImageJ software.The blood glucose of rats was decreased on the 7th, 14th, 21st and 28th day after administration and the retinal thickness was increased on the 28th day after administration . Accordihttps://pubchem.ncbi.nlm.nih.gov/) (https://www.genecards.org/). Meanwhile, the two-dimensional molecules of AS-IV were uploaded into Swiss Target Prediction database (http://www.swisstargetprediction.ch/) as well as into PharmMapper database (http://lilab.ecust.edu.cn/pharmMapper/) respectively for target prediction   , the larediction , with spot.org/)  was usedhttp://www.ncbi.nlm.nih.gov/geo). The GSE12610 dataset contained 5 RNA samples extracted from the retinas of musculus including 3 diabetic samples (streptozotocin induced diabetes at 5 weeks) and 2 normal samples. In GSE28831 dataset, the retinas of Evans rats were extracted and microarray experiments were performed on days 7, 28, and 84 after STZ-induced diabetes. GPL20710 [MoGene-2_0-ST] Affymetrix Mouse Gene 2.0 ST array (mogene20st_Mm_ENTREZG_19) and GPL7294 (Agilent-014879 whole rat genome microarray 4x44KG4131F) were used to analyze datasets GSE12610 and GSE28831. Both datasets identified genes and pathways involved in DR formation. The R programming language was used for statistical analysis and graphing, and Pearson correlation analysis was performed to verify the repeatability of the data within each group. Heat maps were generated to visualize correlations among all samples from the same dataset.We searched the raw data of DR from the Comprehensive Gene Expression Database  (https://omim.org/) (https://www.pharmgkb.org/) (http://db.idrblab.net/ttd/) (https://www.uniprot.org/) (Microarray data linear module Bioconductor  identifids.org/) , OMIM daim.org/) , PharmGkkb.org/) , TTD datet/ttd/)  and Drugot.org/)  and GEO https://string-db.org/)   for inteMolecular docking is a computer structure-based method widely used in drug discovery, which can identify new compounds of therapeutic significance, predict ligand target interactions at molecular level, and depict the structure-activity relationship (SAR) in absence of chemical structure of other target regulators in advance . The higThe collected eyeballs were fixed with 4% paraformaldehyde for 1 h, and then dehydrated with sucrose solution. Subsequently, the eyeballs were embedded in the optimal cutting temperature compound medium  and then stored at −80°C fridge overnight. Retinal frozen sections were prepared at thickness of 10 μm using Leica microtome . These sections were subsequently treated with hematoxylin solution  for 1 min, after washes under running water, and they were continuingly stained by eosin solution  for 2–3 min. After sections dehydrated and transparentized, the fields near the optic nerve (200 μm) were captured for analysis.−ΔΔCt method.Total RNA was extracted from the harvested retinal tissues using the Trizol kit (Biosharp), and cDNA was reversely transcribed using reverse transcriptase kit (Promega). Later, amplification reaction was followed with 20 μL reaction system composed of 10 μL SYBR-Green qPCR Master Mix, 1 μL cDNA Template, 7.8 μL DEPC water and 0.6 μL each of forward and reverse primers. Primers sequences are shown in Retinal tissues were lysed with 100 μL of RIPA tissue lysates, and the supernatant was discarded after centrifugation  at 4°C for 15 min. Then, the protein concentration and purification were determined by BCA method. The protein extracts were separated with agarose gel electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were placed in solution containing 5% fat-free milk powder and blocked with gentle shaking at room temperature for 1 h. Afterwards, these membranes were incubated with primary polyclonal antibodies against p-PI3K and p-AKT  and β-actin monoclonal antibodies  overnight at 4°C. After washing three times, secondary antibodies  were added to incubate for 1 h. After washing, the protein bands were developed with ECL chemiluminescence substrate kit . β-actin was used as a loading control. Finally, densitometry analysis was performed using ImageJ software, and the relative expression levels were calculated by using the gray value of the target strip than the gray value of β -actin.p < 0.05 is considered statistically significant.All data in this study were processed by SPSS21.0 statistical software (IBM) or R programming. Measurement data were expressed by mean ± standard deviation p < 0.01).We firstly verified the effects of AS-IV treatment on retinal condition after DR by employing OCT on rats in the control group, the diabetic group and the treatment group. As shown in the representative images, detailed retinal layer and fundus photographs were observed in all groups, and no significant malformations were shown in any diabetic rats . Howeverth week prior to AS-IV treatment (p < 0.01). There was no significant change in blood glucose of rats in control group as time prolonged. After AS-IV treatment, blood glucose levels decreased a lot within the later 4 weeks, which were significantly lower than that of DM group (p < 0.01).The morphological and histopathological changes of rat retina after DR were observed by HE staining. The retinal surface in the control group was smooth. Each layer presents a clear and complete structure, and the cells in each layer are arranged orderly, evenly and densely . Howeverp < 0.05, logFC>1/logFC <-1), among which 178 down-regulated genes and 182 up-regulated genes were identified  identified 527 differentially expressed genes associated with DR. In addition, we integrated disease targets from DrugBank, GeneCards, OMIM, PharmGKB, and TTD databases, with 3 genes identified in DrugBank, 4079 genes identified in GeneCards, 85 genes predicted in OMIM, 218 genes in PharmGKB and 27 genes in TTD. Together, 4703 disease targets of DR were determined after integration and de-duplication . We matcYersinia infection, Hepatitis B, prostate cancer, Th17 cell differentiation, FoxO signaling pathway  enrichment analysis, 2096 items were obtained, of which the top 10 include T cell activation, epithelial cell proliferation, protein kinase B signaling, regulation of protein kinase B signaling, leukocyte cell-cell adhesion, peptidyl-serine phosphorylation, negative regulation of catabolic process, positive regulation of protein kinase B signaling, tissue remodeling, regulation of reactive oxygen species metabolic process . In the  pathway .In the present study, the possible interactions between 10 hub genes and AS-IV were investigated by molecular docking verification. In the process of molecular model construction, target genes were applied, VEGFA (PDB:1MKG), CASP3(PDB:1QX3), HIF1α (PDB:3HQU), STAT3 (PDB:6TLC), CTNNB1 (PDB:5IVN), SRC (PDB: 4U5J), AKT1 (PDB:5AAR), EGFR (PDB:3G5X), IL1β (PDB: 4X3A), IL6 (PDB:4O9H). Their interaction patterns with key amino acids and their binding at active sites are displayed in p < 0.05, p < 0.01, p < 0.001). On the contrary, AKT1 and CTNNB1 were depressed in diabetic rats, but AS-IV treatment markedly upregulated their expression in DR rats (p < 0.001). These results suggest that the expression of AKT1, CASP3, HIF1α, VEGFA, CTNNB1, IL6, IL1β, SRC and STAT3 might play important roles in the treatment of DR with AS-IV.RT-qPCR was used to detect mRNA expression changes of core targets identified above in DR rats with AS-IV treatment. Among these genes, levels of CASP3, HIF1α, VEGFA, IL6, IL1β, SRC and STAT3 were significantly elevated after DR insult, which were reversed after AS-IV treatment (p < 0.01). After AS-IV treatment, their levels exhibited prominent increase relative to that in DM group (p < 0.01).AKT is a key downstream signal protein of PI3K, and PI3K/AKT pathway is an important signaling pathway involved in the progression and pathogenesis of DR . Thus weAt present, the pathogenesis of DR is complex and remains obscure. This paper mainly combined network pharmacology analysis with experimental verification to explore the molecular mechanism of AS-Ⅳ in the treatment of DR. Our results uncovered that AS-Ⅳ can protect retinal cells against diabetes induced retinopathy, which may act on target proteins AKT1, CASP3 and HIF1α, VEGFA, IL6, IL1β, SRC and CTNNB1, and might be involved in the regulation of PI3K-AKT signaling pathway.Astragalus membranaceus with a wide range of pharmacological effects, including anti-inflammatory and anti-tumor functions  is a bioactive saponin extract of unctions . A recenunctions . In anotunctions . AS-IV hunctions . Consistin vivo. VEGF signaling pathway induces activation of PKC and AKT1, promotes endothelial cell proliferation, and increases neovascularization permeability, which is associated with retinal exudation and macular edema in diabetic retinopathy  stood out after more precise filtering. RT-qPCR validation of 10 core targets revealed that AKT1, CASP3, HIF1α, VEGFA, IL6, IL1β, SRC and CTNNB1 were modulated in the process of AS-IV treatment in DR, suggesting that AS-IV can be used a potential drug to treat DR through multi targets. It has been previously reported that in proliferative diabetic retinopathy, plasma and vitreous VEGFA levels are high , and VEGinopathy . In addiinopathy , and speinopathy . For micinopathy . Reducedinopathy . Activatinopathy . In hypeinopathy . In addiinopathy , which s−/− mice showed significantly lower levels of superoxide production in the retina in response to angiotensin II stimulation than wild-type mice, suggesting a link between IL-6 and oxidative stress and inflammation in retinal pathology (Mounting evidences indicate that immune mechanism involves in the pathogenesis of DR . AKT is athology . IL-1 β athology . Many ofathology .Given current knowledge of these core targets, it can be inferred that AS-IV protects animals against DR through oxidation resistance, anti-inflammation, anti-apoptosis, inhibition of new blood vessel formation and vascular leakage. Meanwhile, the results of GO enrichment analysis on key targets, showed that the regulation of various kinases activity, insulin response and vascular endothelial cells play a major role in the AS-IV therapy of DR process. The enriched KEGG pathways of these crucial genes mainly include PI3K-AKT signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and EGFR tyrosine kinase inhibitor resistance, suggesting that AS-IV may play an anti-inflammatory role by activating PI3K/AKT signaling pathway. Oxidative stress and inflammation may play an important role in delaying the progression of DR . PI3K/AKCollectively, this study identified core targets underlying the therapeutic effects of AS-IV in DR based on comprehensive network pharmacology, and preliminarily unveiled the therapeutic effects of AS-IV might be achieved via activation PI3K/AKT pathway, providing a strong theoretical and experimental basis for the follow-up clinical application of AS-IV in the treatment of DR.


Pasteurella multocida (P. multocida) is an important zoonotic pathogen. In addition to lung lesions, necropsies have revealed macroscopic lesions in the heart in clinical cases. However, most previous studies focused on lung lesions while ignoring heart lesions. Therefore, to investigate the immune response of the P. multocida-infected heart, two murine infection models were established by using P. multocida serotype A (Pm HN02) and D (Pm HN01) strains. Histopathological examination revealed heterogeneous inflammatory responses, including immune cell infiltration in the epicardial and myocardial areas of the heart. Transcriptome sequencing was performed on infected cardiac tissues. To explore the traits of immune responses, we performed the functional enrichment analysis of differentially expressed genes, gene set enrichment analysis and gene set variation analysis. The results showed that the innate immune pathways were significantly regulated in both groups, including the NOD-like receptor signaling pathway, the complement and coagulation cascade and cytokine–cytokine receptor interaction. The Toll-like receptor signaling pathway was only significantly activated in the Pm HN02 group. For the Pm HN02 group, immunohistochemistry analysis further verified the significant upregulation of the hub component MyD88 at the protein level. In conclusion, this study reveals critical pathways for host heart recognition and defense against P. multocida serotypes A and D. Moreover, MyD88 was upregulated by P. multocida serotype A in the heart, providing a theoretical basis for future prevention, diagnosis and treatment research. Pasteurella multocida (P. multocida) .Based on the transcriptome data, ten immune- and inflammation-related DEGs were selected for RT-qPCR verification. Specific primers were designed according to the reference sequences in Genebank with Primer (Version 5.0), and the primer sequences are listed in n = 6) sections. The primary antibodies against MyD88  were diluted 1:400 and then incubated at 4 °C overnight in a humidified container. After three washes with PBS, we added horseradish peroxidase (HRP)-labeled Goat Anti-Rabbit IgG , which was diluted 1:200 and incubated at room temperature for 50 min. After three more washings with PBS, a chromogenic solution was added, and chromogenesis was terminated with tap water. Nuclei were then stained with hematoxylin. Finally, the slices were dehydrated and sealed, and the results were interpreted under a white-light microscope. Twelve fields of view were randomly selected from 400-fold fields (Immunohistochemistry staining was conducted using 4 μm paraffin-embedded heart tissue (d fields . The quat-test in GraphPad Prism (Version 8.0).All data are depicted as means ± SEM, and error bars in graphs indicate SEM. Results are representative of three independent experiments. Statistical significance was analyzed using using two-tailed unpaired Student’s P. multocida infection on the murine heart. In the control group, cardiomyocytes were neatly arranged  signaling pathway, followed by the chemokine signaling pathway C. The unGSEA is a powerful analytical method that focuses on gene sets, which share common biological functions, chromosomal locations or regulatory mechanisms. The results of GSEA A,B reveaGSVA is a method that builds on GSEA and is better than GSEA for characterizing pathways from the data obtained. GSVA can be used to compare differences in pathways between control and infected groups to better determine the activity of each pathway. According to the results of GSVA, the pathways shared by the Pm HN01 and Pm HN02 groups were the NOD-like receptor signaling pathway, the complement and coagulation cascade and cytokine–cytokine receptor interaction. Although there was no marked activation of the Toll-like receptor signaling pathway in the Pm HN01 group, it was significantly activated in the Pm HN02 group A,B.Junb, Iigp1, Gadd45g, C3 and Il4ra) in common pathways, including the TNF and MAPK signaling pathway, the complement and coagulation cascade and cytokine–cytokine receptor interaction, were verified by RT-qPCR to check the accuracy of the transcriptome sequencing results. RT-qPCR verification demonstrated consistent results with RNA-seq (Myd88) and other related DEGs in the pathway to determine their expression trends at the transcriptional level. All of the selected genes showed upregulation in RT-qPCR and RNA-seq. Moreover, the MyD88 signal was detected in the cytoplasm by immunohistochemistry  was significantly upregulated at the genetic level. Additionally, histopathological examination showed myocardial infarction, which was supported by the transcriptome data. Cardiomyocyte marker genes  were significantly downregulated. Furthermore, the functional investigation indicated that NF-κB inhibitor alpha (Nfkbia) [Nfkbia gene was significantly increased. Hence, myocardial infarction may involve cardiomyocyte apoptosis [P. multocida-infected murine model. We next investigated key genes and their related pathways.In bacterial myocarditis, numerous immune cells play critical roles , such as(Nfkbia)  inhibitepoptosis . ThroughNod2 is low, which is consistent with our control group results. However, after infection with Pm HN01 and Pm HN02, the expression of Nod2 was significantly upregulated, which may be attributed to the PGN fragment in the P. multocida periplasmic space [NLRC5, a member of the NLR family, is a negative regulator of the inflammatory pathway. Its expression level was significantly upregulated after LPS stimulation [Nlrc5 was significantly upregulated after PmHN01 and PmHN02 infection in our study, which indicated that the body inhibited the inflammatory response to P. multocida infection by enhancing the expression of Nlrc5. In summary, during infection with Pm HN01 and Pm HN02, genes related to immune inflammation, such as Nod2 and Nlrc5, were generally upregulated and significantly activated the NOD-like receptor signaling pathway. Taken together, the NOD-like receptor signaling pathway is essential for the recognition of Pm HN01 and Pm HN02.As the first line of host defense against bacterial infections , the innic space . NLRC5, mulation . The expP. multocida infection. Moreover, we detected that the hub gene (C3) of the complement and coagulation cascade was upregulated. Thus, these results further indicate that P. multocida activated the complement and coagulation cascade. Inflammation stimulates the high expression of various pro-inflammatory mediators and cytokines in the tissue, which is consistent with the activation of cytokine–cytokine receptor interactions in this study. The increased expression of a cytokine (Il-1β) and cytokine receptor (Il4ra) was validated by RT-qPCR analysis. According to the histopathological examination, inflammatory cell infiltrates were scattered in the myocardium. IL-1β is the central mediator of inflammation [Il-1β, Il4ra, Il-6, Myd88, Nek7 and Nlrp3 were significantly upregulated and significantly activated complement and coagulation and cytokine–cytokine receptor interaction, suggesting that they are crucial for host defense mechanisms to both Pm HN01 and Pm HN02.As downstream pathways of PRRs, complement and coagulation and cytokine–cytokine receptor interaction were significantly activated in this study. Complement and coagulation are pivotal factors contributing to inflammation  and the ammation  and is cP. multocida [Tlr2 and Tlr4 were significantly upregulated after Pm HN01 and Pm HN02 infection, which was consistent with previous test results of P. multocida infection [Myd88  was verified at both the transcription and protein levels. It has been suggested that Pasteurella multocida toxin-induced G protein signaling regulates TLR4-mediated immune responses [Toll-like receptors (TLRs) are transmembrane signal transduction receptors, which activate the innate immune response of the host through PAMPs, thus eliminating pathogens and playing an essential role in immune defense . This stultocida . They arultocida . LPS triultocida . In thisnfection ,47. The nfection . In addinfection  through nfection , which pnfection . Moreoveesponses . Given tP. multocida serotypes A and D. In addition, the significant activation of the NOD-like receptor signaling pathway suggests the importance for the heart recognition of P. multocida. As downstream pathways of PRRs, the significantly activated complement and coagulation cascade and cytokine–cytokine receptor interaction were crucial for the heart innate immune response during P. multocida infection. Moreover, P. multocida serotype A significantly activated the Toll-like receptor signaling pathway and upregulated the expression of MyD88 in the heart. These signaling pathways can be used as prognostic and diagnostic markers. Additionally, signaling pathway inhibitors or activators can be used as therapeutic targets. Together, these findings provide a molecular basis for the prevention and control of Pasteurellosis.To our knowledge, this study is the first to reveal vital pathways by which the heart recognizes and defends against


Background: Cordyceps is a precious Chinese herbal medicine with rich bio-active ingredients and is used for regulating arrhythmia alongside routine treatments. However, the efficacy and potential mechanisms of Cordyceps on patients with arrhythmia remain unclear.Methods: Randomized controlled trials of bradycardia treatment with Cordyceps were retrieved from diverse databases and available data. Dichotomous variables were expressed as a risk ratio (RR) with a 95% confidence interval (CI). Continuous variables were expressed as a standardized mean difference (SMD) with a 95% CI. Network pharmacology was used to identify potential targets of Cordyceps for arrhythmia. Metascape was used for gene ontology (GO) and genome (KEGG) pathway enrichment analysis.Results: Nineteen trials included 1,805 patients with arrhythmia, of whom 918 were treated with Ningxinbao capsule plus routine drugs, and, as a control, 887 were treated with only routine drugs. Six trials reported on bradycardia and the other 13 on tachycardia. Treatment with Cordyceps significantly improved the total efficacy rate in both bradycardia  and tachycardia . Cordyceps also had beneficial secondary outcomes. No serious adverse events occurred in patients treated with Cordyceps. The results of KEGG pathway enrichment analysis were mainly connected to adrenergic signaling in cardiomyocytes and the PI3K-Akt signaling pathway. IL6, TNF, TP53, CASP3, CTNNB1, EGF, and NOS3 might be key targets for Cordyceps in the treatment of arrhythmia.Conclusion: This study confirmed that Cordyceps has a certain positive effect on the treatment of arrhythmia and that its main mechanism may be through the regulation of adrenergic signaling in cardiomyocytes and the PI3K-Akt signaling pathway. Cardiovascular disease takes the lives of 17.7 million people every year, accounting for 31% of all global deaths, and this number will possibly rise to 23.6 million by 2030 . CardiacIn recent years, with the development of evidence-based studies, the effects of traditional Chinese medicine (TCM) have been further confirmed and valued. Chinese herbs and herbal formulas have been used for arrhythmia since the 1970s . Herbal http://www.clinicaltrialresults.org, http://www.clinicaltrials.gov, and http://www.theheart.org), were also considered, covering the period from the earliest possible date up to December 30, 2021. Bradyarrhythmia, which is basically a heart rate below 60 beats per minute, can be due to sinus, atrial or junctional bradycardia, or problems with the conduction system, e.g., sinus bradycardia, sinoatrial block, atrioventricular block, and others , the Wanfang Database, the China Scientific Journal Database (VIP), and the Chinese Biomedicine Literature Service System (SinoMed). Published review articles, editorials, and information from the internet (d others . Tachyard others . The seaThe inclusion criteria were as follows: 1) Study subjects were diagnosed as arrhythmic according to the corresponding guidelines. 2) All patients were randomized to receive treatment with Ningxinbao and contemporary medications, or contemporary medications only. 3) The sample size in each study group was ≥10 cases. 4) Follow-up in each study group was ≥2 weeks. 5) Quantitative measurements of surrogate endpoints and/or adverse cardiovascular events and/or adverse drug effects were available to facilitate outcome analysis.Exclusion criteria were as follows: 1) Studies were non-randomized or non-blinded. 2) Patients enrolled had no definite diagnosis. 3) Different TCM medications were compared. 4) Studies reported only symptomatic changes in patients without objective laboratory measurements. 5) Methodological quality was poor with a Jadad score of <2.p < 0.05. Chi-square and I2 tests were used to test the heterogeneity among trials. Moreover, the fail-safe number was used to estimate the extent of publication bias  was used to synthesize the data. Continuous variables were expressed as mean difference (MD) with a 95% confidence interval (CI), and dichotomous variables as odds ratio (OR) with a 95% CI. Statistical significance was denoted by a two-tailed ion bias .http://tcmspw.com/tcmsp.php), which is a system pharmacology platform designed for studying TCM comprehensively. Our condition was set with compounds OB ≥ 30%, DL ≥ 0.18, while some ingredients that did not meet the criteria, but had significant pharmacological activity were also selected for the next step. We retrieved the targets of tachyarrhythmia , as well as the targets of bradyarrhythmia  from GeneCards (https://www.genecards.org/), which are online servers for target identification. After gathering all the data, we took the intersections of Cordyceps and disease targets using the online drawing tool called Interactive Venn (http://www.interactivenn.net/).The ingredients of Cordyceps were collected from TCMSP (https://string-db.org/cgi/input.pl). The protein interaction network was downloaded from its “string_interactions.tsv” format file. For the list of names, we used common targets of Cordyceps with tachyarrhythmia and bradyarrhythmia. After finishing construction, we hid disconnected nodes in the network. Cytoscape 3.9 was used to build the protein-protein interaction network construction.The PPI network of the genes was constructed using STRING (http://www.metascape.org/). The function activities and the pathways were ranked by their nominal p values with a cut-off of 0.05.For the biological and functional annotation of the genes within each herb-disease-target network and PPI, gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using Metascape (https://pubchem.ncbi.nlm.nih.gov/), and its mechanical structure was optimized by Chem3D software. The Uniprot database (https://www.uniprot.org/) was searched, and the 3D structures regulating the candidate target proteins in the PDB database were downloaded (http://www.rcsb.org/). Autodock tools 1.5.6 (http://autodock.Scripps.Edu/resources/tools) were used for water removal and hydrogenation, for calculating Gasteiger charge, and storing targets and bioactive components as a “pdbqt” format file. The binding conformations were visualized by PyMOL 2.4.0 and Discovery Studio 2019. A flowchart of the network pharmacology study is given in The 2D structural information was downloaded from the PubChem database   and minimal heart rate , compared with the control groups  . Among t2 = 37%)  enrollinl groups .z < 0.00001), atrial arrhythmia , and multiple types of arrhythmias   .z = 0.19; I2 = 0%)  .z = 0.09), ventricular tachyarrhythmia , ventricular flutter , and ventricular fibrillation  (z < 0.00001), IL6 , and TNF  in the Ningxinbao capsule group were better than those in the control group  . One tri = 0.05)  reported = 0.05)  reportedol group .Six trials on bradyarrhythmia  reportedEight trials reported drug-related adverse reactions quantitatively , and a dCordyceps, tachyarrhythmia, and bradyarrhythmia were selected for network pharmacology analysis. In total, 18 main ingredients from Cordyceps were described in the TCMSP database, and 159 related targets were collected. The main ingredients of Cordyceps are listed in In order to speculate on the role of targets in the disease, overlapping bradyarrhythmia and tachyarrhythmia targets were submitted to a string platform to establish the PPI network. Targets and interacting edges were sorted in descending order by degree, and arranged in a concentric circle according to a degree, as shown in p values.GO functional analysis and KEGG pathway enrichment analysis were conducted to elucidate the biological effects on gene functions and signaling pathways of the related targets of Cordyceps in the treatment of arrhythmia. The top 10 GO items and top 20 KEGG pathways were selected based on the c from mitochondria, vasodilation, the adenylate cyclase-activating adrenergic receptor signaling pathway, regulation of the B cell apoptotic process, the adrenergic receptor signaling pathway, and regulation of macrophage differentiation. The cellular components were mainly related to caveolae, the transcription repressor complex, the plasma membrane raft, and the presynaptic active zone. The molecular function was mainly enriched in adrenergic receptor activity, R-SMAD binding, and G protein-coupled amine receptor activity. KEGG pathway results showed that the top 20 pathways were mainly enriched in the PI3K-Akt signaling pathway, adrenergic signaling in cardiomyocytes, fluid shear stress and atherosclerosis, the mTOR signaling pathway, and the Rap1 signaling pathway (For bradyarrhythmia, the biological processes were mainly concentrated in negative regulation of smooth muscle contraction, negative regulation of B cell proliferation, regulation of production of miRNAs involved in gene silencing by miRNA, positive regulation of pri-miRNA transcription by RNA polymerase II, the release of cytochrome  pathway .For tachyarrhythmia, the biological processes were mainly concentrated in the regulation of macrophage-derived foam cell differentiation, protein kinase B signaling, cellular extravasation, prostate gland development, astrocyte differentiation, regulation of cellular response to oxidative stress, regulation of immunoglobulin production, positive regulation of nuclear division, and positive regulation of phosphatidylinositol 3-kinase signaling. In terms of cellular components, the top 10 items were mainly related to platelet alpha granules, caveolae, the clathrin-coated endocytic vesicle membrane, and the plasma membrane raft. The molecular function was mainly enriched in protease binding, nuclear receptor activity, ligand-activated transcription factor activity, and SMAD binding. The KEGG pathway analysis revealed that common genes were mainly enriched in the PI3K-Akt signaling pathway, the AGE-RAGE signaling pathway in diabetic complications, fluid shear stress and atherosclerosis, the MAPK signaling pathway, and the FOXO signaling pathway .To confirm the valid bonding effects between ingredients of Cordyceps and key targets, molecular docking was performed by Autodock. The modes of 11 binding complexes are displayed in In this study, we investigated the efficacy, safety, and potential protective mechanism of Cordyceps in the treatment of arrhythmia. We found that Cordyceps could improve sinus node and atrioventricular conduction in patients with arrhythmia, without obvious adverse reactions. Its possible mechanism of protection is the modulation of adrenergic signaling in cardiomyocytes and the PI3K-Akt signaling pathway .According to previous studies, the petroleum ether extract of Cordyceps improves the arrhythmia caused by aconitine, prolongs the induction time of arrhythmia, and reduces the duration and severity of arrhythmia in rats. The extract of Cordyceps also has an antagonistic effect on arrhythmia induced by barium chloride. The mechanism of its anti-arrhythmic action might involve the regulation of the transport of ion channels in the cell membrane . In our Cordyceps contains a variety of chemical components, such as sterols amino acids, polysaccharides, organic acids, nucleosides, peptides, and trace elements. Caffeine plays an important role in preventing and treating arrhythmia. Previous studies have found that taking a certain amount of caffeine before aerobic exercise can delay the recovery of autonomic control of heart rate in young adults . Low dos2+ handling phenotype. In addition, intraperitoneal injection of IL-6 in rats increased susceptibility to atrial fibrillation (+/K+-ATPase activity and CASP3 protein expression (−/−mice. NOS3 may prevent arrhythmias by modulating Ca2+ transient amplitude (According to the analysis of the PPI network, IL6, TNF, TP53, CASP3, CTNNB1, EGF, and NOS3 may be the key targets of Cordyceps in the treatment of arrhythmia. Previous studies have found that acute administration of IL6 to isolated rat hearts could recapitulate part of the Caillation . TNF⁃α iillation . There iillation . It has pression . Cardiacpression . Previoumplitude . TP53 ismplitude . Experimmplitude .+, K+, and Ca2+ channels of the myocardial cell membrane, which leads to abnormal autonomic activity, triggering activity, and reentry excitement of the myocardium. Adrenergic receptors, as G protein-coupled receptors, play a key role in cardiac physiology in both health and disease. Previous investigations have indicated that cardiac adrenal receptors are directly involved in the control of intercellular electrical communication, which may be a key factor in maintaining normal intercellular conduction and the cardiac electrical network. This may in turn be related to the formation of arrhythmogenic substrates in some heart diseases (2+ release known to date. Stimulation of beta-adrenaline leading to Ca2+ release can induce arrhythmias (2+ signaling, thereby regulating heart rate (+ and Ca2+ currents, intracellular pH, and Ca2+ sensitivity of myofilaments (+ response in the hearts of aging guinea pigs, and thus inhibited those receptors in reducing the occurrence of arrhythmias (The main mechanism of arrhythmia is a change in the physiological characteristics of the Nadiseases . Adeninehythmias . The binhythmias . Enhanceart rate . It has ilaments . Beta-adhythmias .2+ through the cell membrane generates various electrical currents to stimulate action potential, determining the cardiac cycle and cardiac function (2+ overload-induced apoptosis and the PI3K-mediated GRK2/PLC-γ/IP3R signaling pathway to protect against myocardial injury (2+ entry into excitatory cells (2+ channel function CaVβ2 through mediated phosphorylation of CaVβ2 in all excitable cells, including cardiomyocytes, highlights the biological relevance of this mechanism. More importantly, phosphorylation of CaVβ2 is thought to affect calcium influx and myocardial contractility, which is closely associated with the mechanism of arrhythmia (2+ channels in cardiac myocytes. These studies confirmed that PI3Kα is the main regulator of Ca2+ current regulation in cardiomyocytes (2+, in addition to ptdIns-3,4, 5-P3, has been identified as part of a signaling pathway used to regulate the cytoplasmic calcium concentration, suggesting that the functional link between the PI3K-Akt pathway and the calcium signaling pathway may be of broad importance (The movement of Cafunction . Previoul injury . In cardry cells . In addirhythmia . Previoumyocytes . PI3K isportance . The PI3portance . The carportance .Our study presented a relatively strict illustration of the results. However, there were still some limitations. First, all trials included were published in Chinese and their sample sizes were small. Since all studies included were conducted in China, there might be a geographic bias, which limits generalization to the world. Second, all studies included reported positive results, which might reflect publication bias. Further, strictly designed multicenter RCTs are needed to assess the effectiveness and safety of TCM, especially long-term hard endpoints. Finally, in this study, the molecular mechanisms of Cordyceps were predicted only through network pharmacology, so further experiments are needed to confirm these mechanisms.Taken together, based on meta-analysis, network pharmacology, and molecular docking technology, the results of our study suggest that Cordyceps is effective in stabilizing heart rates of patients with arrhythmia. It is hypothesized that Cordyceps may play a role in the treatment of cardiac arrhythmias by modulating adrenergic signaling in cardiomyocytes and the PI3K-Akt signaling pathway. However, the effect of Cordyceps on long-term outcomes in these patients remains unknown. It is hoped that our study will provide more references for the treatment of arrhythmia.