Solanum surattense (S. surattense) is the herbal plant used in many regions of Asia to treat many disorders including various types of cancer. Previous in vitro studies revealed the medicinal importance of S. surattense against hepatocellular carcinoma. However, the exact molecular mechanism of S. surattense against HCC still remains unclear. In vitro and in silico experiments were performed to find the molecular mechanism of S. surattense against HCC. In this study, the network pharmacology approach was used, through which multi-targeted mechanisms of S. surattense were explored against HCC. Active ingredients and potential targets of S. surattense found in HCC were figured out. Furthermore, the molecular docking technique was employed for the validation of the successful activity of bioactive constituents against potential genes of HCC. The present study investigated the active “constituent–target–pathway” networks and determined the tumor necrosis factor (TNF), epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), Bcl-2-like protein 1(BCL2L1), estrogen receptor (ER), GTPase HRas, hypoxia-inducible factor 1-alpha (HIF1-α), Harvey Rat sarcoma virus, also known as transforming protein p21 (HRAS), and AKT Serine/Threonine Kinase 1 (AKT1), and found that the genes were influenced by active ingredients of S. surattense. In vitro analysis was also performed to check the anti-cancerous activity of S. surattense on human liver cells. The result showed that S. surattense appeared to act on HCC via modulating different molecular functions, many biological processes, and potential targets implicated in 11 different pathways. Furthermore, molecular docking was employed to validate the successful activity of the active compounds against potential targets. The results showed that quercetin was successfully docked to inhibit the potential targets of HCC. This study indicates that active constituents of S. surattense and their therapeutic targets are responsible for their pharmacological activities and possible molecular mechanisms for treating HCC. Lastly, it is concluded that active compounds of S. surattense act on potential genes along with their influencing pathways to give a network analysis in system pharmacology, which has a vital role in the development and utilization of drugs. The current study lays a framework for further experimental research and widens the clinical usage of S. surattense.Hepatocellular carcinoma (HCC) is one of the most common malignant liver tumors with high mortality. Chronic hepatitis B and C viruses, aflatoxins, and alcohol are among the most common causes of hepatocellular carcinoma. The limited reported data and multiple spectra of pathophysiological mechanisms of HCC make it a challenging task and a serious economic burden in health care management.  HCC, a complex malignant tumor, is lethal and ranks sixth in mortality worldwide. The prevalence of HCC is still increasing broadly and poses a significant threat to humans . This inHerbal medicine has its origin in ancient traditions and cultures. Herbal medicine involves plants’ involvement to enhance general health and cure disease. These herbal medicines are a rich source of potent ingredients and can be taken as pharmaceutical medications. Even now, many pharmaceutical drugs can be derived from naturally occurring compounds found in plants. Traditional Chinese medicines (TCM) have been practiced in China for thousands of years and are the most prominent complementary and alternative therapy ,7. TCM pS. surattense, family Solanaceae, genus Solanum, is a perennial herb, and it is widely considered the most valuable traditional remedy [S. surattense possesses anti-cancer properties [S. surattense against HCC is still unknown.Consequently, there is increased attention to the usage of herbal medications around the globe . S. sural remedy . After eoperties . BioactiNetwork pharmacology, the next paradigm in drug discovery, is a critical technique for evaluating in silico therapeutic interventions . This inS. surattense and their therapeutic targets responsible for their pharmacological activities and potential molecular mechanisms for the treatment of HCC.Interestingly, network pharmacology has been recognized as a potent and promising approach for developing herbal medicine against HCC. Compared to the western medicine approach based on a one-disease/one-drug methodology, drug discovery still faces various issues including efficacy, safety, and sustainability . This stS. surattense fruit extract at different concentrations (30 to 100 µg/mL) was explored in both liver hepatoma cells (HepG2 cells) and Vero cell lines (African green monkey’s kidney cells) in a dose-dependent manner. Among all of these concentrations of S. surattense fruit extract, including 60, 70, 80, 90, and 100 µg/mL, the results showed a less than 50% cell viability as compared to positive control doxorubicin. S. surattense fruit extract exhibited a non-toxic effect in Vero cells (S. surattense fruit extract (from 60 to 100 µg/mL concentration) is highly effective against liver cancer as analyzed in in vitro analysis with a resultant IC50 value of 62.70 ± 1.14 µg/mL.The toxicological activity of ro cells A,B. Our S. surattense, 20 ingredients were selected as they fulfilled the drug-likeness properties, molecular weight (MW < 500), logarithm of the partition coefficient (logP < 5.6), number of hydrogen bond acceptors (nHA ≤ 10), number of hydrogen bond donors (nHD ≤ 5), and the Lipinski rule of five. The three other compounds  were included in this study because their anti-cancer activity was checked in the lab. However, they did not meet the inclusion criteria -, (AKT1), ESR1, EGFR-43, TNF, HIF1A-, HRAS-, MTOR-41 and apoptosis regulator Bcl-X BCL2L1- showed the highest degree . The higS. surattense targets were discovered using GO annotation and pathway enrichment studies. According to GO enrichment analysis, the key targets were associated with oxidoreductase activity, peptidyl–serine modification, nuclear receptor activity, etc. Using KEGG pathway analysis, relevant signaling pathways related to S. surattense anti-cancer action were identified. GO enrichment and KEGG analysis reveal that hub genes were significantly enriched genes . A network analyzer was used to analyze the network; 34 nodes and 74 edges were found in the compound target pathway network . The 74 S. surattense were docked with the five potential targets of HCC. The 2D structures of these eight active compounds are shown in S. surattense have better binding interaction with HCC targets. For molecular docking, from the Protein Data Bank (PDB) database, MTOR (1AUE), EGFR (1IVO), BCL2L1 (1R2E), ESR1 (1UOM), HRAS (4XVR), HIF1A (5JWP), TNF (5MU8), and AKT1 were considered as the three-dimensional structures of the eight top targets to be selected. Auto Dock (V. 4.2) was employed to validate the best possible binding modes of the interactions. The ligand was supposed to be flexible during docking, whereas the protein was rigid. All eight active components from Calotropis gigantea (Asclepiadaceae) and Xanthium strumarium (Asteraceae) have anti-hepatocellular carcinoma and anti-proliferative (against HepG2 cancer cells) properties, respectively [HCC is among the most common health disorders that significantly impact the world population. According to GLOBOCAN, the highest occurrence rate of HCC has been reported in China, Southeast Asia, Eastern Africa, and Sub-Saharan Western Africa. The interlinked series of multiple pathological pathways comprehending the HCC further intensify the disease and make treating it more challenging . Despiteectively .The medicinal plant may have broad pharmacological actions with multiple targets and pathways composed of multiple components that play a vital role in treating HCC . NaturalS. surattense against HCC. Compounds with a high degree in the compound–target network may consider most S. surattense therapeutic effects on HCC.The network pharmacology (NP) approach, which associates biological systems with in silico technology, might predict potential targets and pathways underlying the anti-cancer effect of HCC. In this current study, we employed NP to reveal the pharmacological mechanism of active constituents in S. surattense, the identified compound with OB 30% and DL index v0.18, was regarded as a potentially bioactive compound because it is probably absorbed and distributed in the human body. In this context, the screening results revealed that glycoalkaloid, steroids, triterpenoids, solanine, and flavonoids were the main phytochemical bioactive compounds of S. surattense that proved potent in the development of HCC by affecting the TP53, GAPDH, AKT1, ESR1, EGFR, TNF, HIF1A, HRAS MTOR, and BCL2L1 gene targets. The PPI network analysis was performed to identify essential proteins associated with the anti-HCC effects of S. surattense, which were indicated by the PPI network’s critical nodes. The interactive nodes showed that S. surattense might interact with representative gene targets directly or indirectly to perform anti-cancer actions. These key targets were mainly involved in biological processes such as tumor cell proliferation, apoptosis, cell cycle control, epithelial–stromal transition, angiogenesis, tumor invasion and metastasis, tumor signal transmission, immunological modulation, drug resistance, and others [The NP approach could help us to screen the putative active ingredients and target ingredients. d others ,24,25. OS. surattense. Quercetin, a natural flavonoid, was the most potent compound that could suppress the growth of HCC cells employed in aerobic glycolysis, and suppress the development of HCC by lowering HK2 protein levels and inhibiting the AKT/mTOR pathway in HCC cells [This study said that quercetin, followed by solanine, was the vital key ingredient of CC cells . The solCC cells ,27,28.S. surattense may reduce HCC cell growth and induce cell death via acting on the PI3K-AKT signaling pathway, which represents the putative mechanism of action of HCC growth. Further studies confirmed that quercetin significantly reduced the expression of p-PI3K p85 (Tyr467), p-PI3K p55 (Tyr199), and p-Akt (Ser473) in HepG2. Quercetin, as a potent flavonoid component in S. surattense associated with an effective anti-cancer drug, may boost the expression of p53 which in turn downregulates the expression of HIF1A, resulting in the proapoptotic process of liver cancer [As shown in GO term and KEGG analysis using enrichment pathways, these findings, mainly linked with the GO results, suggested that r cancer .S. surattense may have inhibition in HCC via directly influencing cancer, metabolic, and immune-related pathways [S. surattense could inhibit the expression of AKT1 and phosphorylation of AKT1. It was predicted that S. surattense controls several targets and pathways in HCC cells based on our findings .The S. surattense extract preparation was performed following the maceration method [S. surattense was shade-dried at room temperature and ground with an electric grinder to obtain a coarse powder. Then, 50 g of S. surattense powder was soaked in 500 mL of 70% ethanol , vigorously agitated 2X/day for seven consecutive days, and filtered using Whitman’s filter paper. The filtrate was dried in a hot air oven at 40 °C to obtain a solid plant extract and stored at −4 °C in amber glass vials until use. There are no reports of such studies on S. surattense to date. Therefore, we wanted to obtain a non-toxic extract that might contain a maximum number of phytochemicals of different classes, and which would not lose its biological activities due to beyond-physiological temperatures. For this purpose, maceration (cold extraction method) was used, although the quantity of phytochemicals might have been compromised by said method, but not the quality. Likewise, 70% ethanol (relatively highly polar and nontoxic solvent) was used instead of methanol (relatively toxic solvent) or a nonpolar solvent to obtain an extract from plant material, so that number of biologically active phytochemicals with their relatively nontoxic properties could be extracted [The n method . In briextracted ,37. The A stock plant extract solution was prepared in dimethyl sulfoxide (DMSO) with a 50 mg/mL concentration, filtered using a 0.22 μm syringe filter, and stored at −20 °C until further experimentation .2 in a carbon dioxide incubator [2) at 37 °C for 2–5 min. Trypsin was inactivated after detaching the cells by adding an FBS-containing medium [Human liver cells HepG2 and Vero  were maintained in a high-glucose DMEM medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin, then cultured at 37 °C in an atmosphere with 5% COncubator ,40. To sg medium , and couS. surattense fruit extract and vehicle (DMSO control) for another 24 h. The cells were washed using a phosphate buffer saline (PBS) following the media aspiration. The WST-8 solution was added (10 μL/well in 100 μL DMEM) [The HepG2 and Vero cell lines were grown in a 96-well cell culture plate up to 70% confluence in respective media for 24 h. Subsequently, the cells were treated with different concentrations  of μL DMEM) ,43, and μL DMEM)  and a reμL DMEM) , and DoxμL DMEM) .Cell viability/proliferation% was evaluated by the following equation:S. surattense were identified using PubChem, Swiss ADME, and canonical SMILES of bioactive compounds were collected from ChEMBL. Using the obtained canonical SMILES, five pharmacokinetic properties of bioactive compounds, including molecular weight, hydrogen bond acceptor, hydrogen bond donor, and the logarithm of partition coefficient, were obtained. The Lipinksi rule of five for drug-likeness was also analyzed to screen for these bioactive compounds. Following the acute accuracy, a comprehensive ADMET analysis was performed using ADMETlab. Bioactive compounds were screened out based on the Lipinksi rule of drug discovery, including the criteria of drug-likeness, molecular weight (MW < 500), the logarithm of the partition coefficient (logP < 5.6), number of hydrogen bond acceptor (nHA ≤ 10), and number of hydrogen bond donor (nHD ≤ 5). Further, SwissTarget Prediction was used to select the potential target of the selected compounds using canonical SMILES. To predict the target of selected compounds, the Homo sapiens species was determined.The active components of Predicting disease-related genes is a first step toward understanding the molecular mechanisms of medicinal herbs for treating various diseases and syndromes. The four disease-related databases, GeneCard, OMIM, CTD, and DisGeNET, were searched and evaluated to predict possible disease-related targets using hepatocellular carcinoma as a keyword. Furthermore, each database contains brief genomic information and functional annotations for all known human genes. All duplicated genes were removed for further analysis.The identification of overlapping disease and chemical targets was made using Venn diagrams. Those are deemed important targets and are subjected to further investigation. Protein–protein interactions (PPI) are significant because of their flexibility, suppleness, and selectivity. STRING is a widely used database of protein–protein interactions that includes information from various sources. The common key targets of active compounds and HCC disease were uploaded to the STRING database for interaction analysis of the key targets at a combined score of 0.4. To visualize the protein–protein interaction network, Cytoscape (version 9) was used .KEGG pathway analysis and GO enrichment analysis was performed using DAVID. The common key targets of disease and plant were subjected to the DAVID database for the functional enrichment analysis. Three GO annotations, cellular components (CC), biological process (BP), and molecular function (MF) were analyzed. KEGG was used for the pathway analysis of the key targets. The top 10 GO annotations and cancer-related KEGG pathways were selected with P-value less than 0.05, and these were discussed in the article in the form of dot plots made by the R ggplot2 package.S. surattense in HCC was studied through network analysis. The active components of S. surattense and the HCC therapeutic target were entered into Cytoscape to form the compound–target network. The network nodes represent the chemical ingredients and targets, while the edges represent their interactions. The “network analyzer” examined the network’s essential parameters.The molecular mechanism of In drug discovery, molecular docking has become a significant catalyst and the most applicable technique. Through molecular docking, it is possible to predict the interaction of ligands to their respective proteins in a crystalline lattice. The crystal structures of proteins MTOR (1AUE), EGFR (1IVO), BCL2L1 (1R2E), ESR1 (1UOM), HRAS (4XVR), HIF1A (5JWP), TNF (5MU8), and AKT1 were retrieved from the RCSB PDB database. The Protein Data Bank (PDB) is a globally interconnected repository for information on the three-dimensional structures of proteins and nucleic acids. Researchers may use these data to better understand various facets of biomedical, from the synthesis of protein to disease. Chimera was used to refine the structure of the protein. Furthermore, molecular docking between key targets and active molecules was done using PyRx software. The docking score between key targets and compounds was used as a primary evaluation factor to filter out potential constituents and their potential targets. The docking analysis was also performed with MOE and Autodock software to validate the docked structures. Then Chimera and Discovery Studio were used to visualize interactions between key targets and active compounds. By measuring the strong affinity between chemicals and their related targets, this stage attempted to investigate the binding energy among compounds.S. surattense such as quercetin control the disorder by suppressing the potential target of hepatocellular carcinoma. The anti-cancer effects of S. surattense on HCC were postulated to be associated with the regulation of tumor cell proliferation, apoptosis, angiogenesis, tumor invasion, and metastasis via multiple signaling pathways, including the AKT1, EGFR signaling pathway, the ESR1 signaling pathway, and the HIF1A signaling pathway.According to a retrospective study, we conclude that herbal medicine (HM) therapy was correlated with a better prognosis in patients with HCC. Furthermore, by predicting active components and molecular targets of herbs, the network pharmacology approach may be used to explore the underlying anti-cancer mechanisms of herbs. A potential research strategy for “precise HM treatment” is the integration of clinical studies with network pharmacology. This study also concludes that the active constituents of


Drosophila melanogaster as a model organism to investigate the protective effect of Flos Puerariae extract (FPE) against sodium dodecyl sulfate (SDS)-induced intestinal injury. Our data showed that FPE had no toxic effect in flies, and significantly extended lifespan in SDS-inflamed flies, reduced stem cell proliferation in the midgut, and maintained intestinal morphological integrity. Furthermore, FPE remarkably recused the altered expression level of genes and proteins in Nrf2/Keap1 signaling, JAK-STAT signaling and Wnt signaling pathways in gut of inflammation flies. Thus, FPE has a protective effect against intestinal injury possibly via increasing the Nrf2/keap1 pathway and suppressing the JAK-STAT and Wnt signaling pathways, which would have tremendous potential for treating IBD.Gut homeostasis is important for human health, and its disruption can lead to inflammatory bowel disease (IBD). Flos Puerariae is a herb with a wide variety of pharmacological activities including antioxidant, antidiabetic, antialcoholismic and anti-inflammatory properties. However, the role of Flos Puerariae on treating IBD remains obscure. Here, we employed  Inflammatory bowel disease (IBD) is a blanket term that refers to a set of immunological disorders affecting the gastrointestinal system that has no recognized cause . There avia a balance of intestinal stem cell (ISC) self-renewal, progenitor differentiation, cell shedding and apoptosis  are known as ‘Flos Puerariae’. It is a perennial herbaceous plant of the Fabaceae family, that is, found across temperate regions of China, Korea, Japan, and India  were obtained from the Bloomington Drosophila stock center . 3–5 day old adult female or male were collected using light CO2 anesthesia and allowed to recover for 2 days before further experimentation.All flies were reared on standard medium at 25 C and approximately 65% humidity under a 12 h light/12 h dark cycle. Flies lines used in this study were as follows: w1118 flies were supplied with different concentrations of FPE in their standard diet from the 1st instar larvae until experiments. 0.4 mm ML385  and 0.6% SDS together were provided to adult females during experiments.Flos Puerariae extract (FPE) was purchased from Shaanxi Ruilin parnell biotechnology Co. Ltd. The dried Flos Puerariae was mixed with 50% (V/V) methanol solution with the solid-to-liquid ratio at 1∶30. The mixture was extracted with ultrasound for 2 h at 70°C. The extracted products were then purified sequentially by petroleum ether, ethanol and chloroform-butyl alcohol, and eluted gradually with mixed mobile phase of methanol-chloroform solution in the silica gel column system. In the end, the isolated ingredients were further analyzed by color reaction, ultraviolet spectrophotometry, high performance liquid chromatography, infrared spectrum and mass spectrum. FPE was diluted with a standard cornmeal-molasses medium to different concentrations . Liquid chromatography coupled to mass spectrometry (LC-MS) was used as a reference standard for the quality control of FPE. LC-MS analysis was performed as previously described . Separat2O2 and 5% sucrose solution, respectively. Filter papers were replaced every 2 days and the number of dead flies was recorded. Four treatments were performed, and each with at least three replicate vials.3–5 days old males or females of the 4 groups  fed the corresponding mediums were cultured for 2 days, and then removed to empty vials for 2 h. 20 flies per vial were transferred into vials containing five layers of filter paper infiltrated by 0.6% SDS and 5% sucrose solution, or 5% HDrosophila  to analyse the intestinal barrier integrity of osophila . Briefly3–5 days old females were starved in empty vials for 2 h at 25°C, then 20 females per vial were transferred to vials containing 5 layers of filter paper on the bottom. Filter papers were wetted with 5% sucrose solution (control) or 0.6% SDS and 5% sucrose solution (experiment). After 16 h, guts were dissected and examined.After fed in the medium containing 5% sucrose with or without 0.6% SDS for 16 h, 15–20 females per group were used to dissect intestines in cold PBS, the isolated guts were fixed with 3.7% formaldehyde for 30 min at room temperature. Following three washes with PBST for 10 min each, the samples were stained with 4’ 6-diamidino-2-phenylindole (DAPI) for 10 min. The tissues were then mounted in cedar wood oil  and observed under an Olympus FV1000 confocal microscope . ISCs were identified by their esg-GFP expression, their size, and their basal location within the intestinal epithelium. The experiments were independently repeated at least three times.TM Optics Module (Bio-Rad Laboratories) using Multiplex PCR Master Mix . Ribosomal gene RP49 was used as the internal control. The primer sequences were listed in Adult females were incubated with 0.6% SDS and 5% sucrose medium or individual 5% sucrose for 16 h, then total RNA of 60 females was extracted with TRIzol reagent  according to manufacturer’s instructions. Total RNA was reverse transcribed using Hieff® reverse transcriptase . Quantitative PCR was performed with a GFX 96 Connectp < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.Statistical analysis was performed using GraphPad Prism software. Comparisons among groups were made by analysis of variance (ANOVA) followed by Dunnett’s t-test. Survivorships among groups were compared and tested for significance with a Log-rank test. Differences between treatment groups were considered to be statistically significant at *The primary constituent of Flos Puerariae is isoflavonoids, including puerarin, daidzein, daidzin, genistein and apigenin . To iden2O2 respectively. SDS interferes with the normal function of the intestinal barrier and stimulates local and systemic inflammation  proliferation to regenerate the damage intestinal epithelium . We nextgstD1 (gstD1-GFP) transgenic flies sensitive to ROS were used   and JAK-STAT pathway (Hop and Socs36E)  . We foun92E-GFP) . The inc92E-GFP) . However92E-GFP) . It indiSocs36E) . SDS sti the gut . mRNA exreatment . It indireatment . We meas the gut . SDS ingrvention . It suggThe intestinal epithelium is a prominent defense barrier against invasion and facilitates host-microorganism interactions , and disDrosophila development is considered to be a high-throughput method for assessing drug safety  to activate JAK-STAT pathway in the gut of flies  protein (Wg in the intestine and activation of the Wnt signaling pathway (Wg and Arm were down-regulated after FPE intake. Together, FPE maintain intestinal homeostasis in inflammatory environment via inhibiting the JAK-STAT and Wnt signaling pathways, and activating Nrf2/keap1 signaling pathway. It is unknown whether there are other pathways that participate in this process. Network pharmacology analysis showed that many targets were enriched in inflammation-related signaling pathways, such as JAK-STAT signaling pathway, NF-kappa B signaling pathway, PI3K-Akt signaling pathway and Toll-like receptor signaling pathway (Drs, Dif and Myd88 induced by SDS in the Drosophila gut (Activation of the JAK-STAT and Wnt signaling pathways are associated with gastrointestinal inflammation and ISCs proliferation, which may accelerate the development of IBD . In Drosr injury . Toxic cof flies . In thisg stress , while t protein . Thus, F pathway . In cont pathway . Dorsal  pathway . Our RT-hila gut , which mPueraria lobata is an abundant source of isoflavones . Here, sCollectively, our study demonstrated that FPE displayed clear protective effects, including anti-inflammatory and antioxidant effects, and the modulation of intestinal epithelial barrier in SDS-induced injury. Further studies revealed that FPE could remarkably attenuate the SDS-induced excessive ISC proliferation and differentiation. Moreover, the protective effect of FPE on IBD is mainly mediated by the Nrf2/keap1, JAK-STAT and Wnt signaling pathways. Therefore, our results provided evidence for FPE as a potential alternative functional foods and medicines for the prevention and treatment of IBD.


Chronic obstructive pulmonary disease (COPD) is a long-term respiratory disorder marked by restricted airflow and persistent respiratory symptoms. According to previous studies, icariin combined with nobiletin (I&N) significantly ameliorates COPD, but the therapeutic mechanisms remain unclear.  The aim of the study is to investigate the therapeutic mechanisms of I&N against COPD using network pharmacology and experimental validation.  The targets of I&N and related genes of COPD were screened and their intersection was selected. Next, the protein-protein interaction (PPI) networks, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Further, a COPD rat model was established to validate the effect and mechanisms of I&N. β, and TNF-α in lung tissues of COPD rats and inhibited the activation of the MAPK pathway and PI3K-Akt pathway.  445 potential targets I&N were obtained from SwissTargetPrediction, STITCH 5.0, and PharmMapper databases. 1831 related genes of COPD were obtained from GeneCards, DrugBank, and DisGeNet databases. 189 related genes were screened via matching COPD targets with I&N. 16 highest score targets among 189 targets were obtained according to PPI networks. GO and KEGG pathway enrichment analyses of 16 highest score targets suggested that these key genes of I&N were mostly enriched in the tumor necrosis factor (TNF) pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphatidyl inositol 3-kinase (PI3K)-protein kinase B (AKT) pathway. Therefore, the treatments of I&N for COPD were connected with inflammation-related pathways. In in vivo experiments, the studies indicated that I&N improved the lung function and alleviated the damage of pulmonary histopathology. Moreover, I&N reduced levels of interleukin (IL)-6, IL-1 Icariin combined with nobiletin has therapeutic effects on COPD by inhibiting inflammation. The potential mechanisms of I&N may relate to the MAPK pathway and PI3K-Akt pathway. Chronic obstructive pulmonary disease is the most common disease of the respiratory system with high morbidity and mortality and endangers public health . Lung anNetwork pharmacology is a strategy based on multidirectional pharmacology, system biology, network analysis, and computational biology, which systematically expounds the potential targets and mechanisms of TCM . In thisIn this study, the targets of I&N and related genes of COPD were screened and the ingredients-disease targets network was established. Then, the potential molecular mechanisms were revealed via gene enrichment analysis and molecular docking. Finally, the COPD rat model was established to verify therapeutic effects and potential pathway of I&N against COPD . Therefohttps://www.swisstargetprediction.ch/), STITCH 5.0 (https://stitch.embl.de/) [https://lilab-ecust.cn/pharmmapper/) [The canonical SMILES of icariin and nobiletin were acquired by searching the keywords of “icariin” (Compound CID: 5318997) and “nobiletin” (Compound CID: 72344) from PubChem . The molmbl.de/) , and Phamapper/) .https://www.genecards.org/) [https://go.drugbank.com/) [https://www.disgenet.org/) [https://www.uniprot.org/) [The related genes of COPD were screened via the keywords of “chronic obstructive pulmonary disease” in GeneCards (ds.org/) , DrugBannk.com/) , and Diset.org/) . Then, aot.org/)  to acquiFirst, we intersected the obtained components targets with the genes associated with COPD and obtained a Venn diagram of the intersected gene symbols. Then, a PPI network was built using STRING  and Cytop value <0.05 was set as a significant difference for KEGG pathway analysis.The GO and KEGG pathways enrichments of the topological potential targets were analyzed in DAVID 6.8 . The p vThe 3D structures of icariin and nobiletin were acquired from PubChem and were transformed from their original constructions into PDB formats using Open Babel 3.1.1. From RCSB Protein Data Bank, the X-ray crystal structures of key proteins were obtained . Seven pKlebsiella pneumoniae (46117-5a1) was purchased from National Center for Medical Culture Collections (Beijing China). Icariin  and nobiletin  were purchased from Chengdu Must Bio-Technology . Doxofylline was obtained from Heilongjiang Fuhe Pharmaceutical Group Co., LTD. . The rat ELISA kits of IL-6 (Cat.No.550319) were purchased from BD Biosciences . The rat ELISA kits of IL-1β (E-EL-R0012c) and TNF-α (E-EL-R2856c) were purchased from Elabscience Biotechnology Co., Ltd . The antibodies for rat of PI3K  and P-AKT  were obtained from Gene Tex, Inc (North America). The antibody for rat of P-p38  was obtained from Cell Signaling Technology . The antibody for rat of GAPDH  was purchased from Proteintech .Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd . Hongqi Canal® Filter tip cigarette was purchased from Henan Tobacco Industry . Klebsiella pneumoniae infection. Specifically, the rats were exposed to CSE (3000 ± 500 ppm) for 40 minutes twice daily for 8 weeks and to Klebsiella pneumoniae  for 5 days once for 8 weeks. The procedures of this study were approved by the Experimental Animal Care and Ethics Committees of the First Affiliated Hospital of Henan University of Chinese Medicine, and the ethical review approval number is YFYDW2019031.A COPD rat model was performed in terms of previous studies . SD ratsD: dose; K: body shape index, K = A/W2/3, A: surface area in m2, W: weight in kg):From week 9, the I&N group rats were given I&N at 2.12 mg/kg/d (the ratio of icariin to nobiletin was 12.5 : 1). The doxofylline is a newer generation xanthine, which is a kind of effective bronchodilator recommended by Global Initiative for Chronic Obstructive Lung Disease (GOLD) [At week 17, 4 group rats were sacrificed after intraperitoneal injection of 2% pentobarbital sodium at 40 mg/kg.th week via the tidal volume (TV), peak expiratory flow (PEF), and 50% tidal volume expiratory flow (EF50) by unrestrained pulmonary function testing plethysmographs .Lung function was detected for all group rats every four weeks from 0 week to 162) = Na/A. Na is the number of pulmonary alveoli in each visual field. A is the area of the visual field. Then, we made a cross (+) under the visual field and counted the number of alveolar septaon the cross. MLI (μm) = L/Ns. Ns is the number of alveolar septa. L is total length of the cross.The lung tissues were soaked in 4% paraformaldehyde solution. Next, the tissues were cut and embedded in paraffin and made slices. Then, the lung tissues slices were stained with hematoxylin and eosin and were observed by a light microscope . The mean linear intercept (MLI) and mean alveolar numbers (MAN) were considered as the degree of alveolar damage. Under microscopy (×200), 6 visual fields were taken in each slice, and the alveolar number and the linear intercept in a fixed area of visual field were measured. MAN , and the concentration was calculated according to the standard curve.The lung tissue was homogenized in PBS solution and centrifuged to collect the supernatant. The secretion of TNF-α , IL-1β , and IL-6  in lung tissues were detected by qPCR.The mRNA levels of GAPDH , TNF-The lung tissues were lysed with RIPA buffer in ice to obtain protein samples. The concentrations of lung tissue protein samples were measured using BCA kits, and the lung tissue protein samples were adjusted to equal concentrations. The lung tissue protein samples with equal concentrations in each group were divided by SDS-PAGE electrophoresis and metastasized to PVDF membranes. 5% skim milk was used to block the PVDF. Next, membranes were incubated with their primary antibodies, including GAPDH (1 : 5000), P-p38 (1 : 1000), P13K (1 : 1000), and P-AKT (1 : 1000), and secondary antibodies (1 : 5000). The membranes were visualized using the Bio-Rad Imaging System .p value of <0.05 was set for a statistically significant difference.The experimental data were analyzed by SPSS v21.0. A comparison among groups was performed by one-way analysis of variance with an appropriate post-hoc test. If the variances were homogeneous, the LSD method was performed. If the variances were inconsistent, Dunnett's T3 test was performed. The mean ± SD were used as the data present presentation. A From PubChem, the 2D structures of icariin and nobiletin were downloaded . 445 genAll of 189 potential therapeutic targets were submitted to STRING database, and they were submitted to CytoScape3.8.2 for constructing and analyzing the PPI network . The PPIThe DAVID 6.8 database was used to perform GO and KEGG analyses on 16 important targets. Positive regulation of transcription from the RNA polymerase II promoter, negative regulation of the apoptotic process, and positive regulation of transcription, DNA-templated, were mostly enriched in BP enrichment analysis; nucleus, cytoplasm, and cytosol were mostly enriched in CC enrichment analysis; protein binding, identical protein binding, and enzyme binding were mostly enriched in MF analysis. . The resTo clarify the potential interaction between two components and the key proteins, molecular docking was performed to reveal the possible binding mode between the 7 highest scoring proteins, including TNF, AKT1, VEGFA, EGFR, JUN, SRC, and MMP9 , and twoAccording to Klebsiella pneumoniae in rats. As described in P < 0.05), and I&N and doxofylline increased the TV, PEF, and EF50 in rats (P < 0.05).To verify treatment of I&N of COPD, we established the COPD model through co-treatment with CSE and P < 0.05); I&N and doxofylline increased MAN and decreased MLI (P < 0.05); and I&N effectively relieved the thickened airway wall in COPD rats (P < 0.05).Lung tissue histopathology analysis indicates that I&N reduced alveolar damage and airway wall thickness . Quantitβ, and TNF-α, and these were decreased with treatment of I&N (P < 0.05) . These results suggested that I&N inhibit inflammatory responses in COPD rats via regulating the PI3K-AKT and MAPK pathways.In the COPD rats, the mRNA levels and protein secretion of inflammatory factor in lung tissues were significantly increased, including IL-6, IL-1 Figures . As in FIt has been verified that TCM has positive therapeutic effects on COPD. BYF, a TCM therapeutic strategy for COPD, has demonstrated that it can inhibit secretion of inflammatory cytokine, recover protease-antiprotease imbalance, and reduce collagen deposition . Due to First, we applied network pharmacology to screen the possible targets of I&N against COPD. 189 targets of I&N in COPD were obtained from 6 databases, and those with BC > 0.0092, CC > 0.5875, degree > 60, and ASPL < 1.7021 were considered as key targets. 16 key targets were screened out from the 189 targets via PPI network analysis, including TNF, AKT, and MAPK1. These key targets were significantly related to inflammation. Furthermore, the 16 key targets were mostly enriched inflammation-related pathway according GO analysis and KEGG analysis, such as TNF, PI3K-AKT, and MAPK signaling pathways. The result suggested I&N may inhibit the inflammatory response in COPD via these proteins and pathways. Then, molecular docking of I&N and these proteins was performed to verify the possibility of interaction, and these proteins, including AKT, TNF, EGFR, and MMP9, had strong binding energy with I&N.α and IL-6 in both the bronchoalveolar lavage fluid (BALF) and serum are decreased via restraining the activation of PI3K-AKT signaling in COPD model rats [α, IL-1β, and HO-1 overexpression following CSE [Inflammation is a key pathological reaction for the development of COPD . The maidel rats . Macrolidel rats . The famdel rats . The MAPwing CSE . Moreovewing CSE . MUC5AC,wing CSE . Accordiwing CSE .α, IL-6 via restraining activation of NF-κB signaling in the LPS-induced acute lung injury mice model and LPS-stimulated A549 cells [β, and TNF-α in lung tissues of COPD model rats were significantly increased and were decreased by I&N and doxofylline. On the other hand, the decline of lung function and emphysema is a common symptom during the development of COPD [It has been reported that icariin inhibits CSE-induced inflammation, ROS production, and airway remodeling via mitigating glucocorticoids resistance in CSE-exposed BEAS-2B cells . In addi49 cells . We had  of COPD . In in vβ, and TNF-α were improved by treatment of I&N in COPD rats. Furthermore, the levels of PI3K, P-AKT, and P-p38 were reduced by I&N. In conclusion, I&N have significant anti-inflammation effects for COPD via the restraining activation of PI3K-AKT and MPAK pathways. However, the complex mechanisms of I&N for treatment of COPD require further exploring.In our research, the therapeutic efficacy and mechanisms of I&N for COPD are verified via the method integrating network pharmacology and experiment validation. 16 key targets of I&N against COPD were screened, including TNF, AKT1, and MAPK1. According KEGG pathway analysis, the activation of the MPAK and PI3K-AKT pathways was a significant mechanism of I&N against COPD. In in vivo experiments, the lung function, pathological damage of lung tissues, and secretion of IL-6, IL-1


Real haptics is a technology that reproduces the sense of force and touch by transmitting contact information with real objects by converting human movements and the feel of the objects into data. In recent years, real haptics technology has been installed in several surgical devices. A custom-made surgical drill was used to drill into the posterior lamina to verify the time required for penetration detection and the distance the drill advanced after penetration. A surgeon operated with the drill and the same aspects were measured and verified. All experiments were performed on female miniature pigs at 9 months of age with a mean body weight of 23.6 kg . There were statistically significant differences in the average reaction time and the distance travelled after penetration between a handheld drill and the drill with the penetration detection function (p < 0.001). The reaction time to detect penetration and the distance after penetration were both significantly improved when compared with those of the handheld surgical drill without the penetration detection function, with mean differences of 0.049 ± 0.019 s  and 2.511 ± 0.537 mm . In this study, we successfully conducted a performance evaluation test of a custom-made haptic interface surgical drill. A prototype high-speed drill with a haptic interface accurately detected the penetration of the porcine posterior lamina. As a treatment for various musculoskeletal diseases, spine surgery involves drilling into the bone near the spinal nerves and vascular organs. This is an extremely demanding procedure because it involves the manipulation of hard tissue in the vicinity of highly vulnerable soft tissue. As a result, intraoperative complications of spine surgery occur at a certain rate. Bone drilling is usually performed with a high-speed drill, and severe complications, such as dural lacerations and spinal cord injury, may occur during the operation. Neurological complications are devastating and can severely diminish a patient's quality of life due to loss of motor function, limiting the patient’s functional ability and causing excruciating neurological pain; every effort should be made to prevent the occurrence of such complications.6. Among these surgical complications, dural lacerations have been reported to affect 2–13% of cases. Most dural lacerations are caused by bone drilling during decompression of the spinal cord7. It has also been reported that the incidence of intraoperative complications, such as nerve injury, is inversely related to the number of years of surgical experience of the surgeon. Imajo et al. found that spine surgeons with less than 5 years of surgical experience had the highest frequency of complications1. It is widely accepted that in spine surgery, when drilling bone using a high-speed drill, the surgeon relies on the feel of the drill to determine whether the bone has been penetrated. Therefore, it is thought that the number of years of surgical experience affects the surgeon’s ability to detect bone penetration, and this directly affects the incidence of complications.Previous reports have shown that the complication rate of spine surgery is relatively high, ranging from 1 to 17%1. In other words, although the number of years of experience in spine surgery influences the complication rate to some extent due to the nature of the procedure, dural lacerations and neurological injuries still occur regardless of the number of years of experience.In contrast, Imajo et al. described the results of a survey of spine surgery complications and concluded that the incidence of intraoperative dural injuries was similar between spine surgeons with less than 10 years of experience and those with more than 10 years of experience9. Real haptics is a technology that reproduces the sense of force and touch by transmitting contact information with real objects and the surrounding environment in both directions by converting human movements and the feel of the objects into data. By converting movements and textures into data, it is possible to program force tactile sensation data into machines, adjust the degree of force, and transmit force tactile sensation over a long distance9. Specifically, by monitoring the drilling force in real time when using the drill, it will be possible to detect penetration, automatically stop the drill, and proceed with the surgery while obtaining detailed information, such as the bone hardness and the identity of the drilling layer. In addition, by extracting intraoperative drilling sensation and movement data, we believe that such data can be applied to develop a training simulator that reproduces a realistic sensation of force and touch and adds an assistance function based on the movements of a skilled surgeon.In recent years, real haptics technology, which has been utilized in various fields, has been installed in high-speed drills to establish a surgical method with greater safetyWe hypothesized that a prototype high-speed drill with a haptic interface could detect the penetration of the porcine posterior lamina more accurately and more reproducibly than experienced spine surgeons could recognize Fig. A,B. In t10. The custom-made drill has a master–slave integrated structure and is driven by a linear motor with two optical encoders and a rotary motor with a rotary encoder10. A schematic drawing of the prototype is shown in Fig. 10. Moreover, when the custom-made surgical drill detects penetration, position control is applied to the linear motor on the slave side to pull in the drill bit while the rotary motor is stopped automatically . The block diagram of the DOB is shown in Fig. The rotary motor is controlled by velocity control. Figure mentclass2pt{minimm, s, Cp(s), and Cf(s) are the master part, slave part, position controller, and force controller, respectively. Bilateral control is applied to the two linear motors to realize transmission of the haptic sensation. The block diagram of the bilateral control is shown in Fig. These equations represent the law of action and reaction. Thus, the haptic sensation between the two linear motors is realized by bilateral control.11. In the drilling state, the reaction force from the cutting object is applied to the slave part. In contrast, when the drill penetrates the object, the reaction force is decreased. Additionally, the velocity of the slave part is increased due to the decrease in the reaction force. Therefore, large displacements of position and reaction force are generated by penetration. Thus, the drill detects the penetration when Eqs.   11. In t by Eqs. .6\documeWhen the custom-made surgical drill detects penetration, position control is applied to the linear motor on the slave side to pull in the drill bit. Additionally, the rotary motor is stopped automatically.After review and approval by the Judging Committee of Experimental Animal Ethics of Keio University School of Medicine, all experiments were performed on female miniature pigs at approximately 9 months old with a mean body weight of 23.6 kg . All animals were purchased from the laboratory  and housed and treated in accordance with rules approved by the Ethics Committee (no. 18047). All experiments were performed in accordance with any relevant guidelines and regulations. This study follows the recommendations in the ARRIVE guidelines.The miniature pigs undergoing surgery were premedicated with midazolam  administered subdermally. Subsequently, inhalant anaesthetics and isoflurane  were used with maintenance at a 2% flow rate after orotracheal intubation. All pigs were killed immediately after surgery by an overdose of isoflurane. After establishing anaesthesia, a standard midline dissection was performed by 1 of 3 board-certified spine surgeons using electrocautery, and the laminae of the thoracic vertebrae and lumbar vertebrae were carefully exposed.The experimental setup is shown in Fig. First, the custom-made surgical drill was used to drill the posterior lamina without the penetration detection function. After penetration was detected, the threshold was set as a control, where − 7 N/s was The time required for penetration detection was defined as the time from the start of the decrease in the reaction force of the slave until The time required for penetration detection.The distance advanced by the drill after penetration.After that, the penetration detection function was turned on, and the measurement results of the drilling of the posterior laminae were used to verify:The distance the drill advanced after penetration was defined as the distance that the slave advanced before the device detected the penetration and started retracting the drill Fig. A,B.FigurThe time required for penetration detection.The distance advanced by the drill after penetration.Next, the surgeon operated the drill without the penetration detection function and verified:The time until the surgeon recognized the penetration detection was set as the reaction time, which was defined as "the time when the drill position started to move forwards rapidly" to "the time when the drill stopped moving forwards (the time when the speed was displaced negatively)", that is, the time taken by the surgeon to detect the penetration and start retracting the drill body. The participants were instructed to move the master in the opposite direction of penetration (retracting the drill).The distance travelled after penetration was defined as the distance travelled from "the position where the drill started to move forwards rapidly" to "the peak of the drill (slave) position", i.e., the distance travelled until the surgeon detected the penetration and started to retract the drill body Fig. C,D.Finally, the haptic drill with the penetration detection function was used by the surgeon, and the same items were measured and verified.Next, to assess if the haptic drill is able to detect the penetration of lamina in an actual surgery setting, the surgeon operated the haptic drill with horizontal movement similar to the actual surgical procedure . A p value less than 0.05 with a CI of 95% was considered statistically significant. A p value less than 0.05 was considered statistically significant. Data were analysed with the Statistical Package for the Social Sciences .The average time to detect the penetration of lamina by the drill with the penetration detection function was 0.015 ± 0.005 s (range 0.01–0.02 s), and the travel distance after penetration was 0.11 ± 0.063 mm , and the distance travelled after penetration was 3.52 ± 1.770 mm . These results clearly showed that even in an experienced surgeon’s hand, it is difficult to recognize the penetration of the lamina early and to stop the drill immediately after detecting the penetration of the lamina during surgery.There were statistically significant differences for both the average reaction time and the distance travelled after penetration between the handheld drill and the drill with the penetration detection function (p < 0.001). The penetration detection time of the device was approximately one-tenth as fast as that of the handheld drill, and the mean difference was 0.154 ± 0.024 s  and 2.862 ± 0.741 mm .Similarly, three spine surgeons drilled the posterior laminae 3 times each until they detected the penetration by the custom-made haptic drill with the penetration detection function. The average reaction time was 0.020 ± 0.012 s (range 0.01–0.04 s), and the distance travelled after penetration was 0.541 ± 0.351 mm . The average reaction time was 0.169 ± 0.041 s, and the distance travelled after penetration was 2.982 ± 1.242 mm when using a handheld drill without the penetration detection function. Additionally, the lack of correlation between the detection time and distance indicated that bone drilling using a handheld drill was less reproducible. However, when using the handheld surgical drill with the penetration detection function, the reaction time and the distance after penetration were both improved. The average reaction time to detect penetration and the distance after penetration were both significantly shorter than those of the handheld surgical drill without the penetration detection function, with mean differences of 0.049 ± 0.019 s and 2.511 ± 0.537 mm, respectively. This result clearly showed that even when the drill was used as a handheld device, the reaction time and the distance after penetration can be significantly shortened if it is equipped with real haptics.Interestingly, analysis of the reaction time and the travel distance after penetration of the handheld surgical drill with the penetration detection function showed no statistically significant differences among the surgeons for their average reaction time and the distance travelled after penetration. According to this result, surgeons can recognize penetration extremely quickly with haptic drills regardless of their years of experience. As we have described in the introduction section, gaining surgical experience has thus far been considered very important in reducing the possibility of neurological complications caused by surgical drills. However, it is difficult to gain experience in actual spine surgery without years of experience performing spinal surgery. Therefore, the establishment of a surgical simulator using real haptics technology will be important for young spine surgeons to gain experience in order to avoid neurological complications caused by surgical drilling.14. Meyer et al. compared the drilling performance of a bone simulator with a haptic system between the trainee resident group and the expert staff surgeon group. They concluded that there were no significant differences in surgical time or the accuracy of the surgical technique13. Thus, the use of a spine surgery simulator with a haptic system may be useful for spine surgery education, which has a steep learning curve. Previous reports showed the utility of real haptics technology only for surgical simulators. This study is the first report indicating the accuracy of the penetration detection function and automatic stop function of haptic drills when performing real surgery.Several previous reports described the availability of haptic technology for surgical simulatorsIn this study, we also proved the accuracy of penetration detection when the surgeon operated the drill in the horizontal direction, which is similar to the actual surgical procedure. It was possible to detect penetration according to the shape of the lamina by recognizing the position by dividing it into 2 mm increments. On the other hand, the position information of the lamina can be moved by the deviation of the spine during the procedure, so overdetection may occur; however, overdetection never causes damage to the spinal cord. Currently, we are planning to add alert systems to the penetration detection system. Further refinement of the penetration detection system will include minimization of the overdetection of penetration.15. These systems are controlled with a haptic device that feeds the exerted drilling force into the hand of the surgeon in real time. Thus, surgeons can detect the differences in tissues and then perform surgeries more safely and accurately. Real haptic systems are also considered to be an essential technology for the development of robotic telesurgery16.Furthermore, the concept of using real haptic technology and virtual reality (VR) for robotic surgery has also been developedWe acknowledge the limitation that we were unable to use human cadavers due to resource limitations. In this study, the drill was used only in a vertical direction against the vertebral lamina of young miniature pigs. However, in actual surgery, the surgeon moves the drill in various directions against the vertebral lamina in various age populations while drilling. Further study for the improvement of this evolutional instrument may require the evaluation of the safety and utility of the haptic interface surgical drill in various bone conditions, including osteoporotic bone.In the present study, a prototype high-speed drill with a haptic interface successfully detected the penetration of the porcine posterior lamina more accurately and more reproducibly than experienced spine surgeons. The integration of haptic technology into spinal drills may be expected to have future applications in accurate, safe robotic surgery and telesurgery.Supplementary Video 1.


Ulcerative colitis is an intestinal condition that severely affects the life quality of a patient. Jiawei Zhengqi powder (JWZQS) has some therapeutic benefits for ulcerative colitis. The current study investigated the therapeutic mechanism of JWZQS for ulcerative colitis using a network pharmacology analytical approach. β, IL-6, and TNF-α were detected in animal experiments. Their effect on the NF-κB signaling pathway and the protective mechanism of JWZQS on the colon by tight junction protein were investigated.  In this study, network pharmacology was used to investigate the potential mechanism of JWZQS in treating ulcerative colitis. The common targets between the two were identified, and a network map was created with the Cytoscape software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of JWZQS was performed using the Metascape database. Protein-protein interaction networks (PPI) was created to screen core targets and main components, and molecular docking was conducted between the main components and core targets. The expression levels of IL-1κB signaling pathway was selected for analysis and verification. JWZQS has been shown in animal studies to effectively inhibit the NF-κB pathway; reduce the expression of IL-1β, TNF-α, and IL-6 in colon tissue; and increase the expression of ZO-1, Occludin, and Claudin-1.  There were 2127 potential ulcerative colitis targets and 35 components identified, including 201 non-reproducible targets and 123 targets shared by drugs and diseases. Following the analysis, we discovered 13 significant active components and 10 core targets. The first 5 active ingredients and their corresponding targets were molecularly docked, and the results showed a high level of affinity. GO analysis showed that JWZQS participate in multiple biological processes to treat UC. KEGG analysis showed that JWZQS may be involved in regulating multiple pathways, and the NF-β, TNF-α, and IL-6, inhibit the phosphorylation of the NF-κB pathway, and alleviate colon injury. JWZQS can be used in clinical, but the precise mechanism of UC treatment requires further investigation. The network pharmacological study provides preliminary evidence that JWZQS can treat UC through multiple components and targets. JWZQS has been shown in animal studies to effectively reduce the expression levels of IL-1 Ulcerative colitis (UC) is an inflammatory condition that primarily affects the mucosa and submucosa of the large intestine (rectum and colon). It is distinguished by large intestinal mucosal ulceration and persistent inflammation . Mucous,In traditional Chinese medicine (TCM), there are several ways to treat UC, which is also known as diarrhea or dysentery. TCM provides a comprehensive understanding of a patient's physical condition based on their pulsation, tongue, signs and symptoms, and modern laboratory tests. Although therapeutic regimens vary depending on constitution, the general approach is to clear heat; expel dampness; strengthen the spleen, kidney, and qi; promote blood circulation; and dispel blood stagnation. JWZQS decoction contains Houpo, Chenpi, Banxia, Danggui, Chuanxiong, Huoxiang, Cangzhu, Xianhecao, and Shengma. The original prescription comes from a Ming Dynasty work by Xue Ji called the “Waike Jingyao.” It has Qi-widening, spleen-energizing, and dampening effects. The current study makes reference to Andrew L. Hopkins' theory of “network pharmacology” and Professor Li Shao's theory of “correlation between TCM and biomolecular network” , 5. NetwThe current study used the network pharmacology approach to construct the “drug-components-targets” network. For the core target and pathway verification, the KEGG and GO enrichment analyses were performed. Molecular docking technique was used to study the affinity between active components and key targets in JWZQS. The flow chart for the study is shown in α, IL-6, and IL-1β in tissues were measured. The phosphorylation of IκB-α and P65 in the NF-κB pathway was detected. Furthermore, the tissue was sliced to examine the damage to the colon. Immunohistochemistry (IHC) detected the expression of ZO-1, Occludin, and Claudin-1 in the mouse colon tissue.We used dextran sulfate sodium salt (DSS) to cause colon injury in mice, developed a UC mouse model, and assessed the clinical symptoms of the mice. Mouse colons were collected, colonic lengths were measured, and the contents and expressions of TNF-The article type was original research.http://tcmspw.com). Following the bioactive constituents criteria, active constituents with drug similarity (DL) not less than 0.18 and oral bioavailability (OB) not less than 30% were screened [We use the search terms “ Houpo, Chenpi, Banxia, Danggui, Chuanxiong, Huoxiang, Cangzhu, Xianhecao, and Shengma” in the TCM database of system pharmacology , OMIM (https://www.omim.org), DrugBank (https://go.drugbank.com), and DisGeNet (https://www.disgenet.org) databases were searched for UC, and human genes were chosen [https://string-db.org) was used to screen out the common targets.TGenecards (e chosen –11. Aftee chosen . String Cytoscape software (3.10.1 version) is used to associate drugs, active ingredients, targets, and diseases and to build an interaction network diagram between components and targets. Edges represent node relationships, while nodes represent active ingredients and targets. The main active components of the drugs were then examined.https://cytoscape.org), and restriction conditions were set for the human species [To establish the interaction between the two proteins, the common target was imported into the Cytoscape database , biological process (BP), and cell component (CC) data. Their sequencing is determined by the number of targets involved in biological processes and signaling pathways.https://www.rcsb.org) in addition to the 3D structures of the small molecules irisolidone, naringenin, nobiletin, quercetin, and wogonin from the PubChem (https://pubchem.ncbi.nlm.nih.gov) database [We downloaded the protein crystal architectures used for docking from the PDB database  according to the procedures (License No.: SYXK2018–0001). Suzhou Xishan Biotechnology Co. LTD. provided male Kunming mice (6~8 weeks old). Thirty Kunming mice were randomly divided into five groups: the blank group (NT), the model group (MOD), the sulfasalazine group (SASP), the JWZQS low-dose group (JWZQS-L), the JWZQS medium-dose group (JWZQS-M), and the JWZQS high-dose group (JWZQS-H). Each group had five mice. For UC model establishment, except for the control group, the other mice were given 3% DSS solution for 7 days, according to the literature . Gavage The disease activity index (DAI) scoring system  was usedα, IL-1β, and IL-6 expression levels in mouse colon tissues were measured using the ELISA kit .TTNF-β, TNF-α, and IL-6 were determined using qRT-PCR as previously described [β, TNF-α, and IL-6 were determined using qRT-PCR as previously described [ΔΔCT− was used to obtain the relative quantitative results.Colon tissues from mice were collected and treated to extract mRNA, and the levels of IL-1escribed . Colon tescribed .  Table μg), which was then transferred to a PVDF membrane . The PVDF membrane was then blocked for 2 h at room temperature with 5% skim milk. PVDF membrane and primary antibody NF-κB p65 (Servicebio GB11997 1 : 300), NF-κB p-p65 (Servicebio GB111421 1 : 300), IκB-α (Abcam EPR20992 1 : 2000), p-IκB-α (ser32 1 : 2000), and GAPDH (Servicebio GB15002 1 : 2000) were stored overnight at 4°C and washed with TBST lotion. The PVDF membrane was then washed in TBS-T washing solution and incubated for 2 h with goat anti-mouse/rabbit secondary antibody (Servicebio GB25301/GB23303 1 : 5000). The trend of protein bands was then detected using an enhanced chemiluminescence solution.The total protein of colonic tissue was extracted in this study by grinding RIPA lysate. BCA (Thermo) reagent was used to measure colonic histamine levels in mice . SDS-PAGE with 12% was used to isolate the protein , Occludin (ab216327 1 : 100), and Claudin-1 (ab15098 1 : 100) and then with the secondary antibodies for 50 min before being examined under a microscope.t-tests. Prism 8.0.20 was used to perform ANOVA  comparisons of more than two groups. Nonparametric tests must be used to evaluate clinical and pathology scores.The data is presented as the mean ± SD. Prism 8.0 was used to compare two groups using unpaired Student's The following nine Chinese medicines were searched in the TCMSP database: Houpo, Chenpi, Banxia, Danggui, Chuangxiong, Huoxiang, Cangzhu, Xianhecao, and Shengma. These compounds' screening criteria were DL (≥0.18) and OB (≥30%). The TCMSP database was used to screen the targets linked to active ingredients, after which the target information was compared, and the gene name was adjusted using the UniProt database . After mUsing the search term “ulcerative colitis,” the human genome annotation databases GeneCards, OMIM, DisGeNet, and DrugBank were searched for disease-related genes. There were 2127 acquired disease targets. The acquired target genes were compared with the genes associated with the aforementioned drug active ingredients. The String website generated a Venn diagram to screen for common genes, as shown in The obtained active ingredient and genetic data will be used to generate “Drug-Components-Targets” and “Drug-Components-Targets-Disease” network maps using Cytoscape software. Figures With the common gene data imported, the String database was used to generate a protein-protein interaction network (PPI) interaction map with the species “Homo Sapiens” as the designation. The TSV file was exported with the lowest interaction score (0.7). The Cytoscape software was used to calculate the major protein interactions. We imported the 123 common targets in the intersection into the String database to generate the interaction map. We import the interaction diagram into Cytoscape software to generate the PPI network diagram .β, VEGFA, TPGS2, CASP3, HIF1A, MAPK3, and other genes, ranked by degree value , IL-6 and quercetin are –7.4 , TNF and nobiletin are –9.1 , and TP53 and wogonin are –6.6 . It is worth noting that AKT1 has a high affinity with naringenin and TNF with nobiletin. Then, we dock all ligands for each docked protein, to observe the interaction between the core targets and other active components , IL-1mponents .β, and TNF-α in the MOD group, effectively controlling inflammation development  and bacterial processes, such as fat polysaccharide activation. These compounds target serine residues for significant degradation mediated by the ubiquitin-proteasome, resulting in the release of NF-κB, nuclear accumulation, and transcriptional activation of target genes [κB differ by cell type. Increased proinflammatory mediator levels are most likely caused by increased NF-κB immunocyte activity, which worsens the inflammation. Therefore, we looked into how JWZQS affected the phosphorylation of P65 and IκB-α proteins in the NF-κB signaling pathway in animals. JWZQS inhibits the activation of the NF-κB signaling pathway, which inhibits the release of inflammatory cytokines IL-1β, IL-6, and TNF-α in a dose-dependent manner.We found that the NF-survival , 29. Dimsubunits –32. IKK et genes , 34. BecIn this study, it was discovered that JWZQS not only had a protective effect on pathological injury and colon shortening throughout the DSS induced UC mouse model but also significantly improved body weight maintenance in mice. JWZQS was dose-dependent in UC mice, according to the findings. The breakdown of the intestinal immune barrier, which is formed by tight connections between cells, is a key factor in the progression of UC. Occludin, Claudin-1, and ZO-1 are intestinal immune barrier-tight junction proteins that are primarily responsible for maintaining the integrity of the intestinal epithelial barrier. Increased permeability of these junction proteins is associated with an increased risk of UC . The stuα, IL-6, and IL-1β in the NF-κB pathway. JWZQS protects DSS-induced UC mouse models by upregulating Occludin, Claudin-1, and ZO-1 in the colon of mice. In addition, many additional signaling pathways may be activated and their mechanisms remain to be investigated.These results suggest that JWZQS treat UC by inhibiting the expression of TNF-κB pathway, reduce the expression of related inflammatory factors, and increase the expression of tight link protein. However, additional research is required to validate the reliability of the research findings. Finally, JWZQS can be used in the clinic. However, the mechanism of JWZQS in treating UC requires more research.In this study, we used network pharmacology and related software system to study the potential mechanism of JWZQS against UC. The affinity between the core target and the active component was verified by molecular docking. Animal experiments revealed that JWZQS could inhibit the NF-


Gout is a common disease with high incidence due to unhealthy diet and living habits. Simiao Powder, as a classic formula consisted of four common herbs, has been widely used in clinical practice since ancient times to prevent and treat gout. However, the pharmacological mechanism of Simiao Powder is still unclear.Based on network pharmacology, Simiao Powder active compounds were identified in TCMSP, ETCM and BATMAN database, used to establish a network of interaction between potential targets of Simiao Powder and known therapeutic targets of gout. Subsequently, the key potential targets are being used for protein–protein interaction, GO enrichment analysis and KEGG pathway enrichment analysis through several authoritative open databases. Molecular docking through AutoDockTools software can verify interaction between molecules. Finally, to validate the predicted results, in vivo experiments based on hyperuricemic-gout mice model were designed and treated with Simiao powder and allopurinol. Serum levels of uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN) and xanthine oxidase (XOD) were determined using a customized assay kit while the expression of PPAR-γ, PTGS1, IL-6 and Bcl2 mRNA were analyzed through qRT-PCR.Disease-target-compound network was visualized basing on the 20 bioactive compounds and the 19 potential targets using Cytoscape software. The results of PPI analysis, GO enrichment and KEGG pathway enrichment analysis indicate that the potential mechanism of Simiao Powder in treating gout may be achieved by regulating immune and inflammatory reactions, improving metabolism and endocrine. The results of molecular docking show that most of the targets and components have good binding activity. In vivo experiments revealed that Simiao powder can decreased serum UA and XOD levels in hyperuricemic-gout mice, and improved renal function. Furthermore, Simiao powder certainly regulates the expression of PPAR-γ, PTGS1, IL-6 and Bcl2 mRNA in ankle tissue in hyperuricemic-gout mice.Collectively, this research predicted a multiple compounds, targets, and pathways model mechanism of Simiao Powder in the prevention and treatment of gout, providing new ideas and methods for in-depth research, via vivo experiments. Gout is a common chronic disease with urate crystal deposition in and around the joints results from longstanding hyperuricaemia , 2. The The treatment of gout mainly includes anti-inflammatory and analgesic treatment in acute arthritis, uric acid control in chronic phase and daily personal life management . MedicatAtractylodes Lancea (Thunb.)Dc, Phellodendri Chinrnsis Cortex, Cyathulae Radix, Coicis Semen, is derived from traditional Chinese medicine and widely used in the clinical treatment of gout in China due to good efficacy and few side effects [At present, it has become the focus of pharmaceutical research to search for drugs to treat gout and reduce hyperuricemia from Chinese herbs and natural materials –17. Rece effects , 19. Mod effects –22. HoweNetwork pharmacology is a new method of drug research proposed based on multi-directional systematic pharmacology and biology –25, whichttp://lsp.nwu.edu.cn/tcmsp.php), ETCM [http://www.tcmip.cn/ETCM/index.php/Home), BATMAN [http://bionet.ncps-b.org.cn/batman-tcm) using “Atractylodes Lancea (Thunb.)Dc”, “Phellodendri Chinrnsis Cortex”, “Cyathulae Radix” and “Coicis Semen”. TCMSP, a unique systems pharmacology database for users to comprehensively study Chinese herbal medicines, contains information on all 500 TCM formulae registered in the Chinese Pharmacopoeia (2010 edition), with a total of 30,069 compounds extracted by literature mining and database integration [To identify the chemical compounds of the four herbs in Simiao Powder, we performed a search in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  , ETCM   evaluation, two ADME-related models, including the evaluation of oral bioavailability (OB) and drug-likeness (DL), were employed to identify the potential bioactive compounds of Simiao Powder. Oral bioavailability (OB) and drug-likeness (DL) are very important pharmacokinetic parameters and key indicators in ADME process . Accordihttps://www.drugbank.ca/) while the gene names were extracted from UniProt Knowledgebase [http://www.uniprot.org). The DrugBank database is a bioinformatics and cheminformatics resource that combines detailed drug data with comprehensive drug target information. The UniProtKB is a central hub for the collection of functional information on proteins, with accurate, consistent and rich annotation, includes widely accepted biological ontologies, classifications and cross-references, and clear indications of the quality of annotation in the form of evidence attribution of experimental and computational data.The protein targets of the compounds were searched from the TCMSP and Drugbank database  (https:/edgebase  (UniProthttps://www.genecards.org), the therapeutic target database [https://db.idrblab.org/ttd/), Pharmacogenomics Knowledge Base [https://www.pharmgkb.org/), and the Online Mendelian Inheritance in Man (OMIM) database [www.omim.org/). The three databases mentioned above are commonly used databases for analysis of genomic known diseases with a genetic component and linked them to the relevant genes in the human genome, and they complement each other with their own characteristics. “Gout” was used to search in all the database as the keyword.Therapeutic targets of gout were collected from four sources, include GeneCards database  and String [https://string-db.org). The two open databases covered the majority of known protein–protein interactions information for human.Based on the prediction targets between compounds and disease, protein–protein interaction (PPI) data were generated through two major existing public PPI databases with the species limited as “Homo sapiens”, including Metascape  (http://d String  (https:/https://david-d.ncifcrf.gov/) and OmicShare platform (https://www.omicshare.com/).The gene ontology (GO) enrichment analysis was conducted to further validate whether the potential targets are indeed a match for gout in three levels, contained biological process analysis, molecular function analysis, cell component analysis. The GO enrichment analysis was performed using the functional annotation tool of DAVID Bioinformatics Resources 6.7   was purchased from Liaoning Changsheng Biotechnology Co., Ltd. . Animals were housed under a normal 12-h/12-h light/dark schedule, room temperature (22 ± 2 °C), relative humidity (55 ± 5%), and given a standard chow and water ad libitum for the duration of the study. All studies were carried out in accordance with the Institutional Animal Care Committee at the Jinan University. Simiao Powder, consisting of four classical herbs: Atractylodes Lancea (Thunb.)Dc, Phellodendri Chinrnsis Cortex, Cyathulae Radix, Coicis Semen,were purchased from Huarun Sanjiu Medicine Co., Ltd. According to the literature , it was Experimental animal model of Hyperuricemic-Gout Mice induced by uricase inhibitor potassium oxonate has been used to study drug action for many years. Mice were divided into 4 groups: Normal + water(group A), Model + water(group B), Model + Simiao Powder(group C), Model + allopurinol(group D). The normal group was intraperitoneally injected with 0.5% CMC-Na solution, while the other groups were intraperitoneally injected with suspension composed of 300 mg/kg potassium oxazinate and 200 mg/kg hypoxanthine at 15:00 p.m. every day for seven consecutive days . Simiao Powder and allopurinol were orally initiated at 16:00 p.m. on the day by intragastric administration. Dosages of Simiao powder and allopurinol were determined based on the conversions from clinical adult dosages and previous studies. The dosage was as follows : allopurWhole blood samples were collected 1 h after final administration on the seventh day, and then allowed to clot for approximately 1 h at room temperature and centrifuged at 3500 rpm for 10 min to obtain serum, stored at − 80 °C until assayed. Simultaneously, ankle tissue was rapidly and carefully separated on ice-plate and stored at − 80 °C for assays, respectively.Serum levels of uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN) and xanthine oxidase (XOD) were determined using a customized assay kit. All assay kits were purchased from Jiancheng Biotech. . Uric acid concentration in serum was determined by the phosphotungstic acid method. Blood urea nitrogen was determined by rapid urease method while Creatinine was measured using sarcosine oxidase method. Xanthine Oxidase was detected by colorimetric method. The operation method of the experiment shall strictly refer to the manufacturer's instructions.Related methods are described in our previous article . BrieflyP < 0.05 was considered statistically significant. Figures were obtained by GraphPad Prism 9 .All data are statistically analyzed using one-way ANOVA analysis with the statistical software SPSS 13.0. Data were expressed as Mean ± Standard Error of the mean(S.E.M.). A value of Atractylodes Lancea (Thunb.)Dc, 4 in Phellodendri Chinrnsis Cortex, 37 in Cyathulae Radix, 9 in Coicis Semen. Among the 59 satisfied compounds, a total of 56 compounds were finally obtained after excluding the duplicates.Based on the criteria of OB ≥ 30% and DL ≥ 0.18, a total of 59 compounds of Simiao Powder were retrieved from the TCMSP, including 9 in According to the target prediction system in TCMSP and Drugbank databases, the 56 potential active components of Simiao Powder collected were input into the databases and 824 putative targets were obtained by pairing. The corresponding target proteins were searched through the UniprotKB database for their corresponding gene symbol names.A total of 756 targets associated with gout were collected from the four open databases, namely, 734 in GeneCards database, 15 in TTD, 4 in PharmGKB and 3 in OMIM database. After removing the overlapping targets, 746 associated targets were obtained.The prediction targets of compounds and disease-related targets were combined and intersected to obtain the possible targets of drug treatment for gout. Subsequently, the result showed that gout share 19 targets with 20 bioactive compounds of Simiao Powder  data were generated for the 19 common targets mentioned above (The confidence score > 0.4). The PPI network showed current interactions among the 19 common targets  enrichment analysis was performed and the results were revealed in three levels via biological process, molecular function and cell component analysis. Figure The KEGG pathway enrichment analysis was performed using the functional annotation tool of DAVID Bioinformatics Resources 6.7 and OmicShare platform. Figure Finally, Compounds-Targets-pathways network for Simiao Powder acting on gout was constructed shown as Fig. The molecular docking software AutoDockTools can apply semi-flexible docking methods on the basis of complex networks to study the interaction between molecules, and then evaluate the protein–ligand binding potential based on its comprehensive characteristics. There were 3 targets with Degree value ≥ 6 in the disease-target-compound network diagram, and top 3 targets in the PPI network diagram. After removing duplicate targets, we obtained 4 targets in total: IL-6, PTGS1, PPARG, BCL2. These targets were searched in the literature respectively and all targets were related to gout. They were respectively molecularly docked with the potential active compounds of Simiao Powder.According to the results of comprehensive molecular docking, the minimum binding energy was -3.31 kcal/mol and the maximum is -7.72 kcal/mol. Among them, the 4 potential active compounds of Simiao Powder  and the amino acid residues of the targets IL-6, PTGS1, PPARG, BCL2 were combined by intermolecular hydrogen bonds. Most of the targets and components had good binding activity because of high binding energy. This indicates that the main characteristic chemical components of Simiao Powder have good binding ability with the targets IL-6, PTGS1, PPARG and BCL2. In the results of molecular docking, there were 31.25% of them binding energy higher than -5 kcal/mol while others were lower than -5 kcal/mol. In addition, there were two of them lower than -7 kcal/mol. Among them, the binding energy of PPARG and stigmasterol is the highest, -7.72 kcal/mol. The results of molecular docking were shown in Table As expected, intraperitoneally injected potassium oxazinate and hypoxanthine significantly elevated serum UA and XOD levels compared with normal mice Fig. a. SimiaoSerum BUN and Cr levels in Hyperuricemic-gout mice were significantly higher than those in the normal group Fig. c-d. SimiThe effects of Simiao powder and allopurinol on mRNA levels of ankle PPAR-γ, PTGS1, IL-6, and Bcl2 in hyperuricemic-gout and normal mice were shown in Fig. Traditional Chinese medicine considers gout to be arthralgia syndrome result from the dysfunction of viscera , which iThis research demonstrates that 20 active compounds and 19 potential genetic targets of Simiao Powder in the treatment of gout were found through network pharmacology. Then, specific functions and mechanisms are revealed through protein interaction network analysis, GO enrichment analysis and KEGG pathways enrichment analysis. The results showed that Simiao Powder, through multi-components-targets-pathways mode, play an important role on the immune regulation, inflammatory response, metabolism and endocrine regulation, cell signal transduction, cell growth and apoptosis, so as to achieve anti-inflammatory and analgesia, uric acid lowering and metabolism improvement. Further classification and analysis showed that the therapeutic effects of Simiao Powder on gout mainly focused on several modules, such as immune regulation and inflammatory response, metabolism and endocrine regulation, and cell function. To visually reflect the important pathway diagram and its key targets, they were compressed and drawn in Fig. Generally speaking, uric acid crystal deposition in articular cavity is the trigger of gout , 13. TheAmong the major putative targets and pathways of Simiao Powder, most of them are related to the immune regulation and inflammatory response for gout. At the gene target level, PTGS1 is the key target with the largest number of junctions, usually responds to hormone stimulation rapidly to produce prostaglandins and continuously regulate physiological processes, such as mediating responses to inflammatory stress . FurtherGout is defined as a metabolic disorder . StudiesOne mechanism of acute gout inflammation is the noninflammatory phagocytosis of macrophages to dying neutrophils , 59. TheThese modules have their own unique functions, but they are connected and affect with each other, forming a dynamic adjustment network. That reflects the characteristics of multiple compounds, multiple targets and multiple pathways of Simiao Powder in the treatment of gout. Furthermore, gout is often accompanied by other comorbidities , 68, 69 Molecular docking reveals the interaction between components and targets in the network, thereby improving the accuracy of the network . The negSimiao powder decreased serum UA and XOD levels in hyperuricemic-gout mice, and improved renal function via regulating serum BUN and Cr levels. It was noted that both Simiao powder and allopurinol can effectively reduce serum uric acid and XOD, but Simiao powder may bring few side effects and improve renal function in the treatment. Our experimental results are consistent with a previous study . In addiOur research still has some limitations. For example, the actual absorption of drugs may be different from the reality, and drug compounds may react with each other to form new unknown compounds. Furthermore, our screening criteria for drug compounds are based on oral absorption and pharmacodynamics, but some compounds may not be absorbed into the blood but act on intestinal microecology, which is conducive to the regulation of immunity and metabolism.Collectively, this research predicted a multiple compounds, targets, and pathways model mechanism of Simiao Powder in the prevention and treatment of gout, providing new ideas and methods for in-depth research, via vivo experiments.


Periodontal disease has been associated with pregnancy complications including preeclampsia. This bioinformatic study is aimed at investigating the possible role of circulating microRNAs (miRNAs) as mediators of the association between maternal periodontal disease and preeclampsia.  Peripheral blood miRNA profiles of periodontitis and controls were sought from Gene Expression Omnibus (GEO), and differential expression analysis was performed. Experimentally validated circulating miRNAs associated with preeclampsia were determined from the Human MicroRNA Disease Database (HMDD v3.0). Venn diagrams were drawn to identify shared circulating differential miRNAs (DEmiRNAs). Significantly enriched target genes, KEGG pathways, and Gene Ontology (GO) terms for the set of shared DEmiRNA were predicted using miRNA enrichment analysis and annotation tool (miEAA v 2.0). Additionally, the shared DEmiRNA-enriched target genes were analyzed for enriched WikiPathways, BioCarta metabolic pathways, and tissue proteins in the human proteome map.  Among 183 circulating DEmiRNA in periodontitis and 60 experimentally validated miRNA in preeclampsia, 9 shared DEmiRNA were identified. The top among 32 overrepresented target genes included MAFB, PSAP, and CDK5RAP2, top among 14 enriched KEGG pathways were renin-angiotensin system and graft-versus-host disease, and that among enriched 44 GO profiles included “positive regulation of epidermal growth factor-activated receptor activity” and “sequestering of calcium ion.” In the overrepresented target gene set, among 10 enriched WikiPathways, the top included “NAD metabolism, sirtuins, and aging” and “regulation of Wnt/B-catenin signaling by small molecule compounds” and PPAR-related mechanisms was top among 13 enriched BioCarta metabolic pathways.  A circulating 9-DEmiRNA set was significantly linked to both periodontitis and preeclampsia. Enrichment analysis identified specific genes, pathways, and functional mechanisms, which may be epigenetically altered and thereby mediate the biological association of periodontitis and preeclampsia. Periodontitis is a chronic, multifactorial inflammatory disease affecting the tooth-supporting structures and a primary cause of tooth loss . GloballAmong infectious diseases, maternal periodontitis has been linked to pregnancy complications and adverse outcomes including gestational hypertension, preeclampsia, preterm delivery, and low birth weight –16. Evid2 transformed, normalized, and assigned precision weights using “vooma” and miRNA with FDR-adjusted p value < 0.05 and logFC > 0 were considered as circulating DEmiRNA in periodontitis. The Human MicroRNA Disease Database (HMDD v3.0) [The Gene Expression Omnibus (GEO) dataset GSE31568 containing whole blood “miRNome” or miRNA expression profiles from multiple cancer and noncancer diseases was downloaded and filtered to retain 18 periodontitis  and 70 hDD v3.0)  was searhttps://ccb-compute2.cs.uni-saarland.de/mieaa2) [p value < 0.5 and 2 minimum required hits per subcategory.Overrepresentation analysis of the shared circulating DEmiRNA set was performed using the “miRNA enrichment analysis and annotation tool” (miEAA 2.0) web interface (/mieaa2) . The spehttps://maayanlab.cloud/Enrichr/) [p value < 0.05 considered as significant.The set of significantly overrepresented genes by the shared DEmiRNA identified in the previous step was subjected to functional enrichment analysis using the webtool “Enrichr” (nrichr/) . The Wiknrichr/)  was utilnrichr/)  was utilp value < 0.05 and logFC > 0  and graft-versus-host disease. Gene polymorphisms in the RAS components modulate inflammation which have been associated with severe periodontitis  and a loγ levels have also been demonstrated in periodontitis [In the following steps, enrichment analysis of the overrepresented target gene set was conducted. The NAD metabolism, sirtuin, and aging pathway was notably enriched. Lowering of multiple sirtuins has been demonstrated in preeclamptic placental syncytiotrophoblast and considered to represent a response to inflammatory and stress stimuli leading to lower energy reserve . Periododontitis . TherefoThese findings highlight several putative key mechanistic pathways that are epigenetically deregulated in a periodontitis state and can thereby contribute to preeclampsia development. However, the present study has limitations. A major limitation is the lack of validation experimental data from animal or cell models or clinical samples. The DEmiRNAs, target genes, and signaling pathways necessitate verification via targeted experimental approaches. Other limitations include the limited number of samples and absence of sampling population diversity, which may curtail the generalizability of these findings. Availability of large datasets with multiple metadata layers can permit more granular and confident understanding of the range of epigenetic mechanisms involved in this context. Furthermore, shared host genetic predisposition including plasminogen activator inhibitor-1 polymorphism , 71 and Nine shared circulatory DEmiRNAs were identified as candidate linkage epigenetic mechanisms between periodontitis. Epigenetic deregulation of target genes MAFB, PSAP, and CDK5RAP2; KEGG pathways renin-angiotensin system and graft-versus-host disease; GO terms “positive regulation of epidermal growth factor-activated receptor activity” and “sequestering of calcium ion”; and target gene-related pathways associated with sirtuins, Wnt/B-catenin, and PPAR signaling were evident as the most important candidate epigenetic mechanisms. These data provide a preliminary basis for experimental research investigating epigenetic mechanisms involved in the association of periodontitis with preeclampsia.