Increased intrahepatic bile acids cause endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) is activated to maintain homeostasis. UPR dysregulation, including the inositol-requiring enzyme 1α/X-box protein 1 (IRE1α/XBP1) pathway, is associated with adult liver diseases but has not been characterized in pediatric liver diseases. We evaluated hepatic UPR expression in pediatric cholestatic liver disease (CLD) explants and hypothesize that an inability to appropriately activate the hepatic IRE1α/XBP1 pathway is associated with the pathogenesis of CLD.We evaluated 34 human liver explants, including: pediatric CLD , pediatric non-cholestatic liver disease controls , adult CLD, and normal controls. We performed RNA-seq, quantitative PCR, and western blotting to measure expression differences of the hepatic UPR and other signaling pathways.Pathway analysis demonstrated that the KEGG ‘protein processing in ER’ pathway was downregulated in pediatric CLD compared to normal controls. Pediatric CLD had decreased hepatic IRE1α/XBP1 pathway gene expression and decreased protein expression of phosphorylated IRE1α compared to normal controls. IRE1α/XBP1 pathway gene expression was also decreased in pediatric CLD compared to AIH disease controls.Pediatric CLD explants have decreased expression of the protective IRE1α/XBP1 pathway and down-regulated KEGG protein processing in the ER pathways. IRE1α/XBP1 pathway expression differences occur when compared to both normal and non-cholestatic disease controls. Attenuated expression of the IRE1α/XBP1 pathway is associated with cholestatic diseases and may be a target for future therapeutics. Cholestatic liver diseases (CLD), including genetic diseases such as Alagille syndrome (ALGS) and progressive familial intrahepatic cholestasis (PFIC), cause significant morbidity and frequently require consideration for pediatric liver transplantation . During A major factor responsible for the lack of effective treatments for pediatric CLD is the gap in our understanding of disease modifying factors, including the protective hepatic responses required to reduce bile acid-induced injury. Cholestasis and the accumulation of hepatic bile acids increase endoplasmic reticulum (ER) stress in animal models and bile acids alter protein folding in vitro –5. ER stOur previous animal studies have demonstrated that UPR pathways regulate liver bile acid synthesis and transporters, and hepatocyte-specific deletion of the UPR protein XBP1 alters bile acid metabolism and susceptibility to liver injury –13. MiceAntibodies are listed in A total of 34 human livers were obtained from biorepositories at Ann & Robert H. Lurie Children’s Hospital (LCH), Chicago, Illinois. All research was conducted in accordance with the Declarations of Helsinki. This study was approved by the LCH institutional review board and written consent or a waiver of consent was obtained following LCH institutional review board regulations at the time of collection. Liver tissue included both pediatric and adult explants (from the affiliated adult transplant center) that were stored at LCH biorepositories. All diseased liver samples, including CLD and autoimmune hepatitis (AIH) samples, were explants from patients undergoing liver transplantation. Normal control livers were explants from deceased persons who had donated their liver for transplantation but were unable to be utilized for transplantation due to recipient issues. Explanted livers were frozen at -80°C. Samples included 9 pediatric (<18 years of age) normal  livers, 10 pediatric CLD livers (pCLD), 5 pediatric AIH livers, 3 adult CLD livers (aCLD), and 7 adult (>18 years of age) normal  livers. Based on prior known clinical and histopathologic diagnoses, the aCLD cohort was composed of 2 patients with primary biliary cholangitis (PBC) and 1 patient with primary sclerosing cholangitis (PSC), and the pCLD cohort was composed of 5 patients with ALGS and 5 patients with PFIC.Total RNA was extracted from frozen liver samples using TRIZOL reagent , and cDNA was synthesized with the qScript cDNA synthesis kit . Quantitative PCR (qPCR) was performed using Power SYBR Green PCR Master Mix  as previously described . Relativhttp://useast.ensembl.org/index.html) [RNA for RNA-seq was submitted for all 34 samples . The stranded mRNA-seq was conducted in the Northwestern University NUSeq Core Facility. Briefly, total RNA examples were checked for quality using RINs generated from Agilent Bioanalyzer 2100. RNA quantity was determined with Qubit fluorometer. The Illumina Stranded mRNA Library Preparation Kit was used to prepare sequencing libraries from 100 ng of high-quality RNA samples (RIN>7). All 34 samples met QC standards and were used for the study. The kit procedure was performed without modifications. This procedure includes mRNA purification and fragmentation, cDNA synthesis, 3’ end adenylation, Illumina adapter ligation, library PCR amplification and validation. lllumina HiSeq sequencer was used to sequence the libraries with the production of single-end, 50 bp reads at the depth of 20–25 M reads per sample. The quality of reads, in FASTQ format, was evaluated using FastQC. Reads were trimmed to remove Illumina adapters from the 3’ ends using cutadapt . Trimmedex.html) . Normaliex.html) . The cutProtein homogenates from frozen liver tissue were isolated using T-PER protein extraction reagent  containing protease inhibitor cocktails and Halt phosphatase inhibitors . Protein quantification was performed using Coomassie Plus protein assay reagent . Equal amounts of protein samples were subjected to immunoblotting for target proteins and immunoreactive bands were visualized using the Amersham ECL western blotting detection kit according to the manufacturer’s protocol . Antibodies for GAPDH or Cyclophilin B were used as loading controls.Sections of frozen liver tissue were thawed overnight in formalin at room temperature, and histology including H&E and Massons-Trichrome staining was prepared by the Pathology Core Facility at Robert H. Lurie Comprehensive Cancer Center of Northwestern University. Two blinded investigators  scored trichrome histology slides for degree of fibrosis (F0-F4) based on METAVIR scoring by reviewing 10 fields of view at 10x magnification .Data was analyzed using Prism Software and shown as mean ± SEM for all experiments. Comparison between two groups was performed using two-tailed student’s t-tests, comparison between 3 or more groups was performed using ANOVA with Tukey’s multiple comparisons, and significance was defined as a p≤0.05.A summary of available demographic/clinical data for the 34 liver samples used in this study is listed in Thirty-two of 34 (94%) available samples had adequate sample size to grade for fibrosis. Pediatric CLD and AIH explant liver samples had similar degrees of fibrosis (3.4 ± 0.2 and 3.9 ± 0.1 respectively), but both groups demonstrated significantly more fibrosis than pNormal samples , and aCLD samples had significantly more fibrosis than aNormal samples  . When ALprotein processing in endoplasmic reticulum” was among the top 5 differentially expressed pathways (-log10(P) = 25.77)  of RNA-seq data is shown in = 25.77) . Heatmap= 25.77) . To conf= 25.77) . In cont= 25.77) . We next= 25.77) .IRE1α expression , and a 67% decrease in XBP1s expression , compared to pNormal livers (ERdj4) and ER degradation enhancing α-mannoside 1 (EDEM1) were also lower in pCLD samples compared to pNormal livers  and ERdj4 was decreased by 50% . Comparing gene expression of liver IRE1α, XBP1s, EDEM1 and ERdj4 of the two pCLD subgroups  showed a trend toward lower gene expression in the PFIC compared to ALGS samples, however, none of these differences were statistically significant and all were lower than the pNormal controls  in pCLD (in both ALGS and PFIC) samples compared to pNormal controls  while phIn order to evaluate which UPR pathway findings in our pCLD were cholestatic liver disease-specific and/or related to progressive liver disease, we utilized explanted livers from patients with AIH as a disease control for advanced, fibrotic non-cholestatic liver disease. RNA-seq data comparing pCLD and AIH disease controls identified 3643 differentially expressed genes. Pathway analysis of the top 3000 differentially expressed genes did not demonstrate any differences related to protein processing in the ER pathways, ER stress or other UPR-related pathways . In contIRE1α, XBP1s, and EDEM1 by 78%, 77% and 67%    , althoug similar . Gene ex p<0.01) . ERdj4 e p<0.01)  similar  p<0.01)  though t p<0.01) , though  p<0.01)  compared p<0.01) .IRE1α, XBP1s, ERdj4 and EDEM1, these differences were not statistically significant  = 11)  and idennificant . Gene ex similar , and the similar .-/-) and control XBP1fl/fl mice have similar initial activation of the UPR, but LS-XBP1-/- mice have impaired resolution of ER stress with resultant apoptosis [Pediatric cholestatic liver diseases commonly require liver transplantation and there are currently no effective medical therapies that reduce the need for liver transplantation in most of these diseases. During cholestasis, high intrahepatic concentrations of bile acids increase ER stress and hepatocellular injury, and an effective UPR is essential to restore cellular homeostasis –5. Dysrepoptosis . Recentlpoptosis . Additiopoptosis . Puri etpoptosis , 25, 26.poptosis . TherefoUnbiased gene expression pathway analysis demonstrated the novel finding that the “protein processing in endoplasmic reticulum” KEGG pathway was decreased in pediatric cholestatic livers compared to normal controls. In addition, 4 other highly significant pathways included ‘NABA CORE MATRISOME’, the reactome pathway: ‘Extracellular matrix organization’, and the GO Biological Processes pathways: regulation of cell adhesion’ and ‘chemotaxis’. These pathways include genes primarily related to extracellular matrix organization including collagens and proteoglycans, as well as genes related to leukocyte migration and cytokines. Given the fibroinflammatory nature of ALGS and PFIC diseases, these pathway changes are not surprising. However, the finding of a significant pathway related to protein processing within pediatric cholestatic liver diseases has not previously been described.We next demonstrated that hepatic IRE1α protein expression and IRE1α/XBP1 pathway downstream target genes were decreased in pediatric cholestatic disease livers compared to normal controls. This impaired or dysregulated hepatic IRE1α/XBP1 pathway expression is consistent with previously reported findings in murine models of cholestasis and murine and human fatty liver disease , 23, 25.In order to determine whether the IRE1α/XBP1 pathway findings in pCLD explant samples were due to the effects of cholestasis and/or due to the advanced liver disease with extensive fibrosis, we next utilized explanted, cirrhotic livers from pediatric patients with AIH. Despite having end-stage liver disease, 4 of 5 AIH patients had serum bilirubin levels of 2mg/dl or lower. We determined that expression of the IRE1α/XBP1s pathway was lower in pediatric cholestatic liver samples than in autoimmune hepatitis disease-control livers, indicating that these changes are likely due to the cholestatic liver diseases, rather than cirrhosis.In contrast, some UPR changes in the pediatric cholestatic liver disease group identified using comparisons to normal livers were also noted when pCLD and AIH cohorts were compared, indicating that these effects are likely due to the presence of cirrhosis and end-stage liver disease. Both pCLD and AIH samples had evidence of increased p-eIF2α expression, which can lead to decreased protein translation, while selectively allowing translation of certain mRNAs including the transcription factor ATF4 . ATF4 prFinally, adult cholestatic liver disease samples had similar trends of the IRE1α/XBP1 and p-eIf2α -ATF4 expression suggesting that the UPR changes in the cholestatic liver groups may not be restricted to pediatric populations. Although not all of these findings achieved statistical significance, the adult sample sizes were small and very limited. Pathway analysis of genes that were significantly downregulated in pCLD samples compared to aCLD samples demonstrated that there were several differentially expressed pathways related to immune functions and cytokine/inflammatory response. Notably, our adult CLD cohort included patients with PBS and PSC which are autoimmune mediated liver diseases, whereas the pediatric CLD cohort included ALGS and PFIC patients which are not primarily immune-mediated liver diseases and therefore these differences may be secondary to the disease specific features of our cohorts.It is unclear if the reduced hepatic IRE1α/XBP1 pathway expression seen in the pediatric cholestatic liver disease group is caused by the cholestasis and elicited only in end-stage liver disease, or whether these patients had an underlying genetic or developmental impairment of hepatic UPR activation which contributed to their progression to end-stage liver disease. Given the spectrum of clinical manifestations and disease progression seen within ALGS and PFIC patients that cannot be attributed solely to the underlying primary gene defects, our data is consistent with additional defects of IRE1α/XBP1 signaling which may impair ER stress resolution and increase these patients’ susceptibility to develop more rapid progression or more severe liver disease.There are several potential limitations to this study. Adult cholestatic liver disease samples had similar trends of the IRE1α/XBP1 and p-eIf2α -ATF4 expression suggesting that the UPR changes in the cholestatic liver groups may not be restricted to pediatric populations. However, given the small number of adult samples, we cannot exclude the possibility that there were age-related differences in pediatric and adult cholestatic liver diseases that we were unable to identify. The ages of our pediatric CLD patients were not available, although LCH does not routinely perform liver transplants on patients above 18 years of age. Additionally, the liver explant samples were obtained from a biorepository which contained a very limited amount of clinical data and no post-transplantation patient outcomes. Since the study was performed in liver explants and analysis is therefore limited to only one time point late in the progression of disease, we cannot determine whether the impaired XBP1 pathway activation was causative for disease progression, or potentially the consequence of it. Nonetheless, several UPR changes observed in livers with cholestatic liver disease differed from our AIH non-cholestatic disease control group, suggesting that they are more specific to the cholestatic nature of these diseases.This study identified that hepatic gene and protein expression of IRE1α/XBP1 signaling and other UPR signaling differs in progressive cholestatic liver diseases compared to normal controls and therefore may be important in the pathogenesis of disease. Although some changes in the UPR are related to cirrhosis, several are unique to patients with cholestatic liver disease. Since hepatic IRE1α/XBP1 pathway expression is protective in murine models of cholestatic liver injury, the attenuated expression of this canonical UPR pathway may be an important factor in disease progression. These data also identify UPR proteins and genes that may serve as putative targets for the development of novel therapeutic interventions.S1 FigA) Demographic data of 34 human liver explant samples. B) Direct bilirubin level (mg/dL) of autoimmune hepatitis (AIH) patients (n = 5) on the day prior to liver transplantation. pNormal = pediatric normal liver, aNormal = adult normal liver, pCLD = pediatric cholestatic liver disease, aCLD = adult cholestatic liver disease, AIH = autoimmune hepatitis, mos = months.(TIF)Click here for additional data file.S2 FigA) Pediatric cholestatic liver disease (pCLD) (n = 9) and autoimmune hepatitis (AIH) livers (n = 4) demonstrated significantly more fibrosis than pediatric normal samples (n = 9) (***p<0.001). B) Adult cholestatic liver disease (aCLD) livers (n = 3) had significantly more fibrosis than adult normal  samples (n = 7) (***p<0.001). C) Alagille (ALGS) (n = 4) and progressive familial intrahepatic cholestasis (PFIC) samples (n = 5) had significantly more fibrosis than pNormal (n = 9) and had similar METAVIR scores to AIH samples (n = 4) (***p<0.001).(TIF)Click here for additional data file.S3 Fig“protein processing in endoplasmic reticulum” (circled in red) was among the top 10 differentially expressed pathways (-log10(P) = 34.28). B) Utilizing the top significantly upregulated genes in pCLD for pathway analysis KEGG pathway hsa04141 “protein processing in endoplasmic reticulum” was not differentially expressed.A) Utilizing all significantly different downregulated genes in pCLD for pathway analysis, the KEGG pathway hsa04141 (TIF)Click here for additional data file.S4 FigA) The top 100 pathways enriched in differentially expressed genes. B) There was no decreased ATF6 gene expression in PFIC (n = 5) and ALGS (n = 5) livers compared to pNormal livers (n = 9) (p = 0.07 and p = 0.5 respectively).(TIF)Click here for additional data file.S5 Fig“protein processing it endoplasmic reticulum” (circled in red) was the top differentially expressed pathway (-log10(P) = 41.54). B) Utilizing all significantly different upregulated genes in AIH samples for pathway analysis. The top 20 differentially expressed pathways are shown.A) Utilizing all significantly different downregulated genes in AIH samples for Metascape pathway analysis, the KEGG pathway hsa04141 (TIF)Click here for additional data file.S6 Fig“protein processing it endoplasmic reticulum” (circled in red) was the top 10 differentially expressed pathway (-log10(P) = 26.74). B) Utilizing all significantly different upregulated genes in aCLD samples  for Metascape pathway analysis, KEGG pathway hsa04141 “protein processing in endoplasmic reticulum” was not in the top 100 pathways enriched in these genes. The top 20 differentially expressed pathways are shown. C) Utilizing all significantly different downregulated genes in pCLD compared to aCLD samples for pathway analysis. The top 20 differentially expressed pathways are shown. D) Utilizing all significantly different upregulated genes in pCLD samples compared to aCLD for pathway analysis. The top 20 differentially expressed pathways are shown.A) Utilizing all significantly different downregulated genes in aCLD samples  for Metascape pathway analysis, the KEGG pathway hsa04141 (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Conclusion: Pharmacological studies based on network indicate that Drynariae Rhizoma may participate in the regulation of several major signaling pathways through direct or indirect action targets and affect the proliferation and differentiation of multiple types of cells, thus playing an anti-ONFH role, which provides a scientific basis for explaining the material basis and mechanism of its anti- ONFH.Through the network pharmacology thought, the action target of the active ingredients of Drynariae Rhizoma was predicted, and the mapping was combined with the related targets of ONFH, and the key nodes of interaction were identified for enrichment analysis, so as to comprehensively explore the pharmacological mechanism of Drynariae Rhizoma against ONFH. The main active ingredients of Drynariae Rhizoma were screened based on pharmacokinetic characteristics in pharmacokinetic database and analysis platform of TCM system (TCMSP). We used the organic small molecule bioactivity database (PubChem) and Swiss target prediction database to predict related targets based on 2D or 3D structural similarity and then mined the known ONFH therapeutic targets through the Human Mendelian Genetic Database (OMIM) and Pubmed texts. Combined with the predicted targets, String database was imported to construct the OP target interaction network diagram of bone fracture therapy. CytoNCA software was used to topology the key nodes of interaction according to relevant node parameters, and String was imported again to construct the protein interaction network diagram. Finally, biological functions and metabolic pathways of key nodes were analyzed through DAVID database. It was revealed that Drynariae Rhizoma may regulate stem cells, osteoblasts, osteoclasts, and immune cells through multiple pathways, including proliferation, differentiation, immunity, and oxidative stress.  Osteonecrosis necrosis of the femoral head (ONFH), one of the most common orthopedic diseases, is a disease caused by blood circulation disorder of the femoral head. Therefore, it is also called femoral head ischemic necrosis. The pathological changes are mainly bone cell blood loss, degeneration, and necrosis. The main changes in imaging are trabecular fracture and femoral head collapse. Long-term pathological changes will cause degenerative changes of articular cartilage and lead to joint inflammation, especially inflammatory lesions of the hip joint, which will cause pain and stiffness of the hip joint, limited activity, claudication, and other symptoms and eventually lead to the loss of hip joint function , 2. The Drynaria fortunei, an orthopedic plant of Polypodiaceae. It has a warm and bitter taste, enters the liver and kidney meridian, and has the function of tonifying kidney and strengthening bone, healing pain, and relieving pain. It is used for the diseases of falling, falling, falling, broken bones, kidney deficiency and waist pain, weak bones and muscles, etc. Modern pharmacological studies have confirmed that Drynariae Rhizoma can promote the proliferation and differentiation of osteoblasts, promote bone regeneration of femoral head, inhibit the activity of osteoclasts, promote bone absorption of calcium, regulate blood calcium and phosphorus levels, and improve the internal environment of femoral head [Drynariae Rhizoma  is the dral head , 5. At tral head , but itsIn 2007, Hopkins proposed the term “network Pharmacology” [http://lsp.nwu.edu.cn/tcmsp.php) were used to search the chemical components of Drynariae Rhizoma. The molecular structure was obtained from PubChem database (http://pubchem.Ncbi.Nlm.nih.gov//) and saved in smiles format.The traditional Chinese Medicine System Pharmacology (tcmsp) and the analysis platform  can accurately predict the target of the active ingredient based on the similarity between the 2D and 3D structure of the molecule and the known ligand [Swiss database (n ligand . SMILES http://www.Disgenet.org/) to obtain the related genes and target proteins of ONFH. Potential targets for ONFH treatment were obtained by mapping with the above targets.Osteoporosis was searched through the Disgenet database  and betweenness centrality. The node size reflects the degree size, and the thickness of the edge reflects the betweenness centrality.http://string-db. org/) has collected a large number of protein interactions obtained through experimental detection and bioinformatics methods, involving a total of 9 643 763 proteins and 1 380 838 440 interactions [The String database  is a high-throughput biological information annotation database, which can provide systematic and comprehensive biological function annotation information for large-scale genes or proteins, so as to mine their biological significance [P < 0.01 and signaling pathways with P < 0.05 were screened and mapped with Origin 2018.The DAVID database  interaction network is constructed through string, with 393 nodes and 5201 relationships. After screening according to DC, BC, CC, EC, NC, lac, and other topologies by cytonca, 97 key nodes and 2332 relationships are obtained, and string is imported again to construct PPI network see .P < 0.05), a total of 320 enrichment results were obtained. It includes 255 items of biological process (BP), 39 items of molecular function (MF), and 26 items of cellular component (CC). The enrichment results of GO in the top five are shown in After go enrichment analysis of key nodes in David database , estrogen receptor 1 (ESR1), cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), matrix metalloproteinase 2 (MMP-2), androgen protein kinase (AR), chemokine receptor 2 (CCR2), and matrix metalloproteinase 3 (MMP-3) are both ONFH targets and predictive analysis targets of the components related to bone fragment. This connection point may be the target of ONFH simultaneous action of bone splice. Eighty-seven index values were independently associated with Drynariae Rhizoma or ONFH, and three of them were neither predictive analysis index values nor ONFH related index values. ONFH can be applied to ONFH using simultaneous or indirect index values. The results of GO aggregation analysis showed that the effect of Drynariae Rhizoma on ONFH might be related to the whole process of BP, such as immunity, inflammation, capillary transformation, and cell necrosis. MF level indicated that cell growth factor, adenosine triphosphate (ATP), transcription factors, and other molecules fully play the role of anti-ONFH. CC level indicates that the system almost covers all the steps of data signaling pathway from production to recovery, such as extracellular, cytoplasmic, nuclear, cytoplasmic, transcription factor complex, Golgi body, and exosome.β-catenin, and the signal of Wnt antagonism increases with time. Estrogen data signaling pathway plays a major role in the production of ONFH after amenorrhea, and the reduction of estrogen data signaling pathway can lead to the promotion of osteoclast and the increase of osteoclast activity on the one hand [KEGG summarized and analyzed the key nodes and further revealed that the osteoclast is based on a variety of methods, from proliferation, differentiation, and immunity, and from multiple levels, such as the regulation of oxidative stress on stem cells, osteoblasts and osteoclasts, and cellular immunity. The classical Wnt data signaling pathway plays a bidirectional role in the differentiation of osteoblasts and osteoclasts. Inflammatory factors play a major role in the ONFH system , 16. Theone hand . On the one hand . Rap1 isone hand . Studiesone hand . Studiesone hand –24. The one hand , 26. Theone hand –30. It fDue to the complexity of the effective components of Chinese medicinal materials and the limitations of experimental research ideas, there is still a lack of investigation reports on the chemical targets and the effectiveness of ONFH alone. This analysis, using network pharmacology, is the first to decipher the potential active ingredients of Drynariae Rhizoma, which can be based on the multitarget, multimode, multisystem important molecular structure in the efficacy of ONFH. As there are totally 16 active ingredients and 55 key targets of Drynariae Rhizoma in treating ONFH , no dataNetwork pharmacology is widely used in life science research in the fields of drugs, target identification, lead compound discovery, mechanism of action analysis, clinical research, efficacy, and safety factors. Based on the prediction and analysis of network pharmacology, we can analyze the relevant chemical substances, potential therapeutic targets, and key signaling pathways for the treatment of ONFH or select reasonable components for molecular docking simulation and then quantitatively analyze the fingerprints to establish a scientific, rational, and easy-to-use basis for multi-indicator evaluation, providing a basis for future relevant scientific research.


The active compounds in DO, their targets, and targets associated with hyperlipidemia were screened across various databases, and the hidden targets of DO in treating hyperlipidemia were forecast. The compound-target (C-T), protein-protein interaction (PPI), and compound-target-pathway (C-T-P) networks of DO were set up with Cytoscape software. The hub genes and core clusters of DO predicted to be active against hyperlipidemia were calculated by Cytoscape. The DAVID database was adopted for Gene Ontology (GO) analysis and KEGG pathway enrichment analysis. Next, we used the high-sucrose-fat diet and alcohol (HFDA)-induced hyperlipidemia rats to evaluate the hypolipidemic effect of DO.  In this study, we obtained 264 compounds from DO, revealed 11 bioactive compounds, and predicted 89 potential targets of DO. The network analysis uncovered that naringenin, isorhamnetin, and taxifolin might be the compounds in DO that are mainly in charge of its roles in hyperlipidemia and might play a role by modulating the targets . The pathway analysis showed that DO might affect diverse signaling pathways related to the pathogenesis of hyperlipidemia, including PPAR signaling pathway, insulin resistance, AMPK signaling pathway, and non-alcoholic fatty liver disease simultaneously. Meanwhile, in the HFDA-induced hyperlipidemia rat model, DO could significantly decrease the level of TC, TG, LDL-c, and ALT in serum, and increase HDL-c as well. The liver pathological section indicated that DO could ease liver damage and lipid cumulation.  In summary, the biological targets of the main bioactive compounds in DO were found to distribute across multiple metabolic pathways. These findings suggest that a mutual regulatory system consisting of multiple components, targets, and pathways is a likely mechanism through which DO may improve hyperlipidemia. Validation experiments indicated that DO may treat hyperlipidemia by affecting NAFLD-related signaling pathways. As an abnormality of lipid metabolism, hyperlipidemia has characteristics such as an increase of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-c), and/or a decrease in high-density lipoprotein cholesterol (HDL-c) in circulating plasma. Hyperlipidemia is associated with etiopathogenesis of different diseases, including atherosclerosis, hypertension, metabolic syndrome, and cardiovascular disease (CVD) . NumerouMonotherapy with pharmacologic agents is inherently challenging for the treatment of hyperlipidemia. Indeed, hyperlipidemia requires a comprehensive approach including changes in diet, exercise, and pharmaceutical therapy. Currently, the commonly used lipid-lowering drugs such as statins and fibrates usually have side effects and contraindications with long-time application, and many clinicians would prefer their patients to regulate dyslipidemia through healthy lifestyle modifications . In contDendrobium officinale (DO) is the most valuable species in the Dendrobium genus, which is mainly observed in the Zhejiang, Yun-nan, and Guangxi Province of China [−/−mice [of China . Its steof China . Recent of China –15. Rema[−/−mice  and in o[−/−mice . Our pre[−/−mice , 18 reveNetwork pharmacology is a new approach of “active compound-target- disease” interaction network based on multidisciplinary integration theory . ComplicTherefore, the molecular mechanism of DO as a treatment for hyperlipidemia was clarified with network pharmacology. Furthermore, we validated experiments in hyperlipidemic rats induced with high-sucrose-fat diet and alcohol (HFDA) to further verify the effect of DO in ameliorating hyperlipidemia. These findings may lay a foundation for further exploration of the therapeutic value of DO as a treatment for hyperlipidemia and other clinical applications.This research adopted network pharmacology and validation experiments to unveil the biochemistry basis and underlie mechanisms of DO as a treatment for hyperlipidemia. http://www.cnki.net), Wanfang Database (http://www.wanfangdata.com.cn/index.html), and PubMed (http://www.ncbi.nlm.nih.gov). Then, analyzing and collating the literature to obtain the chemical constituents of DO were performed.A comprehensive search of DO's chemical compounds was set up with different bibliographical databases such as China National Knowledge Internet (CNKI: https://pubchem.ncbi.nlm.nih.gov/) [http://tcmspw.com/index.php) [All DO's compounds were input into PubChem (ih.gov/)  and Traddex.php)  to get tWe used the TCMSP to analyze the DO compounds collected above, and two silico ADME models were used to obtain the active ingredients in DO, including Per OB  and Per DL (forecast of drug-likeness). The thresholds of the two screening models were set as Per OB ≥ 30% and Per DL ≥ 0.18. The obtained active compounds were adopted as the candidate active compounds .http://www.Uniprot.org/), including name, gene ID, and organism. The active compounds without targets were eliminated; the active compound-target dataset was set up.TCMSP was adopted to screen the targets of candidate active substances in DO. The collected targets were confirmed with the Uniprot protein sequence resource (http://cytoscape.org/) [Cytoscape 3.7.0 platform (pe.org/)  providedpe.org/) .https://www.genecards.org/) [https://omim.org/) [http://www.disgenet.org/) [, and PHGKB database (https://phgkb.cdc.gov/PHGKB/startPagePhenoPedia.action/) [In this study, the disease targets of hyperlipidemia were obtained by searching four databases, including GeneCards database (ds.org/) , OMIM daim.org/) , DisGeNEet.org/) , and PHGaction/) .Disease targets were collected using “hyperlipidemia” or “HLP” as keywords. In GeneCards database, target genes were chosen with the Relevance Score ≥1, and in DisGeNET database, target genes were chosen with the Gene-Disease Score ≥0.1. After duplicates were deleted, hyperlipidemia-related targets were obtained .https://bioinfogp.cnb.csic.es/tools/venny/index.html) to acquire the common targets for DO bioactive compound targets and the hyperlipidemia associated targets.The Venn diagram was set up through an online website (http://string-db.org/) [http://apps.cytoscape.org/apps/cytohubba) [Then, the common target genes were input into the STRING database (db.org/)  to explotohubba)  plugin bhttps://apps.cytoscape.org/apps/mcode).In the complicated biological data network, several genes or proteins are closely associated with each other with the same or similar functions, so they can exert a significant biological coordination effect as a cluster. The data of every node in the network assists in analyzing clusters and constructing functional modules . The funhttps://david.ncifcrf.gov/), which is an integrated functional annotation tool, was used to know the biological significance behind the large gene datasets [As an effective bioinformatics tool, the GO analysis can characterize molecular function (MF), cellular components (CC), and biological process (BP) of genes . The KEGdatasets . In thisWe used Cytoscape software to construct the “Compounds-targets-pathways (C-T-P)” network for DO as a treatment for hyperlipidemia according to the active compounds in DO, intersecting the targeted genes related hyperlipidemia with the DO active compounds through the pathway from KEGG analysis. In this network, every compound, target, or pathway is represented by a node, and every interaction by an edge. At the same time, the plugin Network Analyzer of Cytoscape 3.7.0 was adopted to analyze the degree representing the number of edges interacting with the node.Biochemical reagents as TC, TG, HDL-c, ALT, and AST were purchased from Meikang Biotechnology Co. . Nanjing technology Co., Ltd.  offered hematoxylin and eosin (HE).90 size distribution of 35.01 ± 1.19 μm) and pure water used to prepare with a concentration of 60 mg/mL (crude drug) for reserve. High-sucrose-fat diet  and basic diet were all produced by Zhejiang Academy of Medical Science .Zhejiang Senyu Co., Ltd. (Zhejiang China) offered DO, the origin is Zhejiang , and the growing age is three years old. DO was pulverized into powder (Dn = 30). The hyperlipidemia model of rats induced by high-sucrose-fat diet and alcohol (HFDA) and the specific methods were as follows: the rats were fed with high-sugar and high-fat diet ; at the same time, Red Star (Hongxing) and Erguotou  were added into the drinking water of the rats; 4% alcohol was given for 4 consecutive days at the beginning of modeling, grew to 8% on the fifth day, and then increased by 4% every other 3 days until 22%  offered Sprague-Dawley (SD) rats (ntil 22% . Once thn = 10): (1) normal control group (NG); (2) hyperlipemia model group (MG); (3) DO treatment group . The NG rats received the basic diet and water throughout the whole experiment, and the remaining twenty rats were supplied with HFDA. After modeling for 8 weeks, the HFDA-induced hyperlipidemic rats were randomly assigned to 2 groups (MG and DO) according to the serum TC level, and then the MG and DO groups continued to be supplied with HFDA. NG and MG were given corresponding distilled water by intragastric administration, and DO was given DO . During the experiment, the record of body weight was made every week. After 6 weeks of administration, the blood was collected through orbital vein after 12 hours of fasting. After the experiment, the livers were weighed, and the biggest lobes of livers were fixed with 4% paraformaldehyde, and the rest were kept at −80°C until the next use.Then, SD rats were firstly fallen into 3 groups . Friedewald's formula as LDL-c = TC - (HDL-c + TG/2.2)  was adopLiver histopathology was evaluated with hematoxylin-eosin staining (H&E) and Oil red O staining. The liver specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced at 3 µm thickness, and then, the staining of sections acquired was made with hematoxylin and eosin (H&E) for the histological test . 0.2% OiP < 0.05 was of statistical significance. SPSS 17.0 statistical software was adopted to perform all analyses.All measurements in this research were shown as means ± standard deviation and subjected to one-way analysis of variance (ANOVA). According to comprehensive search by using various bibliographical databases, we obtained about 264 compounds in DO; the main chemical compounds in DO are phenanthrenes, bibenzyls, phenols, acids, esters, amides, saccharides, glycosides, essential oils, and so on  diagram was established on basis of hidden ingredients and targets by Cytoscape software, and it is shown in Through these four databases, GeneCards, DisGeNET, OMIM, and PHGKB, we obtained 802, 69, 102, and 545 hyperlipidemia-associated targets, respectively. Concluding the outcomes of different databases and eliminating duplicate genes, 1210 targets associated with hyperlipidemia were picked up. The details of hyperlipidemia-associated targets were offered in Supplementary (https://bioinfogp.cnb.csic.es/tools/venny/index.html) to acquire the 44 common targets for DO bioactive compound targets and the hyperlipidemia related targets (The Venn diagram was set up by an online website ( targets .We input these common genes into the STRING online website (PPI score >0.4) to create the PPI network, and finally the network made up of 44 interaction nodes and 245 interaction edges were created. As shown in The Hub genes were screened by CytoHubba (the plug-in based on Cytoscape) from the interaction network. And then, we used MCC algorithm to find out the top 6 Hub genes of DO as a treatment for hyperlipidemia , which wOn basis of the MCODE clustering exploration, the key PPI network of DO for hyperlipidemia could be fallen into 3 modules . There wP value, top 10 KEGG enrichment pathways were acquired and framed in a bubble plot (KEGG enrichment exploration was conducted on cluster 1 (McOde1). According to the ble plot . As showTo elucidate the multiple mechanisms of DO in treating hyperlipidemia from an integrated level, GO enrichment analysis was made on the biological process, molecular function, and cellular component of the 44 common targets. As shown in To construct the “Compound-target-pathway (C-T-P)” network as shown in By analyzing C-T-P network, we picked out 5 important signaling pathways that were significantly associated with DO as a treatment for hyperlipidemia. As shown in P < 0.01). After administrating the DO for 6 weeks, the serum TC, TG, and LDL-c significantly decreased, and the serum HDL-c was elevated in a significantly different way (P < 0.05 − 0.01) by comparing with the MG. These results indicate that DO can decrease the serum TC, TG, and LDL-c and increase the serum HDL-c to influence the blood lipid.To examine the effect of DO on the blood lipid degree in HFDA-induced hyperlipidemia rats, serum TC, TG, LDL-c, and HDL-c both were measured before and after DO administration. As shown in P < 0.01) without impact on AST (P < 0.05) (Long-term disturbed lipid homeostasis can lead to hepatic lipid lesions. We detected serum AST and ALT levels after administration of DO to decide the liver function. t on AST . After a < 0.05) , but no  < 0.05)  was showCompared with NG, the liver weight was greatly grown in MG rats, and by comparing with MG, the liver index in treatment group was significantly decreased . The livIn this research, 10 photomicrographs of HE-staining were chosen from each group to calculate the NAS mark at a magnification of ×400. According to liver sections in NG, the structures of tissues were normal in polygonal edge, clear cell boundary, and clear round nucleus . On the Oil-red O staining showed the presence of lipid cumulation in both MG and DO groups compared with NG, but the lipid cumulation in DO group was less than that in MG group Figures .Hyperlipidemia is global threat to public health, contributing to significant annual mortality and enormous health care costs. DO is a tonic herb described in the Chinese Compendium of Materia Medica and has a longstanding history of use as a health food for the folk treatment of diseases related to yin-deficiency for decades, and modern pharmacological studies have confirmed that DO can lower blood lipids in animal models , 17.Network pharmacology approach is a useful approach to research the bioactive compounds and mechanisms of TCM in treating hyperlipidemia. This approach combines insights about the drugs, target proteins, and diseases to form drug-target-disease networks, which are similar to the TCM principles of multi-component, multi-pathway, and multi-target synergy . This reIn this research, we obtained 11 bioactive compounds in DO from various bibliographical databases and predicted 89 potential targets, through the network  and KEGG pathway analyses, and specifically found that naringenin (MOL004328), isorhamnetin (MOL000354), and taxifolin (MOL004576) might be the main compounds in DO that are responsible for its effects on hyperlipidemia. These compounds may act by modulating the above targets . The pathway exploration in our study suggests that DO may effect multiple signaling pathways related to the pathogenesis of hyperlipidemia, including the PPAR signaling pathway, insulin resistance, AMPK signaling pathway, nonalcoholic fatty liver disease (NAFLD), and thyroid hormone signaling pathways simultaneously. The GO enrichment analysis of targets revealed that the ingredients of DO may have a synergistic effect on the treatment of hyperlipidemia, mainly by regulating cholesterol homeostasis, affecting cholesterol metabolic processes, and aiding in the clearance of low-density lipoprotein particles. Meanwhile, in the HFDA-induced hyperlipidemia rat model, DO could significantly decrease the level of TC, TG, LDL-c, and ALT, increase HDL-c in serum as well, and ease liver damage and lipid cumulation.Several of the active compounds predicted to be active by the analyses in this study have actually been shown to be effective in reducing serum TC and lipid deposition.In vitro experiments have demonstrated that taxifolin inhibits cholesterol synthesis in a dose- and time-dependent manner. Mechanistic studies have suggested that taxifolin inhibits the activity of HMG-CoA. Further, cellular cholesterol esterification and synthesis of triacylglycerol and phospholipids were also significantly suppressed in the presence of taxifolin [For example, naringenin is a regulator for cholesterol efflux that enhances lipoprotein profiles and protects against cardiovascular disease, and that regulation is mediated by the ATF6 branch of the ER stress and PI3K/AKT pathway . Importaaxifolin . Meanwhiaxifolin , which pAfter exploring the PPI network of DO for hyperlipidemia, the key targets of DO for hyperlipidemia included AKT1, TNF, PPARG, ADIPOQ, and APOB. These targets are primarily involved in regulating cholesterol homeostasis, cholesterol metabolic processes, and clearance of low-density lipoprotein particles.AKT is a serine/threonine protein kinase that has been implicated in numerous cellular processes . AKT1 haα inhibits cholesterol efflux by causing overexpression of micro-RNA-101and inhibiting the expression of ATP-binding cassette transporter A1 (ABCA1) [As an inflammatory element, TNF is generated by activating monocytes or macrophages. While oxidizing low-density lipoprotein (ox-LDL), TNF can grow the binding of ox-LDL to endothelial cells and further improve the expression of ox-LDL receptor . Existin (ABCA1) .PPARG is a significant regulator of lipid homeostasis. Activating or inhibiting PPARG expression causes changes in the activity of proadipogenic and antiadipogenic regulators. Lipid mobilization is controlled by these processes to adipocytes by boosting adipogenesis and controlling the expression of adipocyte-secreted proteins and adipocytokines including leptin and adiponectin, decreasing adipotoxicity .Adiponectin  is a unique adipocytokine that includes growing insulin sensitivity, boosting fatty acid oxidation, stopping inflammatory reactions, and inducing endothelium-dependent, nitric oxide-mediated vasodilation . ResearcApolipoprotein B (APOB) is a main structural protein in very low-density lipoprotein, intermediate-density lipoprotein, LDL, and lipoprotein (a) , which cIn summary, AKT1, TNF, PPARG, ADIPOQ, and APOB may be targets for the action of DO in the treatment of hyperlipidemia.Based on the KEGG enrichment and C-T-P network analysis, DO has been hypothesized to influence several key pathways, which are important in antihyperlipidemia. Those signaling pathways include the PPAR signaling pathway, insulin resistance, the AMPK signaling pathway, and nonalcoholic fatty liver disease (NAFLD).α and PPAR-γ mainly regulate lipid metabolism, insulin sensitivity, bile acid (BA), and glucose homeostasis [β/δ regulates lipid metabolism, glucose homeostasis, anti-inflammatory effects, and fatty acid oxidation, which are keys areas for the action of drugs for hyperlipidemia. Experimental data have suggested that naringenin regulates the activity of nuclear receptors PPAR , resulting in decreased production of cholesterol and bile acid [The PPAR signaling is important in many diseases such as obesity, diabetes, and atherosclerosis. PPAR-eostasis . Similarile acid .Insulin signaling is connected to Type 2 diabetes and related diseases like obesity, hyperlipidemia, and atherosclerosis that are caused by insulin resistance . PublishAMPK activation promotes the activation of signaling pathways related to fatty acid oxidation and inhibits anabolic ATP-consuming processes such as gluconeogenesis and synthesis of lipids and proteins . AMPK haα, and FAAs, and also associated with NAFLD progression. NAFLD also presents with atherosclerotic dyslipidemia, postprandial lipemia, and HDL dysfunction [The development of NAFLD is tightly tied to other clinical developments such as obesity, dyslipidemia, diabetes, and metabolic syndrome , associafunction .Through preliminary network pharmacology analysis, we used PPI and KEGG analysis to get potential targets and key signaling pathways of DO against hyperlipidemia. The results indicated that NAFLD signaling pathway and other NAFLD-related signaling pathways are involved in the treatment of hyperlipidemia by DO. Then, we established a HFDA-induced hyperlipidemia rat model and supplied with DO to verify its antihyperlipidemic effect. The outcomes displayed that DO could significantly reduce TC, TG, and LDL-C, grow HDL-C in hyperlipidemia model rats, and improve the damage caused by liver and lipid metabolism disorder.Dyslipidemia often co-existed with NAFLD/NASH, and the relationship between dyslipidemia and NAFLD is bidirectional. In this study, liver pathological section implied that DO can ameliorate hepatocyte ballooning, steatosis, and inflammation to stop NAFLD caused by lipid metabolism disorders. The lipid droplets in liver were stained with Oil red O, and it was found that DO reduced the lipid droplets, which showed that DO better influences serum TC and liver function; it is further verified that DO may treat hyperlipidemia by affecting NAFLD-related signaling pathways.This research made network pharmacology and validation experiments to reveal the biochemistry foundation and underlying mechanisms of DO as a treatment for hyperlipidemia. There are multiple active compounds in DO that act on multiple distinct targets through a variety of biological processes and pathways to treat hyperlipidemia; meanwhile, validation experiments indicated that DO may treat hyperlipidemia by affecting NAFLD-related signaling pathways.However, these conclusions are limited by their purely bioinformatic design and preliminary experimental verification. But these findings suggest that DO, a botanical treatment from TCM, may have a true pharmacological basis for a clinical effect. As such, we hope that these findings may motivate future network pharmacology-based investigations of other Chinese herbs that may be promising treatments for against hyperlipidemia and related disorders.


Background: Epimedii Folium, as a natural botanical medicine, has been reported to have protective effects on intestinal diseases by modulating multiple signaling pathways. This study aimed to explore the potential targets and molecular mechanisms of Epimedii Folium extract (EFE) against cisplatin-induced intestinal injury through network pharmacology, molecular docking, and animal experiments.Methods: Network pharmacology was used to predict potential candidate targets and related signaling pathways. Molecular docking was used to simulate the interactions between significant potential candidate targets and active components. For experimental validation, mice were intraperitoneally injected with cisplatin 20 mg/kg to establish an intestinal injury model. EFE  was administered to mice by gavage for 10 days. The protective effect of EFE on intestinal injury was analyzed through biochemical index detection, histopathological staining, and western blotting.Results: Network pharmacology analysis revealed that PI3K-Akt and apoptosis signaling pathways were thought to play critical roles in EFE treatment of the intestinal injury. Molecular docking results showed that the active constituents of Epimedii Folium, including Icariin, Epimedin A, Epimedin B, and Epimedin C, stably docked with the core AKT1, p53, TNF-α, and NF-κB. In verified experiments, EFE could protect the antioxidant defense system by increasing the levels of glutathione peroxidase (GSH-Px) and catalase (CAT) while reducing the content of malondialdehyde (MDA). EFE could also inhibit the expression of NF-κB and the secretion of inflammatory factors, including TNF-α, IL-1β, and IL-6, thereby relieving the inflammatory damage. Further mechanism studies confirmed that EFE had an excellent protective effect on cisplatin-induced intestinal injury by regulating PI3K-Akt, caspase, and NF-κB signaling pathways.Conclusion: In summary, EFE could mitigate cisplatin-induced intestinal damage by modulating oxidative stress, inflammation, and apoptosis. Cisplatin is a broad-spectrum and highly effective platinum-based antitumor drug, which is widely used in the treatment of various solid tumors such as lung cancer , gastricReactive oxygen species (ROS) react with biological macromolecules , resulting in oxidative damage to cell membranes and lipid peroxidation, thus causing extensive tissue damage . CisplatEpimedium sagittatum Maxim, Epimedium koreanum Naka, Epimedium brevicomu Maxim or Epimedium pubescens Maxim , is a well-known Chinese herb that has been used in functional foods and complementary medicines for more than 2000 years. Epimedii Folium has the effects of tonifying the kidney, strengthening the yang, dispelling wind, and removing dampness in the theory of traditional Chinese medicine (TCM). Hence, it is applied to prevent and treat sexual dysfunction, osteoporosis, rheumatism, neurasthenia, chronic nephritis, and cardiovascular diseases. Modern pharmacological studies have proved that Epimedii Folium has a variety of pharmacological activities, including antioxidant  Database. Based on the Oral Bioavailability (OB) ≥ 30% and Drug-Like (DL) ≥ 0.18 as thresholds, the potential active compounds of Epimedii Folium were screened by ADME analysis.The active ingredients of Epimedii Folium have collected through the Traditional Chinese Medicine Systems Pharmacology , PharmGkb (https://www.pharmgkb.org), and GeneCards (https://www.genecards.org/) database. Potential target genes of Epimedii Folium for the treatment of intestinal injury were obtained through the Veeny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/) intersection. Then we used Cytoscape (v.3.8.0) software to build the active compound-target-intestinal injury network.All the possible targets against intestinal injury have retrieved from the OMIM (https://string-db.org/). Then, the PPI network results were imported into Cytoscape v.3.8.0 (www.cytoscape.org/) for network generation and further analysis. Moreover, the median of three topological parameters, “Betweenness Centrality,” “Closeness Centrality,” and “Degree”, were calculated to evaluate the topological importance of nodes in the PPI network.PPI network of acquired drug targets was created using the STRING database , biological process (BP), and cellular components (CC). KEGG enrichment analysis was performed to screen out the potential signaling pathways of Epimedii Folium in the treatment of intestinal injury diseases.To explore the role of essential target genes through bioinformatics description and annotation, GO and KEGG pathway enrichment analysis on shared target genes was performed using R software with Bioconductor package under the conditions of http://www.rcsb.org/) in PDB format. The chemical structures of Icariin, Epimedin A, Epimedin B, and Epimedin C were obtained from the PubChem database (https://pubmed.ncbi.nlm.nih.gov/). The 3D structures of active compounds were constructed and optimized by ChemBio3D Ultra 14.0.0.117 software, and their energy was minimized using the MM2 algorithm. PyMol software removed water molecules and organic compounds from receptor proteins. The target protein receptor molecules were hydrotreated and charged by AutoDockTools-1.5.6 software, and the compounds and target protein receptors were converted to PDBQT format. Finally, Auto Dock Vina software was used to verify the molecular docking of potential targets and components. Each group of molecular docking was run three times, and the ionization energy was recorded. The visualization of docking results with the best binding ability was presented using BIOVIA Discovery Studio (2019) Visualizer.To validate the association between compounds and targets, molecular docking simulations were performed using AutoDockTools-1.5.6 software. Crystal structures of crucial target proteins were downloaded from the Protein Data Bank database  were provided by Beijing Hua-Fu-Kang Biotechnology Co., Ltd., license No. SCXK (Beijing) 2019-0008. Mice were allowed free access to food and water in a rearing chamber free of specific pathogens and a 12-h light/dark cycle. The temperature controlled at 22.0°C ± 2.0°C, and the humidity maintained at 60.0% ± 10.0%.n = 10): normal group, model group, cisplatin + EFE (100 mg/kg) group and cisplatin + EFE (200 mg/kg) group. Since there is currently no therapeutic agent for cisplatin-induced myocardial injury in the clinic, a group of positive drugs was not set up in the present work. EFE was dissolved in 0.05% carboxymethylcellulose sodium (CMC-Na) and administered to mice by gavage for ten consecutive days. On the 7th day of EFE administration, except the normal group, mice in the other groups were intraperitoneally injected with cisplatin 20 mg/kg to establish an acute intestinal injury model .Epimedium brevicomu Maxim by Professor Han Mei from the College of Chinese Medicinal Materials, Jilin Agricultural University. Epimedii Folium extract was prepared and quantified according to previous reports , and identified as  reports . High-pe reports . The quaThe use of cisplatin in chemotherapy often leads to severe intestinal toxicity. Our research group has fully proved that cisplatin can cause intestinal toxicity in the body at a dose of 20 mg/kg in previous studies . CisplatBlood samples were collected from mouse ocular venous plexus and centrifuged at 3,500 rpm for 10 min to separate serum samples. The levels of DAO, TNF-α, IL-1β, and IL-6 in serum samples were determined using ELISA kits according to the manufacturer’s instructions. 10 µl serum samples were added to 96 well plates coated with matrix and incubated at 37°C for 30 min, then chromogenic agent was added, and the absorbance of samples in each group at 450 nm was measured within 15 min. Finally, the content of biochemical indicators was calculated through the concentration-absorbance curve.Tissue samples were accurately weighed, 0.9% sterile saline was added according to weight (g): volume (ml) = 1:9, then homogenized with a tissue homogenizer, and finally centrifuged at 3,500 rpm for 10 min to collect the supernatant for subsequent biochemical parameter detection. The levels of MDA, GSH-Px, and CAT in tissue homogenate were determined using corresponding commercially available detection kits according to the instructions provided by Nanjing Jiancheng Bioengineering Institute.2∼3 cm fresh mouse duodenum, jejunum, and ileum were immersed in 10% neutral buffered formalin for at least 48 h, routinely dehydrated and deparaffinized, embedded in paraffin, and then cut into 5 µm thick sections. Paraffin sections were stained with H&E solution, and then pathological changes were observed with a light microscope, and images were collected. The degree of intestinal injury was assessed according to the histopathological scoring system.5 μm thick tissue sections were stained with Hoechst 33258 staining solution according to the kit instructions. After standing for 5 min, the sections were washed twice with phosphate buffer  for 3 min each time and then sealed with anti-fluorescence quenching sealant. Finally, nuclear apoptosis was observed and photographed under a fluorescence microscope . Hoechst 33258 staining results were quantified by Image-Pro Plus 6.0 software.Paraffin tissue sections were deparaffinized and hydrated with xylene and gradient ethanol solution and then repaired with citrate buffer  under microwave conditions at medium-high temperature for 8 min. After returning to room temperature, 5% bovine serum albumin (BSA) was added dropwise to block the sections for 20 min, and then NF-κB P65 (1:100) primary antibody solution was added and incubated overnight at 4°C. Biotinylated goat anti-rabbit IgG solution was added and incubated at 37°C for 30 min. Diluted Cy3-SABC (1:400) solution was added dropwise to the sections and incubated at 37°C in the dark for 30 min. 4, 6-diamino-2-phenylindole (DAPI) solution was added for nuclear staining. Finally, the slices were sealed with anti-fluorescence quenching sealant. The fluorescence expression intensity of the antibody was observed under a fluorescence microscope  and photographed. Immunofluorescence quantitative analysis was performed using Image-Pro Plus 6.0 software.Total protein samples were obtained by lysing mouse intestinal tissues with RIPA lysis buffer supplemented with protein phosphatase inhibitors. Total protein concentration in the tissues was determined using the BCA protein quantification kit according to the instructions provided by the manufacturer. The protein samples were separated on 15% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes by electrophoresis. Then, the PVDF membranes were sealed with 5% skimmed milk at room temperature for at least 2 h and incubated with p-PI3K , PI3K , p-Akt , Akt , p-p53 , p53 , Bax (1:2000), Bcl-2 (1:2000), cleaved caspase-9 , caspase-9 , cleaved caspase-3 , caspase-3 , cytochrome c , and β-actin (1:2000) primary antibody solutions at 4°C overnight. The membranes were then incubated with the HRP-conjugated secondary antibody solution at room temperature for 1.5–2 h. Finally, the intensities of protein bands were quantified by Quantity One software .p < 0.05 or p < 0.01 was considered statistically significant.All experimental data were expressed as mean ± standard deviation (Mean ± S.D). Statistical significance was analyzed by SPSS version 19.0 software. The statistical histograms were made by GraphPad Prism 8.04 software . In all cases, Using the keyword “Epimedii Folium,” 23 active compounds were retrieved from TCMSP database. The potential active ingredients were collected under the screening conditions of OB ≥ 30% and DL ≥ 0.18. We searched through TCMSP, combined with Chinese and foreign literature supplements, deleted the active ingredients without targets, and finally obtained 25 active ingredients of Epimedii Folium, as shown in The identifiable candidate targets of Epimedii Folium associated with intestinal injury were introduced into the STRING database to set up the PPI network diagram. Subsequently, the PPI network of candidate targets was inputted into Cytoscape-v3.8.0 software for visualization. We excluded disconnected nodes from the PPI network and finally obtained 197 nodes and 3,491 edges . The netR software. A total of 2768 GO terms (p < 0.05) were obtained from GO enrichment analysis, including 2,437 biological process (BP) terms, 104 cellular components (CC) terms, and 227 molecular function (MF) terms. The top 10 markedly enriched biological processes of the 200 core targets were depicted in p < 0.05), and the finally sorted-out top 30 vital signaling pathways are shown in To explore the biological functions and relevant pathways of Epimedii Folium associated with intestinal injury, GO and KEGG pathway enrichment analysis of the 200 candidate targets was carried out after calculation with The PPI network screened out the core targets of Epimedii Folium against intestinal injury. To verify the reliability of candidate targets in the PPI network, four active compounds from Epimedii Folium were docked with AKT1, p53, TNF-α, NF-κB caspase 3, and caspase 9 using Auto Dock Vina software. The Vina score  represented the binding affinity between the target protein and compounds. The lower the Vina score, the more stable the ligand binding to the receptor, and the greater the possibility of molecular interaction. As the score for molecular docking, the binding energy less than -5 kcal/mol indicates a more vigorous binding activity. Interestingly, the results showed that Icariin, Epimedin A, Epimedin B, and Epimedin C readily bind to AKT1, p53, TNF-α, NF-κB, caspase3, and caspase9 due to their very low binding energies . These rp < 0.01). However, EFE administration could dramatically inhibit the increase of the biochemical index (p < 0.05 or p < 0.01), indicating that EFE could effectively alleviate the intestinal damage caused by cisplatin.As can be seen from the line chart in Gastrointestinal Morphology of mice in each experimental group was observed . It was p < 0.01). Interestingly, the intensity of fluorescence expression was significantly attenuated after EFE administration (p < 0.05 or p < 0.01). Moreover, as depicted in p < 0.01). However, the secretion levels of proinflammatory factors were suppressed considerably after EFE administration, especially in the high-dose EFE group (p < 0.05 or p < 0.01). These results suggested that EFE might effectively ameliorate cisplatin-induced intestinal injury through anti-inflammatory effects.To verify whether EFE could ameliorate cisplatin-induced intestinal injury through anti-inflammatory effects, the expression levels of NF-κB p65 in mouse intestinal tissues were detected by immunofluorescence staining. As shown in stration  (p < 0.0p < 0.01). Interestingly, EFE could reverse the changes of these oxidative stress indices (p < 0.05 or p < 0.01).To evaluate whether EFE could protect intestinal tissues from oxidative stress damage caused by cisplatin, the levels of MDA, GSH-Px, and CAT in intestinal tissues were detected using corresponding kits . The resp < 0.05 or p < 0.01).As shown in p < 0.01). However, the levels of these proteins were substantially reversed after EFE administration, and especially the high-dose EFE showed better effects (p < 0.05 or p < 0.01).To further explore the protective mechanism of EFE on cisplatin-induced intestinal apoptosis, western blotting was used to detect the expression levels of apoptosis-related proteins in mouse intestinal tissues . The res effects  (p < 0.0Escherichia coli by regulating the p38 MAPKs signal pathway (Epimedii Folium is a critical Chinese herbal medicine widely used to treat various malignant diseases. According to literature reports, flavonoids extracted from Epimedii Folium could ameliorate intestinal damage by regulating multiple signaling pathways. For example, icariin, the main flavonoid in Epimedium, effectively alleviated LPS-induced impairment of intestinal goblet cell function by modulating oxidative stress, inflammation, and apoptosis . Icariin pathway . Icariin pathway . Cisplat pathway . While E pathway .Chinese herbal medicines show good prospects in the treatment of complex diseases owing to their fewer side effects and multi-target effects. Due to the complexity of the ingredients of traditional Chinese medicine, it is difficult to comprehensively explore the potential pharmacological activities of the drugs through the research model of animal experiments. Network pharmacology is a novel method to reveal the active components and action mechanisms of Chinese herbal medicines . In lighOxidative stress is thought to play a crucial role in the mechanism of cisplatin-induced toxicity . From thOxidative stress and inflammation are closely related in biological systems . In addiPrevious studies have demonstrated that cisplatin-induced toxicity is closely related to oxidative stress-induced apoptosis . When ciPI3K-Akt is a crucial intracellular signal transduction pathway that plays a crucial role in cell growth, proliferation, and migration . It is aMitochondrial DNA is one of the main targets of cisplatin . CisplatIn conclusion, combing with network pharmacology, molecular docking, and animal experiments, our study demonstrated for the first time that EFE could significantly attenuate cisplatin-induced intestinal damage by regulating oxidative stress, inflammation, and apoptosis. The molecular mechanism of action might be mainly related to PI3K/Akt, p53, and NF-κB signaling pathways . These f


To predict the target of Seabuckthorn polysaccharides in the prevention and treatment of cervical cancer, and to explore its multi-target and multi-pathway mechanism.Using the Swisstarget database, a total of 61 potential targets of polysaccharide active components were obtained. Cervical cancer related targets were obtained from the GeneCards database. The correlation score was greater than 5 targets for 2727; 15 intersection targets of active ingredients and disease were obtained by Venn diagram. Cytoscape3.6.0 software was used to construct the Polysaccharide composition-Target-Disease Network and Protein-Protein Interaction Networks (PPI). Cytoscape3.6.0 software was used for visualization and network topology analysis to obtain core targets. Kyoto encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) were analyzed using Metascape database. SailVina and PyMOL software were used for molecular docking to verify binding strength.A total of 15 core targets were obtained for cervical cancer. These targets are significantly enriched in HIF-1 signaling pathway, Galactose metabolism, EGFR tyrosine kinase inhibitor resistance, growth factor receptor binding, carbohydrate binding, protein homodimerization activity and other GO and KEGG entries; Molecular docking showed that ADA and GLB1 were well bound to Glucose, D-Mannose, and Galactose.The effect of seabuckthorn polysaccharides on the prevention and treatment of cervical cancer is characterized by multi-component, multi-target and multi-pathway, which provides scientific basis for further research on the activity of seabuckthorn polysaccharides. The normal cells in the tumor organism have undergone qualitative changes under the long-term action of numerous internal causes  and external causes , thus having the ability of excessive proliferation. Malignant tumors are called cancers, cervical cancer is a type of cervical malignancy with the highest incidence among gynecological malignancies, cervical cancer is the most common cause of cancer-related deaths in women in 42 low-income and lower-middle-income countries , 2. In rCarbohydrates are mainly monosaccharides, disaccharides, polysaccharides and other types according to their structure. Monosaccharides are the simplest sugars. In general, human digestive enzymes will not break them down into smaller sugars. Polysaccharides are macromolecular compounds that are polymerized from more than 10 monosaccharide molecules. Its characteristics are very different from monosaccharides, disaccharides and oligosaccharides, etc. Polysugars are generally insoluble in water, sweet, can’t form crystallization, no reducing property . SeabuckNetwork pharmacology is a branch of pharmacology of bioinformatics and high-throughput omics analysis. It can be used to study the mechanism of action of TCM prescriptions and monomers on diseases. In this study, network pharmacology was adopted to study the multi-target and multi-pathway action characteristics of seabuckthorn polysaccharide, and to explore the active substance basis and action mechanism of seabuckthorn polysaccharide in the prevention and treatment of cervical cancer.Seabuckthorn contains flavonoids, tannins, terpenoids, polysaccharides, vitamins and other active components. Flavonoids are the main functional compounds of seabuckthorn. At present, more than 50 kinds of flavonoids have been isolated and identified from seabuckthorn plants, mainly using isorhamnetin, quercetin, and kaempferol as aglycones, which are combined with glucose, rhamnose, arabinose and rutin to form glycosides. There are more than 20 triterpenoids in sea-buckthorn, mainly including oleanolic acid, ursolic acid, corosolic acid and other triterpenoids . Polysac1 screening Cervical Cancer related targets , limited the study species as “homo sapiens,” obtained the PPI network, obtained the protein interaction information, and utilized Cytoscape 3.9.0 software to draw the PPI network, and screened out the core targets according to the node degree  enrichment analysis and Kyoto encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. According to the analysis .3 to lose screening of the main active ingredient, download a small molecule ligands 3D structure. Input the screened core target proteins in the PDB 4 database, download the 3D structure of the target proteins, prepare the ligand files and receptor files, use SailVina and PyMOL software for molecular docking verification, the smaller the docking fraction, the stronger affinity. A docking fraction less than −5.0 indicates a strong affinity between the docking compound and the target  can be found in the article/supplementary material.XF and ZS conceived the project and revised the draft. XF designed the methodology. AT and PG collected and cleaned the dataset. WP and RZ interpreted the results and drafted the manuscript. All authors have read and approved the final manuscript.


Fermented egg-milk peptides (FEMPs) could enhance the colon-intestinal barrier and upgrade the expression of zonula occludens-1 and mucin 2. Besides, the underlying biological mechanism and the targets FEMPs could regulate were analyzed in our study.Herein, the immunofluorescence technique and western blot were utilized to evaluate the repair of the intestinal barrier. Network pharmacology analysis and bioinformatics methods were performed to investigate the targets and pathways affected by FEMPs.via hydrogen bond interactions. Our study combined the experiment in vivo with the method in silico and investigated the interaction between peptides and targets in a pattern of multi-targets and multi-pathways, which offered a new perspective on the functional validation and potential application of bioactive peptides.Animal experiments showed that FEMPs could restore intestinal damage and enhance the expression of two key proteins. The pharmacological results revealed that FEMPs could regulate targets related to kinase activity, such as AKT, CASP, RAF, and GSK. The above targets could interact with each other. GO analysis indicated that the targets regulated by FEMPs could participate in the kinase activity of the metabolic process. KEGG enrichment revealed that the core targets were enriched in pathways related to cell apoptosis and other important procedures. Molecular docking demonstrated that FEMPs could bind to the key target AKT  To protect the body from the environment, the intestinal tract—one of the largest luminal interaction areas—contributes more to the body. The intestinal barrier was reported to be vital in regulating the immune system, improving nutrition absorption, and maintaining intestinal health . Among tA reference has confirmed that the changing of the intestinal barrier might be a pathological cause of obesity. A type of intestinal microbial metabolite called short-chain fatty acids was found to regulate intestinal dysfunction and ameliorate obesity . AnotherOn the basis of the consequences of the intestinal barrier on the health of the body, people have become increasingly interested in functional and effective food ingredients. Protein derived from egg white was claimed as the feature of repairing the damaged intestinal barrier . BioactiIn extensive research on the mechanism of bioactive peptides regulating the intestinal barrier, researchers mostly mentioned a single target and pathway. Unlike the traditional idea, network pharmacology, a novel, and powerful tool provide evidence for the possible mechanism of action of multi-target and multi-pathway disease regulation . It has via the idea of network pharmacology and the method of molecular docking. This study combines in vivo assessment with in silico evaluation, which could provide a new perspective for intestinal barrier enhancement and colonic disease treatment and broaden the horizons of egg products' functional applications.In our study, the DSS-induced colitis mouse model was applied to evaluate the repair of the intestinal barrier. The ZO-1 and MUC-2 were focused on as the factors. Besides, the potential pharmacological mechanism under the FEMPs effect on the intestinal barrier was performed FEMPs were acquired in our lab according to the previous method. DSS  was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai). Biodewax and clear solution, 4′,6-diamidino-2-phenylindole (DAPI), and ethylenediamine tetraacetic acid (EDTA) (pH 8.0) antigen retrieval solution were bought from Wuhan Servicebio Technology Co., Ltd. Ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd, and BCA protein quantitative detection kit, β-actin, and TBS buffer solution were bought from Wuhan Servicebio Technology Co., Ltd.A total of 60 Balb/c mice  were obtained from Beijing Charles River Co., Ltd  and housed in the lab in the Animal Model Laboratory Building at Jilin University. The housing condition was adjusted to 20–23°C of temperature, 40–70% of humidity, and a light-dark cycle of 12 h per day. Before the beginning of the experiment, the mice were made to acclimate to the environment and were given food and water freely. All the animal procedures in the whole experiment were implemented in accordance with the guidelines for laboratory animal care and use at Jilin University. The animal experiments were reviewed and confirmed by the Jilin University animal ethics committee . The entire experiment's anesthetic-required procedures were performed under the influence of isoflurane.For the allocation of the 60 mice (22–24 g), the five groups were settled, which were named CK (the short name of control check), CK + FDP (control check and drink FEMPs freely), DSS + FDW (dextran sulfate sodium and drink water freely), DSS + GP (DSS and gavage FEMPs), and DSS + FDP (DSS and drink FEMPs freely). The integral experiment period was divided into the intestinal damage-making period  and the treatment period . During period 1, the DSS, the DSS + GP, and the DSS + FDP groups were made to drink 3% DSS solution instead of making them drink water to run the intestinal-damaged colitis model. Meanwhile, the CK and the CK + FDP groups were provided with free food and water. During period 2, the DSS + GP group was asked to obtain 200 mg/kg/day FEMPs by gavage. The CK + FDP and the DSS + FDP were required to drink FEMPs freely for 4 h and drink water for the other 20 h per day. The DSS + FDW group was asked to drink water. All the groups were given free mouse food, and the body status of all the mice was recorded during the experiment periods. The mice were euthanized according to animal procedures and guidelines. The colons were harvested and weighted, then cryopreserved at −80 °C.Incubate sections were deparaffinized and rehydrated with xylene and absolute ethanol before being placed in an EDTA antigen retrieval buffer with a pH value of 8.0. Maintained at a sub-boiling temperature for 8 min, we washed the sections three times with PBS (pH 7.4). Then, 3% BSA was added here to cover the marked tissue and block non-specific binding in 30 min. After that, the primary antibody and the secondary antibody were provided. DAPI counterstain was carried out in the nucleus, threw away the liquid carefully, then the slips were covered with the anti-fade mounting medium. The incubated sections were detected and envisioned by fluorescent microscopy.The western blot analysis was developed under the protocol of a previous study with some modifications . The keyhttps://web.expasy.org/peptide_cutter/, was used here to collect the FEMPs' information. All the proteins and peptides in fermented egg milk and the sequences of LC-MS/MS results were collected based on our previous study  was utilized here to filter the Homo targets. Intestinal barrier-related targets were obtained from the GeneCard database (https://www.genecards.org). Besides, the targets obtained from references linked to intestinal health were used to supplement the targets set.The sequence information for FEMPs was based on the database in our lab and silicon digestion. The ExPASy PeptideCutter program, a database available at us study  and uploegulated . The tarhttp://string-db.org/)  , a powerhttps://metascape.org) .To understand the underlying effect and mechanism of intestinal barrier functions of the core targets, we performed the GO and KEGG enrichment analysis based on the Metascape software (ape.org) . In the https://www.genome.jp/kegg/pathway.html). The targets FEMPs could influence in the pathway were marked in a different color, and the figure was visualized via Adobe Illustrator software.FEMPs' regulation of the pathway was analyzed by Metascape software, and the related genes were marked by the website of the KEGG pathway (10.2210/pdb1UNQ/pdb) was downloaded from the database of the RCSB Protein Data Bank (http://www.rcsb.org/pdb), and the organism of the protein was selected as “Homo.” All the molecules of water were excluded from the crystal structure of AKT (1UNQ) to prepare for the molecular docking process. The binding site detection was according to the reference. In this study, a grid box of 25 × 25 × 25 Å was established at the surrounding binding site location, and the grid was generated suitably for peptide docking. The flexible style of the peptides was selected. The hydrogen bonds and cooperative interactions between the AKT residues (1UNQ) and ligands were visualized and analyzed.Based on the computer calculation, molecular docking, a considerable tool widely used to reveal the relationship and interaction between receptors and ligands, was used here to analyze the action of FEMPs on the targets. The calculation has been extensively utilized and confirmed to predict the interaction energy between different molecules. In the current research, molecular docking was performed based on the Autodock Tools and Autodock Vina software. Based on the node degree analyzed by the PPI network , the AKTPrevious references have confirmed the significance and effect of TJs in the occurrence and preservation of UC , 25. ZO-The results of immunohistochemistry staining revealed the two vital proteins locally. As To further determine the relationship between the observed proteins and FEMPs treatment in experimental colitis, an experiment with the western blot was performed in our study. The proteins were extracted from the colons of the mice. As shown in p-value of this network was 1.0e−16  were contained in the FEMPs targets database. The STRING website was applied to perform the PPI network. After that, a network containing 52 nodes and 223 edges was received, and the enrichment  1.0e−16 . In the  1.0e−16 .To further demonstrate the information on interactions and functions of the PPI, the interaction analysis of the edges was calculated by the STRING database. The edges with a combined score larger than 0.90 are listed in The GO and KEGG enrichment analyses of the above targets were carried out by Metascape software to further find out their potential biological functions. The parts of BP, CC, and MF were performed. The top 35 results  are shown in p-value of 4.41e−10, was enriched with the largest count of 10. These results showed that the targets could participate more in the PI3K-Akt signaling pathway. VEGF signaling pathway and EGFR tyrosine kinase inhibitor resistance were listed here with an enrichment score of 59.06 and 51.46, respectively. Meanwhile, many pathways related to intestinal health, such as the TLR signaling pathway  was downloaded from the RCSB Protein Data Bank (http://www.rcsb.org/pdb) of the “Homo” organism  could produce hydrogen bonds in conformation with other atoms.According to the result of the PPI network analysis, the target AKT revealed the highest degree, which indicated that this target might play a significant role in the PPI network and could affect other targets easily. Thus, the AKT target was chosen as the receptor in the molecular docking process. A widely known and powerful method, molecular docking, was developed here to demonstrate the interaction between the target and ligands. There have been many studies that have utilized molecular docking to define the interactions between different targets and peptides , 32, 33.organism . The watorganism . As the In recent years, the intestine's role as a protective organ of the body has brought it to the forefront of medical research. In addition, intestinal immune capacity has become a hot and well-known topic in recent research , 35. As Bioactive peptides derived from the raw material or other food ingredients were reported as having intestinal protective functions and features. Our previous research found that fermented egg-milk peptides could alleviate intestinal inflammation symptoms and establish the gut microbiota in colitis mice . HoweverAkkermansia muciniphila on two important evaluation markers of the intestinal barrier, ZO-1 and Occludin .SL: writing—original draft preparation. QY, XD, SL, and XL: data curation and formal analysis. ZD, TZ, and XS: writing—reviewing and editing. MX: sample collection. JL and FP: supervision. TZ, JL, and FP: conceptualization. All authors contributed to the article and approved the submitted version.


Background: Osteoarthritis (OA) is a degenerative disease which serious affects patients. Ligusticum chuanxiong (CX) has been shown to have a certain curative effect on osteoarthritis in traditional Chinese medicine therapy. This study is based on network pharmacology and molecular docking technology to explore the potential mechanism of CX.Methods: Components of CX to treat osteoarthritis were screened in the TCMSP database and targets were predicted by the PharmMapper database, the osteoarthritis targets were collected from the GeneCards database, and intersection genes were found to be the possible targets of CX anti-OA. The STRING database and Cytoscape software were utilized for protein-protein interaction analysis and further screening of core targets. The Metascape database was used for KEGG and GO enrichment analyses. Then, the top 10 pathways were selected to construct “drug-compound-target-pathway-disease” network analysis. Finally, molecular docking was used to analyze the binding affinity of seven compounds with core targets and TNF-α.Results: Seven compounds with 253 non-repetitive targets of CX were screened from the TCMSP database and 60 potential intersection targets of CX anti-OA were found. PPI network analysis showed that the core targets were ALB, AKT1, IGF1, CASP3, MAPK1, ANXA5, and MAPK14, while GO and KEGG pathway enrichment analyses showed that the relevant biological processes involved in the treatment of osteoarthritis by CX might include the MAPK cascade and reactive oxygen species metabolic process. The KEGG pathway analysis result was mainly associated with the MAPK signaling pathway and PI3K-AKT signaling pathway. We further docked seven ingredients with MAPK1 and MAPK14 enriched in the MAPK pathway, and TNF-α as the typical inflammatory cytokine. The results also showed good binding affinity, especially FA, which may be the most important component of CX anti-OA.Conclusion: Our research revealed the potential mechanism of CX in the treatment of OA, and our findings can also pave the way for subsequent basic experimental verification and a new research direction. Osteoarthritis (OA) is a degenerative disease of the joints, it mainly occurs in knee joints and the clinical manifestations are pain, swelling, and limited movement . The patThe research of traditional Chinese medicine (TCM) on osteoarthritis can be traced back to thousands of years ago . It was The medicinal value of Ligusticum chuanxiong (CX) is in its roots and stems. It has been used in traditional Chinese medicine for thousands of years which was first mentioned in “Shennong’s herbal classic” . It is oCX, as with other traditional Chinese medicines, has complex action mechanisms and multi-channel interaction . In the Active components of CX were screened and predicted by network pharmacology. By constructing the “drug-component-target-pathway-disease” interactive network and utilizing GO and KEGG pathway enrichment analysis and molecular docking technology, the potential molecular mechanism of CX in the treatment of osteoarthritis was explored. Our research may provide guidance for subsequent basic experimental research.http://lsp.nwu.edu.cn/tcmsp.php) (The TCMSP database (msp.php)  was utilmsp.php) . We usedmsp.php) .http://www.lilab-ecust.cn/pharmmapper/) (https://pubchem.ncbi.nlm.nih.gov/) and uploaded to the PharmMapper server. Species “Homo sapiens” was selected in uniport (https://www.uniprot.org/) (https://www.genecards.org/). The keyword “osteoarthritis” was used to collect potential genes. We selected a correlation score >1 of OA targets, norm fit >0.6 of CX component targets for further analysis, and the overlapping area of the Venn diagram represented the potential targets of CX anti-OA.The reverse pharmacophore localization database PharmMapper (mapper/)  was usedot.org/)  to standhttp://string-db.org; Version 11.5) (Homo sapiens” being selected in Cytoscape software (version 3.8.1) further. The MCODE (molecular complex detection) and Cytohubba plug-ins were used to collect the core targets of CX anti-OA to construct the regulatory network.The STRING (on 11.5)  databasehttp://www.metascape.org/) , cell composition (CC), molecular function (MF), and KEGG pathways. The results were imported into bioinformatics software (http://www.bioinformatics.com.cn/). Sequentially, the highest pathway in the KEGG mapper (http://www.kegg.jp.org/)   databasejp.org/)  was highCytoscape (version 3.8.2)  softwarehttp://www.swissdock.ch/) (www.rcsb.org) website (The Swiss dock (ock.ch/)  database website , and che website . The spe website .The flaw chart of this study as displayed in A total of 60 predicted targets were imported into STRING for PPI network analysis . The netp < 0.01), included 871 BP items, 40 CC items, and 59 MF items. We further selected the top 10 BP, CC, and MF catalogs for visualization (p < 0.01)  , the topd in red .The D-C-T-P-D network of CX for the treatment of osteoarthritis is displayed in Finally, we employed molecular docking to determine the possibility of binding between the core target and the CX active compounds via applying the Swiss dock website . The preOsteoarthritis is a common degenerative joint disease and the incidence rate of middle-aged and elderly people is increasing every year, which negatively impacts the health and quality of sufferers’ lives . CurrentWith the emergence of modern bioinformatics, people are beginning to pay more attention to the combination of computer science and biology. Data mining can help us find valuable information from big data and guide us to carry out meaningful research . Based oThrough the D-C-T-P-D network, it was determined that FA, wallichilide, and senkyunone were the most important potential components of CX anti-OA. Previous studies have indicated that the folate receptor is overexpressed on activated macrophages, which are closely related to osteoarthritis . Some liThen we applied a PPI network. The results showed that there were abundant interactions among targets, which made it easier to produce cascade effects. The core targets were ALB, AKT1, IGF1, CASP3, MAPK1, ANXA5, MAPK14, CAT, and IGF1R. These targets corresponded to multiple components in CX; this point fully reflected the characteristics of multi-target and multi-component traditional Chinese medicine. Some studies have shown that the osteophyte formation of OA in AKT1-knockout mice was blocked in the animal model of osteoarthritis. Further studies have found that the mechanism was the inhibition of the expression of calcification inhibitor nucleotide pyrophosphatase/phosphodiesterase 1 . And IGFWe discovered that the “PI3K-Akt signal pathway”, “MAPK signal pathway”, “osteological differentiation”, and “IL-17 signal pathway” were the potential key mechanisms of CX in the treatment of OA. In our study, MAPK1 and MAPK14 may be involved in the main mechanism of CX in the treatment of OA, as the most potential core molecules in the MAPK pathway. At the same time, the molecular docking results also proved that the active components of CX were well docked with MAPK1 and MAPK14. A variety of stimulating factors or toxic injury can activate the PI3K-Akt signaling pathway, and then regulate basic cell functions such as transcription, proliferation, and survival . ERK MA. And theIn conclusion, our study used network pharmacology to elaborate the potential mechanism of CX in the treatment of osteoarthritis, and intuitively verified its effectiveness through molecular docking. Our study showed that CX can act on chondrocytes, synovial cells, and other cells closely related to the occurrence and development of OA. Meanwhile, CX was effective in the treatment of osteoarthritis by multi-target and multi-channel properties. However, there are still some deficiencies in this study, and the reliability of these conclusions needs further basic research to verify. Our research provided a new direction for the further research of CX in the treatment of osteoarthritis.Our research, based on bioinformatics, found that Ligusticum chuanxiong was effective in the treatment of osteoarthritis with the characteristics of multi-component, multi-target, and multi-pathway properties. Our results also guide directions for subsequent basic experiments.