Acanthopanax senticosus. This study aims to investigate the underlying mechanism of the anti-osteoporosis action of EE through network pharmacology, molecular docking and gut microbiota.Eleutheroside E (EE) is an anti-inflammatory natural compound derived from the edible medicinal herb in vivo anti-OP effects of EE.Network pharmacology was used to explore the potential core targets and main pathways mediated by EE in osteoporosis (OP) treatment. Molecular docking was exploited to investigate the interactions between the active anti-OP compounds in EE and the potential downstream targets. Following the multi-approach bioinformatics analysis, ovariectomy (OVX) model was also established to investigate the In vivo experiments demonstrated the protective effect of EE treatment against accelerated bone loss, where reduced serum levels of TRAP, CTX, TNF-α, LPS, and IL-6 and increased bone volume and serum levels of P1NP were observed in EE-treated mice. In addition, changes in gut microbiota were spotted by 16S rRNA gene sequencing, showing that EE treatment increased the relative abundance of Lactobacillus and decreased the relative abundance of Clostridiaceae.The top 10 core targets in PPI network were TP53, AKT1, JUN, CTNNB1, STAT3, HIF1A, EP300, CREB1, IL1B and ESR1. Molecular docking results that the binding energy of target proteins and the active compounds was approximately between −5.0 and −7.0 kcal/mol, which EE has the lowest docking binding energy with HIF1A. Enrichment analysis of GO and KEGG pathways of target proteins indicated that EE treatment could potentially alter numerous biological processes and cellular pathways. In vivo, EE prevents the onset of OP by regulating gut microbiota and inflammatory response and is therefore a potential OP drug.In summary, these findings suggested that the characteristics of multi-target and multi-pathway of EE against OP.  PostmenAcanthopanax senticosus (A. senticosus) which has been extensively used as nutritious food and oriental medicine in Asia belongs to the Araliaceae family and is widely distributed in China, Korea, and Japan. A. senticosus has been used to treat rheumatoid arthritis, diabetes, and hypertension for a long time and exhibited in vivo immunomodulating activities  is clinically more compelling over current anti-OP medications due to its safety and effectiveness. And TCM has a long history acting as a complementary and alternative treatment for OP patients , 6. Acantivities –9. Eleutce model . HoweverIn the past decade, ever-growing evidence has shown a strong association between the gut microbiota and many human diseases , 12. For22.1https://tcmspw.com/tcmsp.php). The compounds were screened according to oral bioavailability (OB) ≥30% and drug likeness (DL) ≥0.18 (https://pubchem.ncbi.nlm.nih.gov). Then it was imported into STP (http://swisstargetprediction.ch/) to obtain the predicted targets of the chemical components. The active ingredient targets were obtained after screening by probability>0. Target information from the STP and TCMSP database was integrated to get the potential targets of EE components (https://www.uniprot.org) was used to convert each target into genes (https://www.genecards.org), OMIM (https://www.omim.org), and TTD databases (http://db.idrblab.net/ttd/) with osteoclast differentiation and osteoclastogenesis as the keywords. GeneCards were filtered above the median relevance score (The active components of EE were extracted from the TCMSP (L) ≥0.18 . SDF formponents . UniProtce score . The int2.2https://cn.string-db.org/). The protein species was set to “Homo Sapiens” and the interaction score was set to 0.4. Meanwhile the unconnected nodes in the network were hidden to obtain and the interaction results were saved as TSV format files. The files were imported into Cytoscape software to draw PPI network diagrams, and topological parameters were analyzed by applying the network analyzer of Cytoscape’s built-in network analysis plug-in (Protein-protein interaction (PPI) underlies most biological processes in living cells and is essential for understanding cellular physiology in normal and disease states . The pot plug-in . Accordi2.3https://metascape.org) was used to perform GO term enrichment analysis and KEGG pathway enrichment analysis on the intersection targets , and then the core proteins were dehydrated and delighted with PyMol 2.4.0 software. The structures of the core components were retrieved by PubChem, downloaded in SDF format, and converted to pdb format with OpenBabel 3.1.1. AutoDock 4.2.6 software was used to routinely process protein receptors and small molecule ligands and save them in pdbqt format. Molecular docking and binding energy calculation scripts were run using AutoDock Vina 1.1.2  were acquired from the Guangdong Medical Laboratory Animal Center. These animals were placed in a 12 hours light/dark cycle. After acclimation for 7 days, all mice were randomly divided into four groups (n=5 per group): Sham group, OVX group, EE-L (20 mg/kg) group, and EE-H (40 mg/kg) group. All animals had free access to food and water. EE was dissolved in 1% carboxymethyl cellulose sodium (CMC-Na). Conversely, the Sham and OVX group received intragastric administration of equal volume CMC-Na. After 12 weeks, mice were sacrificed and collected their left femur, serum and feces. The Ethics Committee of Guangdong Pharmaceutical University had reviewed and approved the experimental protocol according to the Guide for the Care and Use of Laboratory Animals.2.6Micro-CT  was used to scan the morphological structure of the left distal femur of mice, and the bone trabeculae in the area 0.8 mm above 0.5 mm from the growth plate were selected as the region of interest (ROI) for 3D reconstruction. Bone mineral density (BMD), bone volume fraction (BV/TV), bone surface/bone volume (BS/BV), number of trabeculae (Tb.N) and trabecular separation (Tb.Sp) of the ROI were analyzed with evaluation analysis software. The analysis ranges were kept consistent for all specimens.The serum was obtained from anesthetized animals by retroorbital puncture at the end of the study. The levels of TRAP, CTX, P1NP, TNF-α, LPS and IL-6 were measured by ELISA according to the manufacturer’s instructions.2.7After collecting the mice feces, the feces were placed in sterile cryopreservation tubes, rapidly frozen in liquid nitrogen, and stored in an ultra-low temperature refrigerator at -80°C for gut microbiota diversity analysis. The E.Z.N.A.®soil DNA kit  was used to extract DNA from each mouse fecal sample, and the extracted DNA was detected by 1% agarose gel electrophoresis. Then all primers were designed based on the conserved region. 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used for PCR amplification of the V3-V4 region of the 16S rRNA gene to perform the illumina deep sequencing. PCR products were detected by 2% agarose gel electrophoresis. The purified PCR products were sequenced using the NEXTFLEX®Rapid DNA-Seq kit. The PCR products were sequenced through Illumina’s Miseq PE300 platform .2.8± SD. The ANOVA test was used for the data with normal distribution, and the Mann–Whitney test was used to analyze data that did not meet the assumptions of the ANOVA . When P<0.05, the differences were considered to be statistically significant.All data were represented as mean 33.1The 2D structure of EE is shown in 3.2The interaction relationships of potential EE targets affecting osteoclast were obtained from the STRING database , response to hormone (GO:0009725), gland development (GO:0048732), response to growth factor (GO:0070848) . The levels of BV/TV, BS/BV, and Tb.N were increased significantly, while Tb.Sp was presented an opposite trend in EE-H (P<0.05). The value of BMD was enhanced but not significantly different (P>0.05) , a sharp increased in P1NP by EE (P<0.05), moreover, the contents of TRAP, CTX, TNF-α, LPS and IL-6 were significantly increased in the OVX . Treatment of EE reversed the increased serum levels of TRAP, CTX, TNF-α, LPS and IL-6  (Serum P1NP levels of mice in the OVX was significantly decreased compared with the Sham (3.6We analyzed the number of common and unique OTUs among the different groups to show that four groups had different compositions (Bacteroidia, Bacilli, and Clostridia were the predominant in all groups. At the family level, compared to the Sham, OVX reduced the relative abundance of Lactobacillaceae, while the relative abundance of Clostridiaceae significantly increased. Compared to the OVX, EE increased the relative abundance of Lactobacillaceae and decreased the relative abundance of Clostridiaceae. At the genus level, OVX decreased the relative abundance of Lactobacillus significantly, while increased the relative abundance of Clostridium_sensu_stricto_1. EE treatment increased Lactobacillus and decreased Clostridium_sensu_stricto_1 significantly. At the species level, the contents of Uncultured_bacterium_g_norank_f_Muribaculaceae and Uncultured_bacterium_g_Clostridium_sensu_stricto_1 in the OVX were higher than those in the Sham, whereas the contents of Lactobacillus_murinus decreased. Compared to the OVX, EE reduced the relative abundance of Uncultured_bacterium_g_norank_f_Muribaculaceae and Uncultured_bacterium_g_Clostridium_sensu_stricto_1, while the relative abundance of Lactobacillus_murinus significantly increased.To study the specific changes in bacterial communities, cluster histograms were drawn to show the changes in gut microbiota at the class, family, genus and species levels in each sample (4in vivo.OP is a systemic skeletal disease that results in deteriorated bone structure and low BMD, which can compromise bone strength and increase the risk of fractures . ExcessiIn vivo experiment revealed that mice lacking p53 displayed elevated osteoclastogenesis and bone resorption (in vitro (Latest advancements in network pharmacology allow researchers to visually analyze disease targets and action pathways, assisting to investigate the mechanism of TCM’s actions . The PPIsorption . AKT1 issorption . A studysorption . JUN famsorption . CTNNB1 sorption , and hassorption . In inflsorption . HIF1A pin vitro . EP300 iin vitro . CREB1 ain vitro . IL1B isin vitro .GO and KEGG enrichment analysis are important bioinformatic tools for understanding high-degree relations between genes/proteins and biological processes/signaling pathways . GO analLactobacillus supplementation has been shown to benefit human gut microbiota (Clostridiaceae is negatively correlated with human bone density, and that a decrease in the level of Clostridiaceae can prevent bone loss caused by estrogen deficiency (Lactobacillus was increased and Clostridiaceae was decreased by EE treatment, which may be the key mechanism of EE beneficial to OP. A healthy intestinal epithelial barrier can prevent tight hyperpermeability (Previous studies have established connections between intestinal microbiota, gut barrier integrity, chronic inflammation, and the HIF-1 signaling pathway . PD’Amelcrobiota , 47, andcrobiota , providificiency . Througheability , which ceability . LPS coueability . EE redu5in vivo validation. The network pharmacology analysis and molecular docking revealed that EE exerted anti-OP effects via modulating multi-targets and multi-pathways, and the in vivo results showed that EE regulated gut microbiota and inflammatory responses to alleviate the progression of OP. Of note, our conclusions are only based on limited experimental data, and more comprehensive and in-depth analyses are needed in the future.In summary, to decipher the molecular mechanisms of EE in the treatment of OP, network pharmacology analysis and molecular docking was performed coupled with The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found below: NCBI; Accession to cite for these SRA data: PRJNA1025301.The Ethics Committee of Guangdong Pharmaceutical University had reviewed and approved the experimental protocol according to the Guide for the Care and Use of Laboratory Animals. The study was conducted in accordance with the local legislation and institutional requirements.TZ: Writing – original draft. YZ: Writing – original draft. DG: Writing – review & editing. YX: Writing – review & editing. JW: Writing – review & editing. LT: Writing – review & editing. QD: Writing – review & editing. PS: Writing – review & editing.


Apigenin, luteolin, diallyl trisulfide, silibinin and allicin had the highest percentage of C-T interactions while, AKT1, CASP3, PTGS2, NOS3, TP53 and MMP9 were found to be the most enriched genes. Moreover, the most enriched pathways were pathways in cancer, fluid shear stress and atherosclerosis, relaxin signaling pathway, IL-17 signaling pathway and FoxO signaling pathway. Additionally, Curcuma longa, Allium sativum, Oleu europea, Salvia officinalis, Glycyrrhiza glabra and Silybum marianum had the highest number of P-C-T-P interactions. Furthermore, molecular docking analysis of the top hit compounds against the most enriched genes revealed that silibinin had the most stabilized interactions with AKT1, CASP3 and TP53, whereas luteolin and apigenin exhibited the most stabilized interactions with AKT1, PTGS2 and TP53. In vitro anti-inflammatory and cytotoxicity testing of the highest scoring plants exhibited equivalent outcomes to those of piroxicam.Most synthetic immunomodulatory medications are extremely expensive, have many disadvantages and suffer from a lot of side effects. So that, introducing immunomodulatory reagents from natural sources will have great impact on drug discovery. Therefore, this study aimed to comprehend the mechanism of the immunomodulatory activity of some natural plants via network pharmacology together with molecular docking and  The disorder known as autoimmune illness occurs when the immune system unintentionally targets its own body cells. If the immune system's error is not fixed, the attack may spread to the heart, lungs, and other crucial organs. Uncertainties surround the mechanisms behind immune response dysregulation. It is conceivable that excessive exposure to heavy metals, pathogenic bacteria and parasites, leaky gut syndrome and dietary imbalances can tax the immune system to the point where it becomes dysregulated2. Despite the fact that there are more than 100 different forms of autoimmune diseases; rheumatoid arthritis, multiple sclerosis, lupus, psoriasis, type 1 diabetes and others are the most prevalent ones3. The global autoimmune disease therapeutics cost is very huge, where the overall cost of autoimmune diseases therapeutics worldwide in 2021 was evaluated by 127 billion dollars and is projected to reach 149 billion dollars by 20234. Medication associated with autoimmune disorders treatment is one of the most multifaceted and challenging areas of modern medicine. This evokes pharmaceutical companies for the continued research for new drug candidates in this area. Different varieties of drugs including NSAIDs, anti-inflammatories, immune suppressants, corticosteroids, interferons and others, are approved for autoimmune disorders treatment. For example, the anti-inflammatory drugs can regulate the immunity by interfering with immune cell regulation, pro-inflammatory cytokines’ synthesis, and gene expression. It is believed that the balance between pro-inflammatory cytokines  and anti-inflammatory cytokines  is a key factor in inflammation and immune response homeostasis, which underlies many diseases5. In addition, the inflammatory signaling regulation is aided by their capacity to alter the expression of number of pro-inflammatory genes, including multiple cytokines, nitric oxide synthases, lipoxygenase, and cyclooxygenase, as well as to their anti-oxidant properties like ROS (reactive oxygen species) scavenging activities7. However, immunosuppressive medications have many disadvantages and suffer from a lot of side effects as it should be administrated for long term, accompanied by adverse metabolic disturbances and toxicities, in addition to the high risk of infection, cancer incidence and lack specificity8.The human immune system plays a vital role to protect the body from totally different attacks of any morbific microorganisms. Immune system is classified into two categories which include humoral or antibody mediated immunity and cellular mediated immunity10. In addition to observational research, there is evidence from human intervention trials that several plant diets may have anti-inflammatory properties. At the level of bioactive substances present in plant diets, carotenoids and flavonoids in particular seem to affect inflammatory as well as immunological processes. For example, a randomized, double-blind, controlled clinical trial was carried out on herbal formulation consisted of “turmeric extract, ginger, and black pepper” versus Naproxen drug for chronic knee osteoarthritis and the results demonstrated that ginger which is the most prevalent herbal product used, with active ingredients like gingerols, can reduce the inflammation and therefor relieve the pain of osteoarthritis11, where turmeric with curcumin as the most active component was found to be a potent anti-inflammatory agent and can reduce the complications of rheumatoid arthritis13. The results of studies also, indicate the therapeutic effects of black pepper in reducing inflammation14 due to the alkaloid called pyrene. In conclusion, there is strong evidence that plant diets and their non-nutritive by-products influence inflammatory and immune responses15. So that, introducing immunomodulatory reagents from natural plant sources will have great impact on drug discovery as this will provide safer bioavailable candidates with decreased production costs. Moreover, the discovered compounds will not have the undesirable side effects reported for the synthetic chemicals currently available. Even so, herbal medications are expected to be quite complex taking in consideration their chemical components, which may interact with a number of protein targets to have a therapeutic effect16. Such great complexity also elicits numerous pharmacodynamics interactions such as synergistic effects17, that arise between these various ingredients, which makes it challenging to pinpoint the precise mechanism of action of such complex substances.Epidemiological evidence indicates that dietary patterns have a significant impact on inflammatory processes. Primarily consuming fruits, vegetables, and whole grains is the best way to get valuable molecules like polyphenols, resveratrol, epigallocatechin gallate, capsaicin and curcumin. These secondary plant metabolites have powerful anti-inflammatory characteristics and can modulate the signaling pathways that modify how pro-inflammatory genes like cyclooxygenase, lipoxygenase and phospholipase A2, are expressed20. Given that medicinal plants are multi-component and the network pharmacology approach emphasizes the idea of “network target, multicomponent therapeutics,” in a holistic manner analogous to the complex matrices of medicinal herbals, this strategy is thought to be appropriate for understanding the mechanism of action of medicinal plants24. Network pharmacology can be used to forecast the protein targets of the active ingredients of the plants and, consequently, the disease pathways that will be affected. Predicting the primary active ingredients and probable target genes of medicinal plants involves the application of multiple targets identification process using network pharmacological analysis, which is being employed more and more27.Recent years have seen the successful use of network pharmacology for the building and visualization of disease-gene-target drug networks, which can aid in the assessment of the molecular mechanisms of drugs from a multi-dimensional perspectiveThe aim of this work is to comprehend the mechanism of the immunomodulatory activity of some natural plant sources via network pharmacology analysis. The identification of protein targets and the related pathways followed by gene enrichment analysis were carried out. In vitro anti-inflammatory testing and docking analysis were further performed to verify the potential immunomodulatory activity of natural plant sources extract. This study displays deeper insights about natural plant sources molecular mechanisms of action in the control of autoimmune disorders utilizing an integrated strategy of network pharmacology, docking analysis and in vitro testing.An in-house database of 2154 phytochemical constituents was constructed depending on a previous literature analysis of the chemical makeup of 32 selected immunomodulatory plants.28. After filtration of database, only chemicals that obeyed at least three of the aforementioned characteristics were retained. Oral bioavailability (OB) of phytoconstituents was also estimated29. It shows how much of a pharmacological dosage taken orally still has an unaltered effect at the therapeutic site of action. In the database, only compounds with OB ≥ 30% were kept. All the database compounds that satisfied the specified requirements were maintained for further network pharmacology-based analysis  if it has a molecular weight less than 500 Da, fewer than 10 hydrogen-bond acceptors (Hacc), fewer than 5 hydrogen-bond donors (Hdon), 10 or fewer rotatable bonds (RBN), and has a measured log P (ClogP) less than fiveis Table .in-silico analysis approach was tried in this study to provide a quick, effective and high throughput technique to identify the potential immunomodulatory molecular targets of the constructed phytochemicals database. A constituent-target (C-T) network dependent on 600 plant constituents and putative targets was created utilising the screening results received from the STITCH 5.0 public database in order to uncover the mechanisms of action of the chosen plant constituents and immunomodulatory-associated target genes. STITCH is a huge database that holds a big data on chemical interactions. It connects 1.5 million genes across 373 genomes with over than 68 000 compounds, including 2200 drugs, and offers information on their interactions30. The function of each discovered target gene and its relationship to immunomodulation were determined using the Universal Protein Resource database (UniProt). In-depth information about more than 550 000 proteins and their functions can be found in UniProt, a resource for protein sequences and functional annotation31. The interactions between chemicals and genes are given "combined scores" in the STITCH 5.0 database, with stronger interactions displaying higher scores. In this investigation, only substances with interaction scores greater than 0.4 were kept  and 759 edges made up the constructed constituent-target (C-T) network Fig. , with anα or RAC-alpha serine/threonine-protein kinase) is a crucial signaling node in controlling the activation of adaptive immune cells32 because it controls a variety of activities such as metabolism, cell survival, growth, proliferation, and angiogenesis33. Additionally, it has been shown to have substantial anti-inflammatory effects through inhibiting NF-κB-mediated transcription34. Meanwhile, CASP3 (Caspase-3) is implicated in the activation cascade of caspases necessary for apoptosis execution35. Prostaglandin-endoperoxide synthase 2 (PTGS2) is an essential enzyme for inflammatory prostaglandins biosynthesis and production36, while tumor suppressor protein (TP53) affects the innate immune system by secreting factors that modify macrophage activity to prevent tumour development. Additionally, it inhibits some pro-inflammatory proteins like NF-κB and STAT3, to support tissue homeostasis and prevent overreacting immune systems. Moreover, nitric oxide synthase-3 (NOS-3) is hypothesised to play anti-apoptotic roles and engaged in the suppression of genes implicated in cell proliferation. It also reduces the expression of several cytokines such as INF-γ, IL-1β, IL-6 and TNF-α, in a variety of immune cells including monocytes, eosinophils and lymphocytes. The nitrosylation of several transcription factors, including JAK/STAT and NF-κB, mediates this action37. On the other hand, TP53 helps to recognise non-self-antigens, which in turn triggers anti-tumor immunity through a variety of pathways40. Matrix metalloproteinase 9 (Mmp9) is a secreted gelatinase that participates in the remodelling of the extracellular matrix by degrading the components of the extracellular matrix. It also controls the pro-inflammatory cytokines TNF-γ and IL-1 to control the inflammatory response41. A protein–protein network revealed significant gene–gene interactions  is a database tool for deriving high-level biological system functions and applications from molecular-level data, particularly from wide-ranging molecular datasets produced by genome sequencing and other high-throughput experimental techniquesut Table . It can ut Table A that thut Table B was buiκB and COX-2 inhibitor), was found to be valuable in lupus therapy and also for preventing inflammation in other Th17-mediated inflammatory diseases like Crohn’s disease, rheumatoid arthritis, and psoriasis as well as in prevention of inflammation-based tumors overexpressing COX-2 in colon and breast as it was found to inhibit the autoantigen-presenting and stimulatory activities of the APCs (antigen presenting cells), which are required for the activation and development of lupus-associated Th1 and Th17 cells as well as B cells and causes the overactive lupus APCs, B cells, and T cells to undergo apoptosis, most likely by suppressing the expression of NF-κB-regulated anti-apoptotic molecules, particularly COX-2 and c-FLIP, which are chronically overexpressed by the lupus immune cells47. In addition, luteolin was reported to inhibit the overexpression of inflammatory mediators and catabolic factors, probably via preventing NF-κB activation. Additionally, it prevents T cell growth and their AKT/mTOR signaling. Thus, luteolin is an emerging immunosuppressant as a novel mTOR inhibitor. It also ameliorates inflammation and Th1/Th2 imbalance by regulating the TLR4/NF-κB pathway50. Meanwhile, diallyl trisulfide (DAT) were proved to have a potential anti-inflammatory activity by possessing an antagonistic action on TLR4 (Toll-like receptor 4/nuclear factor-κB pathway) as it inhibits the production of myeloid differentiation factor 88. DAT was reported also to increase the stabilization of IκBα by preventing its phosphorylation by IκB kinase (IKK) complex, and inhibit the NF-κB transcriptional activites52. Moreover, silibinin was declared to dramatically decrease the demyelination and inflammation signs in experimental autoimmune encephalomyelitis by upregulating the release of the anti-inflammatory Th2 cytokines and downregulating the pro-inflammatory Th1 cytokines. It also showed anti-carcinogenic and anti-inflammatory properties as a result of NF-κB transcription factor suppression. Additionally, through ERβ binding, silibinin triggers apoptosis, slows proliferation, and inhibits the production of the pro-inflammatory cytokines TNF-α and IL-17 in CD4 + T cells54. Also, allicin was proclaimed to suppress the chemokines IL-8, IP-10, and MIG, as well as the release of IL-1β from intestinal epithelial cells. This action was at least partially mediated by down-regulating the mRNA levels and the involved inhibition of activation of the NF-κB pathway55.The PubMed database was searched for publications linking the hit compounds to the various immunomodulatory pathways in order to validate the findings from the network pharmacology-based study Table . For insCurcuma longa (29%), Allium sativum (19%), Oleu europea (17%), Salvia officinalis L (16%), Glycyrrhiza glabra (10%) and Silybum marianum (9%) had the highest number of P-C-T-P interactions which would suggest that these plants have more active substances that can act as immunomodulatory agents. In reality, there are numerous reports on the anti-inflammatory action of Curcuma longa in traditional medicine. In Indian medicinal system, Curcuma longa is used in wound healing and as an anti-inflammatory and anticancer plant56. Meanwhile, Allium sativum has been utilized traditionally since ancient times due to its biological properties including anti-tumour, anti-inflammatory, antioxidant, tuberculosis, arthritis, rhinitis, bronchitis, and immunomodulatory activities58. Moreover, essential oil extract of Oleu europea fruit is applied topically in Italy to alleviate rheumatism and to boost the circulation59. Infusion of fresh leaves of O. europaea is utilised in Tunisian traditional medicine as an antioxidant, anticancer and as a remedy for various types of inflammation60. In addition, Salvia officinalis L has been used to cure rheumatism and inflammation in Latin American and Asian folk medicine61. It also, has been utilized to treat throat and skin inflammations in European traditional medicine62. Numerous investigations have been carried out in recent years to identify novel biological effects of Salvia officinalis L and to document its traditional usage. Numerous pharmacological actions, such as antioxidant, anticancer, anti-nociceptive, anti-inflammatory, and antimutagenic properties, have been identified by these research63. While Glycyrrhiza glabra has long been utilised traditionally in Japan as an antioxidant, anti-inflammatory, cancer preventative, and anti-arthritic agent65. Finally, Silybum marianum also was reported to exhibit immunomodulating and antioxidant activities by scavenging free radicals, in addition to its ability to reduce inflammation and inflammation-related pain66.After looking at the 759 C-T interaction distributions on the selected plants, a combined plant-constituent-target-pathway network was created. The cytoscape combined score of C-T interactions was used to rate the plants. Figure 67. By importing the selected targets into the DAVID bioinformatics tools and restricting the annotations to Homo sapiens, GO enrichment analysis was performed on the targets, revealing the most enriched pathways and GO keywords with the greatest log P value and gene counts. As demonstrated in Fig. in_vivo,in addition to 12 BIOCARTA pathways named: insulin signaling pathway, IL-2 receptor beta chain in T cell activation, IL 2 signaling pathway, Erk1/Erk2 Mapk signaling pathway, The 4-1BB-dependent immune response, PTEN dependent cell cycle arrest and apoptosis, IL 6 signaling pathway, IL 3 signaling pathway, IL12 and Stat4 dependent signaling pathway in Th1 development, WNT signaling pathway, mTOR signaling pathway and integrin signaling pathway. Furthermore, 35 KEGG pathways including pathways in cancer, TNF signaling pathway, ErbB signaling pathway, FoxO signaling pathway, prolactin signaling pathway, focal adhesion and PI3K-Akt signaling pathway. In addition, 7 REACTOME pathways named ROS and RNS production in phagocytes, signaling by EGFR, negative regulation of the PI3K/AKT network, gastrin-CREB signaling pathway via PKC and MAPK, RAF/MAP kinase cascade, VEGFR2 mediated cell proliferation and CD28 dependent PI3K/Akt signaling were identified  assigns several GO concepts to a single query sequence and classifies gene products into three distinct categories: biological process, cellular component, and molecular function68. The results were found to fulfil all the required criteria settled for network pharmacology analysis evaluation.The evaluation process including the three aspects—reliability, standardization, and rationality, was carried out according to the method described previously by LiCurcuma longa, Allium sativum, Oleu europea and Salvia officinalis L, were performed utilizing colorimetric tests. The definitive goal of these in vitro investigations is to verify the network pharmacology based analysis results through assessment of some plants-targets binding affinities. Saturosporine was employed as a positive control for Akt1 inhibitory assay since it has been showed to be a strong inhibitor of Akt1 gene69, where Ac-DEVD-CHO, obtained from caspase 3 colorimetric assay kit supplied by Sigma, was used as a positive control for caspase-3 gene. The results demonstrated that among the screened agents, Curcuma longa was found to have the higher inhibitory activities on both Akt1 and caspase-3 assays with IC50 of 475 µg/ml ± 0.031 and 500 µg/ml ± 0.053, respectively. The Akt1 inhibitory activity of other tested inhibitors was in the order of Allium sativum (IC50 = 690 µg/ml ± 0.044) followed by Oleu europea (IC50 = 715 µg/ml ± 0.014) and finally Salvia officinalis L (IC50 = 740 µg/ml ± 0.063), respectively. Whereas, the caspase-3 inhibitory activity of other tested inhibitors was in the order of Salvia officinalis L (IC50 = 590 µg/ml ± 0.045) followed by Allium sativum (IC50 = 625 µg/ml ± 0.072) and finally Oleu europea (IC50 = 800 µg/ml ± 0.021), respectively, , which is the concentration of the drug necessary to reduce the viability of cells by 50%, was established for the extract and the standard anti-inflammatory drug; piroxicam utilizing MTT test. The Allium sativum, Oleu europea, Glycyrrhiza glabra and Salvia officinalis extracts demonstrated higher IC50 values  than that of piroxicam (100 μg/ mL) illustrating that these extracts are less toxic than piroxicam , demonstrated comparable effective anti-inflammatory concentrations (EAICs) with piroxicam (1.21 ± 0.1 μg/mL), indicating the potential anti-inflammatory activities of these extracts. Real-time polymerase chain reaction (PCR) was used to assess the gene expression of four pro-inflammatory mediators  in both untreated and lipopolysaccharide (LPS)-treated WBCs in order to ascertain the genetic basis of the anti-inflammatory effect . Meanwhile, LPS induced the TNF-α expression by 2.66 ± 0.01-folds that was diminished by the Glycyrrhiza glabra and Curcuma longa extracts to 0.016 ± 0.001 and 0.02 ± 0.03-folds, respectively. This level was nearly close to what piroxicam had achieved (0.03 ± 0.0003-folds). However, the expression of IL-6 was increased by LPS by 5.4 ± 0.2-folds, and this was reduced to 0.9 ± 0.02 and 1.3 ± 0.01-folds, respectively, by Allium sativum and Curcuma longa extracts. This amount was very comparable to what piroxicam had accomplished (1.1 ± 0.1-folds). It was noticed that Curcuma longa extract showed no statistically significant differences between it and the known synthetic anti-inflammatory drug, piroxicam.As unveiled from the prior network pharmacology study, the highest scoring plants, were retrieved to be β, INF-γ, TNF-α and IL-6 to a similar degree  (PDB ID 5F1A) included one hydrogen bond between the 4' hydroxyl group and His90 residue. Along with these interactions, there were polar interactions with His90, Gln192, Ser530, Ser353 and Thr94 as well as hydrophobic interactions with Leu384, Lue531, Leu352, Phe518, Phe381, Met522, Ile517, Ala527, Ala516, Trp387, Val349, Val523, Tyr355, Tyr385. A charged positive interaction with Arg513 was also detected , in which apigenin was particularly integrated, were hydrophobic. Pro151, Pro223, Pro222, Cys220, Cys229, Trp146, Val147 and Leu145 were all found to interact hydrophobically with apigenin, and the 5, 4'-hydroxyl groups were particularly integrated in two hydrogen bonds with Cys220 and Asp228. This may have a significant impact on apigenin increased stability in the binding pocket of the target protein. Polar interactions with Thr150, Thr155, Thr230, and Ser149 were also present, in addition to charged negative interactions with Glu221, Asp228 and Asp148  measurements were performed for the shifts of ligands with respect to residues at the binding sites, and root mean square fluctuation (RMSF) measurements were made from the MD trajectory to explain fluctuations in protein structure71. As given in Fig. Two hundred (200) ns duration molecular dynamics (MD) simulation was performed to analyze the in silico stability of TP53 & apigenin, AKT1 & silibinin, and PTGS2 & luteolin protein–ligand complexes obtained from the molecular docking study72. As shown in Fig. Schematic 2D diagrams of protein–ligand interactions at 200 ns are given in Fig. The study design is summarized as shown in Fig. 73 and summarized briefly in Table http://dnp.chemnetbase.com/faces/chemical/ChemicalSearch.xhtml;jsessionid=0A16BF52515E734B15A96DCBAE7788B9), National Centre for Biotechnology Information's PubChem database (https://pubchem.ncbi.nlm.nih.gov/), ChEMBL (https://www.ebi.ac.uk/chembl/) and Chemdraw software  were used to provide 2D structures of these compounds in (.sdf) format. Schrodinger software (2017A) was utilized to transform these 2D structures to SMILES format.Depending on our previous study of the chemical makeup of 32 selected immunomodulatory plants, 2154 phytochemical constituents were obtained for compilation of an in-house database as illustrated in Table 28. In order to determine if the examined compounds would be good therapeutic candidates, a variety of physio-chemical attributes were estimated. In this consider, compounds with an anticipated oral bioavailability (OB) ≤ 30 were not included in this investigation. In expansion, compounds that met fewer than three of Lipinski's five requirements were also disregarded.Using the Qikprop software (Schrodinger suite 2017A), plant constituents obtained from the database were filtered out through the application of ADME system and Lipinski's rule of fivehttp://stitch.embl.de/, ver. 5.0) using the 'Homo sapiens' species setting25. Genes information like genes ID, names, organism and function were obtained from UniProt (http:// www.uniprot.org/)26. Only the ‘Homo sapiens’ proteins connected to immunosuppressive disorders were kept. Then, using STRING database (https://string-db.org)36, the protein–protein interaction network (PPI network) was created.The target genes associated with the chosen chemical constituents were identified utilizing STITCH DB (http://www.cytoscape.org/). The nodes in these networks stand for genes, constituents and pathways, while the edges denote the interactions existing between them. The Cytoscape network analyzer plug-in was used to compute the network parameters. The importance of nodes in each built network was evaluated utilizing Cytoscape combined score of interactions.For further investigation of the multi-level mechanisms of action of plant components in the treatment of immunosuppressive disorders, different types of networks including plant-constituent, gene-pathway, constituent-target gene and constituent-gene-pathway networks—were built using Cytoscape 3.8.2 (https://david.ncifcrf.gov/) were searched to learn more about gene ontology and to identify the canonical pathways, cellular components, biological processes and molecular functions that were closely related to the target genes105. The only selected pathways had P-values ≤ 0.05. P ≤ 0.05 was considered to be statistically significant.The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and the Database for Annotation, Visualization and Integrated Discovery (DAVID) ver. 6.8 , were bought, from a reputable local market in Alexandria, Egypt. Professor Sania Ahmad, a professor in Faculty of Science at Alexandria University examined the tested samples macroscopically and microscopically in order to be able to authenticate them. Voucher specimens were registered in the Pharmacognosy Department herbarium, Faculty of Pharmacy, Alexandria University under the following codes: , respectively. Our study complies with relevant institutional, national, and international guidelines and legislation. The plants were firstly air dried before being individually double macerated on 70% ethanol for extraction. The obtained extracts were filtered and then evaporated using a rotary evaporator , at reduced pressure. To make a stock solution from each sample, 1 mg of each tested extract was added to a separate 10 mL volumetric flask, dissolved in 1 mL of dimethyl sulfoxide (DMSO), and finally the volume was completed to 10 mL using distilled water. The assay buffer solution  was then used to dilute the stock solutions of each sample under examination to produce different concentrations of each sample.According to the results of the network pharmacology study, the best-scoring plants  inhibitory activity using spectrophotometric assay.106. The ADP assay kit offers a straightforward method for detecting ADP in a wide range of samples, specifically those containing reducing agents that may interfere with oxidase-based assays. ADP concentration was evaluated using a coupled enzyme colorimetric assay (450 nm) that was proportional to the quantity of ADP present. The bioassays were performed as described by Al-Sha’er et al.107 and mentioned in supplementary material. Saturosporine, an Akt inhibitor, was tested as a positive control, while the negative controls were prepared by adding the substrate after terminating the reaction.The Akt1 inhibitory activity of the top four plants resulted from network pharmacology analysis was measured using a colorimetric assay of released adenosine diphosphate (ADP)108 supplied by Sigma, and described by Ros et al.109 as in supplementary materials.The inhibitory activity of the top four plants resulted from network pharmacology analysis on caspase 3 gene was quantified as illustrated on caspase 3 colorimetric assay kit (CASP-3-C)β, TNF-α and INF-γ were determined using real-time polymerase chain reaction (PCR). The means ± standard deviations of three distinct replicates were used to express the results. The method described by Darwish et al.111, as given in the supplementary data, contains the specific details about the procedures. Statistical analysis of the results was performed using One-way ANOVA test- Tukey post-test options implemented on GraphPad Prism 5 program.The MTT test was used to evaluate the cytotoxicity of the various extracts in comparison to piroxicam. Each extract's effective anti-inflammatory concentrations (EAICs) were calculated in a culture of lipopolysaccharides (LPS) activated human WBCs obtained from human volunteers with ethical approval from ethical committee (IRB NO: 00012098). Expression levels of IL-6, IL-1Crystallographic structure of the most enriched target genes , identified through network pharmacology analysis, were obtained from the Protein Data Bank (PDB). Each protein crystal structure was chosen based on the highest resolution possible. Through the use of the protein preparation wizard (OPLS 3 force field) module run in the Schrodinger suit, the preparation and energy minimization of crystal structures of the target proteins were carried out. The protein optimization was followed by the assignment of hydrogen bonds and bond order. At pH 7, zero order bonds to metals and disulphide bonds were also constructed. Next, all water molecules that were farther than 5°A away from the active site were eliminated. The grid boxes were constructed using the residues implicated in interactions with the co-crystallized ligands. The compounds 2D structures were loaded as (.sdf) format into the Lig Prep 2.3 module  to build the least energy 3D structure for each compound and search for alternate conformers. The ionisation states were adjusted to create all conceivable states at pH 7. Molecular docking analyses were performed utilizing the Glide docking program of Maestro molecular modelling package executing extra-precision (XP-Glide) module. The ligand–target interactions such as hydrogen bond, hydrophobic interactions, ion pair interactions together with the binding modes of the identified compounds were demonstrated using Maestro interface.112. The CHARMM-GUI server was used to prepare the necessary input files113. Using AMBER99SB force fields, proteins and compounds’ topology files were created114. The TIP3 water model was used to dissolve the protein–ligand complexes, using a rectangular box type 10 Å away from the complexes. Then, 0.15 KCl salt was added to neutralize the solution. The created system was minimized to 5000 nsteps with the steep integrator. Nose–Hoover and Parrinello-Rahman algorithms were used to equilibrate it using NVT/NPT ensemble steps of 0.3 ns duration at 300 K and 1 atm pressure. 200 ns MD simulation was run and 1000 frame was recorded. The root mean square deviation (RMSD) and the root mean square fluctuation (RMSF) were measured with gmx scripts116. RMSD and RMSF plots were created with QtGrace v2.0.6. The PyMol Molecular Graphics System version 2.5.2 software was used to make MD trajectory videos and protein–ligand binding poses visualization.To test the stability of protein–ligand complexes derived from molecular docking studies with Glide, MD simulations were run with Gromacs v2020.1Curcuma longa, Allium sativum, Oleu europea, Salvia officinalis L, Glycyrrhiza glabra and Silybum marianum had the highest number of P-C-T-P interactions which would suggest that these plants have more active substances that can act as immunomodulatory agents. However, the main drawback in the current network pharmacology analysis studies is the insufficient scientific verification as it needs extensive in-vitro, in-vivo and clinical investigations68.Given that medicinal plants are multi-component and the network pharmacology approach emphasizes the idea of “network target, multicomponent therapeutics,” in a holistic manner analogous to the complex matrices of medicinal herbals, this strategy is thought to be appropriate for understanding the mechanism of action of medicinal plants. In this study, network pharmacology-based analysis of 2154 phytochemical constituents obtained from 32 selected immunomodulatory plants was carried out. A total of 37 constituents and 32 targets were found to be involved in 40 immunomodulatory-associated pathways. Apigenin, luteolin, diallyl trisulfide, silibinin and allicin had the highest percentage of C-T interactions, where the target genes AKT1, CASP3, PTGS2, NOS3, TP53 and MMP9 were found to be the most enriched ones by possessing the highest combined scores, suggesting that they may be the key nodes in C-T network. KEGG analysis illustrated that pathways in cancer, fluid shear stress and atherosclerosis, relaxin signaling pathway, IL-17 signaling pathway and FoxO signaling pathway had the largest number of observed genes and the smallest false discovery rate. Additionally, a combined plant-constituent-target-pathway network demonstrated that Furthermore, molecular docking analysis of the top hit compounds; apigenin, luteolin, diallyl trisulfide, silibinin, and allicin, against the active sites of the most enriched immunomodulatory target genes; AKT1, CASP3, PTGS2, NOS3 and TP53 was carried out. The results revealed that silibinin had the lowest binding energy with RAC-alpha serine/threonine-protein kinase (AKT1) followed by caspase-3 and finally tumor suppressor protein TP53, whereas luteolin and apigenin exhibited the highest stabilized interactions with RAC-alpha serine/threonine-protein kinase followed by prostaglandin-endoperoxide synthase 2 and then tumor suppressor protein TP53. The extracts of the highest scoring plants retrieved from network analysis, were then subjected to in vitro anti-inflammatory and cytotoxicity testing exhibiting outcomes that are equivalent to those of piroxicam. These plants are suggested as prospective sources of immunomodulatory agents by this study, which also offers a thorough explanation of the mechanism underlying the extracts’ supposed anti-inflammatory action. To support our findings, additional in vivo and clinical research are advised.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


We propose a hypothesis about pathogenic mechanism on RIAISs and proofed it through western-blot. Our results indicated significant dysregulation of metabolic pathways in RIAIS patients. Most importantly, we found that RIAISs were associated p53 and PI3K-AKT pathway.Cervical carcinoma (CC) is the fourth most common cancer in females and radiotherapy is always as the definitive therapy for cervical cancer patients who are not suitable for surgery. Radiation-induced acute intestinal symptoms (RIAISs) occur in 50-80% of cervical cancer patients. Some research shows that RIAISs may relate to inflammatory reaction by radiotherapy but the action mechanism is also not clearly and the details of the molecular mechanism are still urgently needed. In this paper, basing on  During the therapeutic process of patients with cervical cancer and patients who are adverse surgical candidates, radiotherapy is always the ultimate treatment . With adThe intestine is particularly sensitive to ionizing radiation and causes side effects such as vomiting, anorexia, weight loss, diarrhea, dehydration, and infection . PatientCervical carcinoma (CC) is the fourth most common cancer in females, especially in developing countries . HoweverMetabolomics is becoming an important component of systems biology . Current1H NMR based metabonomic analyses was employed to detect the change of metabolins before and after radiotherapy between different groups. The data integrated with bioinformatics analysis results were used to identify potential targets or pathways and novel therapeutic regimens. As a result, we built a profile of RIAISs which based on metabolins and bioinformatics. We found that radiotherapy mainly affected the PI3K-AKT and P53 pathway. The activation of these pathways will lead to an inflammatory reaction and these results could reveal the nosogenesis of RIAISs. The result will aid in our understanding of the pathobiology of the disease.In this study, feces, serum, and urine of CC patients were used as samples. An Chemiluminescent HRP substrates were purchased from Millipore Corporation . Primary antibodies against AKT (33748), p-AKT (11054), NFKB (48498), p-NFKB (11166), GAPDH (40493), goat anti-rabbit IgG secondary antibody (L3012) were purchased from Signalway Antibody .Detail information are as the same as our previous study . A summaOn treatment of patients, our previously published treatment protocol was followed for this study . The detSamples were grouped as follows:Group I-samples of RIAIS group before radiotherapy.Group II-samples of the control group before radiotherapy.Group III-samples of RIAIS group after radiotherapy.Group IV-samples of the control group after radiotherapy.1H NMR analysis are shown in the supporting information. The workflow of the whole study is shown in This section was according to our previous work . The det1H NMR analysis are shown in the supporting information.This section was according to our previous work . SpectraThis section was according to our previous work . The dethttp://hmdb.ca) and Compound Discoverer 3.0 software was used as the search database. Next, we used the DAVID tool (https://david.ncifcrf.gov/) to analyze protein function and pathway enrichment. To identify the key targets, we used Venny 2.1.0 to intersect two parts, including all proteins from differential pathways by metabonomic analysis, proteins associated with RIAISs by GeneCards database. Then, the key target was uploaded to the STRING database (https://www.string-db.org/cgi/input.pl), and the minimum required interaction score was set to 0.4 to obtain a protein–protein interaction (PPI) network of key targets and proteins related to the key targets. Finally, the constructed networks were merged, and the duplicated edges were removed and visualized in Cytoscape3.2.1.To identify the differential metabolites and pathway ascription, a corresponding metabolomic database . For single comparisons, significant differences between the means were determined by one way ANOVA test. A p-value less than 0.05 was considered to indicate significant differences. All data were processed using GraphPad Prism 5.01 software.1H NMR results into MestReNova software for phase correction and baseline adjust. Import 1H NMR-based metabolomic strategy was first used to identify differential metabolites in CC patients after radiotherapy. As a result, 733 metabolites were identified with a false discovery rate (FDR) ≤ 0.01. After further data analysis by HMDB database, a total of 524 metabolites were found to be significantly different among different groups. With the criteria of p-value < 0.05, only 151 metabolites were found. These proteins were annotated in 20 different pathways in the KEGG database. Among these findings, the PI3K-Akt, MAPK, Thermogenesis, Choline metabolism in cancer, IL-7 metabolic pathways, p53, and other pathways were involved the main biological processes of RIAISs  and RIAISs disease-associated proteins (1316) obtained from GeneCards, were used in the integrated analysis. As shown in the Venn diagram in To further predict the molecular mechanism of RIAISs caused by radiotherapy, a PPI network was built. As According the results about the network analysis above, we choose the downstream proteins in PI3K-Akt signaling pathway and p53 signaling pathway and tried to test and verify the phosphorylation levels of AKT and NFKB. The changes in the phosphorylation levels of AKT and NFKB in the urine samples are shown in Radiotherapy is commonly used as the ultimate treatment for patients with locally advanced cervical cancer or poor surgical candidates . For cer1H NMR spectroscopy is a powerful tool for chemical structural elucidation and chemical dynamic studies and analytic instrument to get information of biomarkers and help us reveal the mechanism of disease more accurately [curately . Furthercurately .However, although the accuracy of instruments is developing rapidly, but single-omic results still have some limits, which mainly focus on the one-sidedness description of the entire life system. Bioinformatics now offered us more and more useful tools and databases, they could be a supplement of single-omic results and they will help us describe the vital process in a integrity view. In this study, a reasonable analysis strategy which based on metabolins and bioinformatics was used . We founP53 is a tumor suppressor gene and is one of the primary cellular factors determining the nature of growth arrest and/or cell death after exposure to ionizing radiation. P53 pathway plays an important role in damage surveillance and as such has been dubbed the guardian of the genome. Upon DNA damage, P53 pathway was activate, downstream proteins (like NFKB) accumulation was increased and translocated to the nucleus where it binds to DNA, acting to regulate the transcription of a number of genes . AccordiPI3K-AKT signaling plays a significant role in oxidative stress and inflammation . In addi1H-NMR metabonomic and bioinformatics analysis, we proposed a hypothesis about pathogenic mechanism on RIAISs and proofed it through western blot. Our results indicate significant dysregulation of metabolic pathways in RIAIS patients. Most importantly, we found that RIAISs were associated p53 and PI3K-AKT pathway.In summary, this study demonstrated distinct differences in the spectra acquired between the feces of RIAIS patients before and after radiotherapy, and between RIAISs and controls. Basing on a


Previous studies have reported conflicting results about the association of vitamin D (VD) level with coronary artery disease (CAD). We aimed to study the association of VD with atherosclerotic CAD in Egyptian individuals.p < 0.001. VD was correlated with the number of diseased coronary arteries and lipid profile . By multivariate analyses, VD was an independent predictor of CAD , and the number of diseased coronary arteries, p < 0.001, especially three-vessel disease .We prospectively enrolled 188 consecutive CAD patients with a median age of 55(50–62) years; 151(80.3%) were male. All patients were diagnosed by cardiac catheterization and were compared with 131 healthy controls. VD levels were measured in serum samples of all participants. Compared to controls, CAD patients had a significantly lower median VD level, 14.65 (9.25–21.45) versus 42.0 (32.0–53.0) ng/mL, We have shown that low VD should be considered a non-traditional risk factor for CAD in Egyptian individuals. Low VD was correlated with coronary atherosclerosis, especially in patients with multivessel effects.The online version contains supplementary material available at 10.1186/s43044-023-00419-5. Coronary artery disease (CAD) is a worldwide health problem. The identification of risk factors such as male sex, old age, smoking, diabetes mellitus, hypertension, dyslipidaemia, chronic kidney disease, and positive family history is important for the primary and secondary prevention of CAD. However, not all patients with CAD have traditional risk factors, suggesting other reasons for their atherosclerosis. Large cross-sectional studies with long-term follow-up of more than seven years have found that low vitamin D (VD) was associated with a high risk of cardiovascular events, myocardial infarction, and mortality –3. MoreoAt the cellular level, VD receptors are expressed in many tissues of the cardiovascular system. They may control cell proliferation, apoptosis, and cell adhesion. They also downregulate proinflammatory cytokines and metalloproteinases and upregulate anti-inflammatory cytokines and inhibitors of metalloproteinases. In contrast to previous findings, the association between VD and cardiovascular disease is still controversial. Studies have shown conflicting results on the association of VD level or supplementation and CAD, endothelial function, vascular calcification, and carotid intima-media thickness . AdditioPrevious results on the effects of VD on CAD were chiefly derived from other parts of the world. This study aimed to investigate the effect of VD levels on atherosclerotic CAD and its severity in Egyptian patients who underwent diagnostic cardiac catheterization. This study was preceded by a laboratory study that consisted of immunohistochemical measurements of VD receptors in human and animal arterial tissues, which revealed deficient VD receptors in atherosclerotic specimens .This study was the clinical phase of a study on VD levels and atherosclerosis in Egypt. The first phase was a laboratory study that measured VD receptors in human atherosclerotic aortic specimens derived from 50 patients with myocardial infarction during surgery. That study also investigated the effects of supplementation with variable doses of VD  on the aortas of 40 male Wistar rats. All the rats were fed a high-fat diet to induce vascular atherosclerotic processes. They were subdivided into 4 groups by their VD supplementation. The first group did not receive VD supplementation, while the rest of the 3 groups received small, medium and large doses of VD, respectively. The 4 groups were compared with a control group, 10 rats, which received a normal diet. The study found that VD receptors were low in all human aortic specimens. In animal groups, VD administration showed a dose-dependent effect on vascular VD receptors and atherosclerotic foam cell formation: Compared to the control group, the high dose VD group had marked reduction in atherosclerotic foam cell formation and the highest expression of VD receptors, while VD receptor expression and foam cell formation were also different in other VD groups (lower VD receptors and marked foam cell formation), suggesting a protective effect of VD administration . The tubes were centrifuged at 3000 rpm for 10 min. Sera were separated and kept at − 20 °C until the time of analysis. For measurements of total serum 25-hydroxyVD (25-OH VD), we used a total 25-OH VD EIA kit supplied by Epitope Diagnostics, Inc. San Diego, CA 92121, USA . Serum total cholesterol was quantified by an enzymatic colorimetric technique provided by Spectrum Diagnostics,  . Serum tt tests and ANOVA. Mann–Whitney U and Kruskal–Wallis tests were used for variables not normally distributed. Categorical variables are presented as frequency (%). The chi-square with exact test was used for comparisons. Odds ratios (OR) were calculated using Cochran’s and Mantel–Haenszel statistics. Correlations between normal variables were measured by the Pearson test, while the Spearman test was used for nonparametric correlations. To input data into parametric tests, the data were log-transformed. Multivariate analyses were performed to identify variables independently associated with CAD, and the number of diseased coronary arteries  by using binary and multinomial logistic regression analyses, respectively. Linear regression analysis for VD with the forward stepwise method was also performed. Variables in the equations were sex, age, diabetes mellitus, dyslipidaemia, smoking, hypertension, family history, renal impairment , total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, VD, and left ventricle ejection fraction. ROC curves were drawn to identify optimal cut-off values. All tests were performed by using SPSS package version 25 . All results were two-sided and p < 0.05 was considered significant.Continuous variables are presented as mean ± SD for normally distributed variables and median  for variables not normally distributed. Comparisons of means were done by using A total of 188 consecutive CAD patients who underwent cardiac catheterization were included prospectively. Our CAD population had a median (IQR) age of 55 years (50–62) with a male prevalence of 80.3% and dyslipidaemia in 87.2%  vs. 42 (32–53),5–21.45 vp < 0.001 versus controls.Figure p = 0.003]. Its cut-off value was 30 ng/dL, which had a sensitivity of 81% and a specificity of 81.4%, with an area under the curve of 0.92 (p < 0.001) , while the protective HDL-cholesterol was significantly lower (p < 0.001). Importantly, individuals who had low VD more frequently had multivessel disease , and more often had ACS , especially acute myocardial infarction , than individuals who had high VD.Table r = − 0.7), total cholesterol (r = − 0.6), LDL-cholesterol (r = − 0.6), HDL-cholesterol (r = 0.5), triglycerides (r = − 0.4), left ventricle ejection fraction (r = 0.4), smoking habit (r = − 0.3), and age (r = − 0.2) (p < 0.001 for each correlation) , renal impairment , and sex .Low VD was correlated with the number of diseased coronary arteries (p = 0.003) but also the number of diseased coronary arteries (p < 0.001) , p = 0.011; two-vessel disease, OR 0.84 (95% CI 0.73–0.96), p = 0.012; and three-vessel disease, OR 0.83 (95% CI 0.72–0.95), p = 0.008]. These results were confirmed by a separate linear regression analysis on VD concentrations, which revealed a significant association with CAD  and the number of diseased vessels .By multivariate analyses, low VD measurement was an independent predictor of not only CAD p = 0.00 but alsoThe chief findings of this study are that low VD concentrations in the blood were significantly associated with CAD and its severity in Egyptian patients. This prospective study found that low VD levels independently predicted atherosclerotic coronary artery lesions in ischaemic patients. Moreover, it was an independent predictor of multivessel coronary affection diagnosed by cardiac catheterization. The study complemented the laboratory stage of this work that previously revealed the existence of low VD receptor expression in human and animal atherosclerotic arterial strips . Taken tp < 0.001]. It acted concurrently with the atherosclerotic lipid profile  on the coronary arteries to cause atherosclerosis. Zittermann et al. illustrated how low 25-OH VD levels could result in atherosclerotic changes . The rupture of vulnerable plaques was thought to be responsible for most of these events. Vulnerable plaques are characterized by rich lipid content, a thin fibrous cap and high inflammatory activity. Although the molecular function of VD signalling is still not clear [p < 0.001] [Most [103 (57.3%)] of our patients low VD (< 30 ng/mL) had ACS, including unstable angina, ST-elevation myocardial infarction and non-ST-elevation myocardial infarction. The odds of low VD in patients with ACS were higher than they were in patients with high VD  , 21, 25.p < 0.001). Taking into consideration that other variables, such as diabetes mellitus, left ventricular ejection fraction, LDL-cholesterol, total cholesterol and smoking, were important, a low VD level was an independent predictor of multivessel disease of coronary arteries in our population. However, in peripheral arterial disease, conflicting results of VD levels have been reported [Multivessel disease of coronary arteries would carry an increased risk of future cardiovascular events in CAD patients. Two- and three-vessel disease were prevalent in the majority of patients with low VD, 109 patients (60.6%). The OR of having more than single-vessel disease in the population with low VD versus those with high VD was 4.43  yielded no significant differences because too few patients were in each group. The study was not powered to investigate the association of VD and ACS.VD level was significantly lower than normal in Egyptian patients with CAD. In addition to the traditional cardiovascular risk factors, low VD was associated with coronary artery atherosclerosis and its severity. Prospective trials to demonstrate the effect of VD supplementation to treat CAD patients are encouraged, especially in those with multivessel disease and acute coronary syndrome.Additional file 1: Fig. 3A Correlation between log vitamin D and log total cholesterol . B Correlation between log vitamin D and log LDL-C . C Correlation between log vitamin D and log HDL-C . D Correlation between log vitamin D and log TG . .


Aspergillus flavus and Penicillium notatum and used a thorough bioinformatics analysis to characterize the toxicity and predict genes and protein targets for it. The predicted median fatal dosage (LD50) for citrinin was 105 mg/kg weight, and it belonged to toxicity class 3 . Citrinin was found to be well absorbed by human intestinal epithelium and was a Pgp nonsubstrate (permeability glycoprotein), which means that once it is absorbed, it cannot be pumped out, hence leading to bioconcentration or biomagnification in the human body. The main targets of toxicity were casp3, TNF, IL10, IL1B, BAG3, CCNB1, CCNE1, and CDC25A, and the biological pathways implicated were signal transduction involved in DNA damage checkpoints, cellular and chemical responses to oxidative stress, DNA damage response signal transduction by P53, stress-activated protein kinase signaling cascade, netrin–UNC5B signaling, PTEN gene regulation, and immune response. Citrinin was linked to neutrophilia, squamous cell carcinoma, Fanconi anemia, leukemia, hepatoblastoma, and fatty liver diseases. The transcription factors E2F1, HSF1, SIRT1, RELA, NFKB, JUN, and MYC were found to be responsible. When data mining was performed on citrinin targets, the top five functional descriptions were a cell’s response to an organic cyclic compound, the netrin–UNC5B signaling pathway, lipids and atherosclerosis, thyroid cancer, and controlling the transcription of the PTEN gene.The mycotoxin citrinin, which can contaminate food, is a major global concern. Citrinin is regarded as an inevitable pollutant in foods and feed since fungi are widely present in the environment. To identify contentious toxicity and lessen its severity by understanding the targets of citrinin in the human body and the impacted biosynthetic pathways, we analyzed the production of citrinin from  Monascus pigments, fermented sausages, wheat, corn, and rice [Penicillium citrinum in 1931. Citrinin has been linked to human genotoxic, embryotoxic, teratogenic, carcinogenic, and mycotoxin nephropathy consequences. Citrinin is known to have a variety of bioactivities in vitro, including antibacterial, antifungal, and possible anticancer and neuroprotective properties, in addition to its toxic effects. Despite having antibiotic properties, it was never used as an antibiotic because it showed toxic effects on mammals, particularly nephrotoxicity [13H14O5. Its IUPAC name is -6-hydroxy-3,4,5-trimethyl-8-oxo-3,4-dihydroisochromene-7-carboxylic acid. Citrinin pollutants have been found in human biological fluids as well as agricultural commodities and feedstuffs such as cheese, sake, and soy sauce [Monascus pigments, fermented sausages, wheat, corn, and rice [Aspergillus flavus and Penicillium notatum. We then used a thorough bioinformatics analysis and toxicity characterization to determine the disease-causing targets for citrinin. To further elucidate citrinin’s potential role in human toxicity, its cheminformatic aspects were studied. Computer models have been promoted as a viable alternative to experimental methods when knowledge about compounds is limited.Citrinin pollutants have been found in human biological fluids as well as agricultural commodities and feedstuffs such as cheese, sake, and soy sauce ,2. Citriand rice ,4,5,6. Mand rice ,8. Mycotand rice ,11,12. Tand rice ,16. Citrand rice . Over thand rice . Althougand rice ,21. The and rice . The negand rice . Citriniand rice . Given tPenicillium notatum and Aspergillus flavus fungal cultures were maintained and grown on Petri dishes at 25 °C for 10 days. The spores were suspended and harvested by adding 10 mL of sterilized distilled water to each plate. The spore suspension thus obtained was filtered using cheesecloth. The filtered biomass was then dried at 40 °C for 24 h, and the dry weight of mycelium was determined. Filtrates were stored at 4 °C for carrying out citrinin extraction, and the weight of the biomass was recorded. Each experiment was performed in duplicate. To validate the production of citrinin, thin-layer chromatography (TLC) was performed on the acquired culture filtrates. Citrinin was extracted three times with chloroform (1:1 v/v) added to the filtered solution for the TLC. The lowest layer of the separated mixture was collected. At 40 °C, the fractions were dried in a dry oven. The synthesis of citrinin was verified by TLC under UV light after dilution of citrinin extracts with 2 mL of chloroform. Briefly, sample extracts and a standard (containing 0.5 g/mL) from Sigma Chemical Co. were applied to TLC plates . In glass tanks lined with aluminum foil, TLC plates were produced in a solvent composed of toluene, ethyl acetate, and formic acid (6:4:0.5 v/v). After development, plates were dried for 30 min to one hour, and when viewed under wavelength (365 nm), a bright yellow spot corresponding to citrinin was seen. Each experiment involved duplicates. Potato dextrose agar (PDA) (39 g) was dissolved in distilled water to make a one-liter solution (1L). After autoclaving for 15–20 min at 121 °C, 50 mg/L streptomycin was added after cooling, and 20–25 mL portions were dispensed into sterile 15 × 100 mm Petri dishes. https://tox-new.charite.de/protox_II/, accessed on 15 January 2023) was used. ProTox-II incorporates molecular similarity, pharmacophores, fragment propensities, and machine-learning models. The predictive models are based on data from both in vivo instances and in vitro assays, such as the Tox21 assay, the Ames bacterial mutation assay, the hepG2 cytotoxicity assay, and the immunotoxicity assay . The models have performed well and have been validated on separate external sets. The predicted median fatal dosage (LD50) was obtained in mg/kg weight along with the toxicity class, and the prediction accuracy [For the prediction of various toxicity endpoints of citrinin (PubChem CID: 54680783), such as acute toxicity, hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity, immunotoxicity, adverse outcome pathways (Tox21), and toxicity targets, ProTox-II  [http://ctdbase.org/, accessed on 15 January 2023). The SMILES for citrinin were obtained from (PubChem CID: 54680783). The database contains data on multiple genes and proteins that differ between various species and respond to environmental toxins such as citrinin. Comparative Toxicogenomics Database (CTD) has significantly broadened its scope to accurately reflect a trio of hand-selected chemical–gene, chemical–disease, and gene–disease connections [Fast yet robust predictive models for physicochemical properties such as BOILED-Egg, iLOGP, and bioavailability radar were generated from SwissADME (ry 2023) , with anry 2023) . SwissADry 2023) . Furthernections .https://genemania.org/, accessed on 15 January 2023) to analyze the gene sets interacting with one another for physical interaction, genetic interaction, co-expression, co-localization, shared protein domains, or predicted interactions. A real-time multiple association network integration approach was used to forecast gene function. GeneMANIA was used to discover any additional genes connected to a group of input genes. GeneMANIA creates gene networks by scoring nodes using functional genomic data and label propagation. The network can then be subjected to any analytic feature available in Cytoscape or Metascape. Gene function networks were shown by using colors based on the network nodes by function (GO annotation), and a color key was given in the legend [https://metascape.org/gp/index.html#/main/step1, accessed on 15 January 2023) to find biological pathways and protein–protein interaction enrichments. It uses a variety of functional databases, such as GO, KEGG, and UniProt, to analyze data from a variety of other species in addition to data from the literature. It also evaluates not just a single dataset but also many gene sets simultaneously. Gene identities (Entrez Gene IDs) were automatically converted into human Entrez Gene IDs by Metascape using the EggNOG and Homologene databases, and all the transcription factors and pathways implicated with the appropriate p values were reported. Hypergeometric accumulative distributions recorded p-values to collect datasets and enrichment factors [The homosapiens option was used to analyze the target genes implicated in SwissADME, a comparative toxicogenomics database, and the ProTox-II tool for citrinin in GeneMANIA  for citrinin was 105 mg/kg weight ; Class II, which is also fatal if swallowed (LD50 ≤ 50), Class III, which is toxic if swallowed (LD50 ≤ 300), Class IV, which is harmful if swallowed (LD50 ≤ 2000), Class V, which may be harmful if swallowed (LD50 5000), and Class VI which is nontoxic (LD50 > 5000). In our preliminary investigation, it was estimated (93–94% accuracy) that citrinin should be classified in category III . ConsumpWe mined functionally related genes from SwissADME, ProTox-II, and the Comparative Toxicogenomics Database (CTD) and created a network of protein–protein interactions through the GeneMANIA database. According to data in the plasma proteomic signature of healthy adults, functional enrichment revealed that the hub genes and functionally related genes that were mostly implicated in the CTD database were casp3, TNF, IL10, IL1B, BAG3, CCNB1, CCNE1, and CDC25A A. Benzo. Citrinip-values. Hypergeometric accumulative distributions recorded p-values to collect datasets and enrichment factors. Citrinin targets the DNA damage response, positive regulation of miRNA transcription, modulation of the netrin–UNC5B signaling pathway, leukocyte proliferation regulation, etc. Multiple malignancies have been linked to UNC5B signaling, which boosts cell survival when its ligand netrin-1 is present and promotes cell death when it is not. Additionally, it has been noted that blocking the ligand reduces tumor invasiveness and angiogenesis [Our results are consistent with the findings from previous studies on cell lines reporting that the inhibition of the JNK signaling pathway improves cell viability in response to oxidative stress . To fullogenesis . Citrinip-values from the various route and process enrichment experiments were retained as the functional descriptions of the various MCODE components (The molecular complex detection (MCODE) method was used to identify intricately connected network components in networks with 3 to 500 proteins. Circle plots are used to display their overlap and shared term relationships. The five terms with the greatest mponents . The top five clusters and their representative enriched terms are presented in the table with functional descriptions obtained with citrinin targets. These were a cellular response to an organic cyclic compound, the netrin–UNC5B signaling pathway, lipids and atherosclerosis, thyroid cancer, and regulation of PTEN gene transcription. Citrinin was found to change a state or activity in a cell in terms of movement, secretion, enzyme production, gene expression, etc. as a result of an organic cyclic compound stimulus. Citrinin’s mechanism of toxicity exposure has been elucidated, and it has been observed that citrinin targets several genes and proteins that are implicated in the diseases mentioned, and the transcription factors regulating these genes have also been elucidated. Previous studies reported enzymes including malate dehydrogenase and glutamate dehydrogenase as well as the ATP synthase complex involved in the respiratory chain implicated with citrinin exposures . Other sThis study investigated the pharmacological mechanisms underlying the toxicological activity of citrinin to provide a platform for future research and development. The lethal dose of citrinin has been predicted . Citrini


Forsythia suspensa (Thunb.) Vahl, which has been found to be effective in treating cholestasis. However, its key component for alleviating cholestasis has not been determined. In this study, four representative active ingredients in forsythia fruit were selected. Through network pharmacology and molecular docking technology, we tried to find the key component for its treatment of cholestasis. Furthermore, the model of cholestasis in mice was established to verify the protective effect of the key component on cholestasis. Network pharmacology and molecular docking showed that forsythoside A (FTA) is the key component of forsythia fruit in the treatment of cholestasis. In vivo experiments revealed that FTA treatment could alleviate liver injury, dysfunction, and collagen deposition induced by cholestasis in mice. At the same time, FTA treatment inhibited inflammatory factor release and fibrosis-related factor expression. In addition, FTA treatment also reduced MMP-2, TLR4, MYD88, NF-κB p65, and p-NF-κB p65 protein expression. In conclusion, FTA, a key component of forsythia fruit, alleviated liver damage and fibrosis caused by cholestasis via inhibiting the TLR4/NF-κB pathway, extracellular matrix accumulation, and inflammatory cytokine expression. The research results could provide a scientific reference for the development of forsythia fruit as a drug or functional food to prevent and treat cholestasis.Forsythia fruit, edible fruit of  Cholestasis or cholestatic liver disease (CLD) is the accumulation of excess bile acid and abnormal metabolites in the body induced by bile duct injury, leading to apoptosis and necrosis of bile duct cells and hepatocytes. If allowed to develop without intervention, it may eventually develop into liver fibrosis and cirrhosis, or even liver failure ,2. ChronForsythia suspensa (Thunb.) Vahl (FS), is an aromatic and edible fruit. In the 2020 edition of the Ch.P, the fruits were identified as the medicinal part of FS. Extensive evidence shows that FS can effectively exert hepatoprotective effects through its anti-inflammatory, antioxidant, anti-fibrosis, and bile-promoting properties [Herbal remedies are the first drugs used by humans due to the fact that plants can produce a variety of secondary metabolites that have medicinal effects on humans  ,10,11. Aoperties ,15,16. Foperties . Clinicaoperties . HoweverDue to the inclusion of multiple bioactive compounds in medicinal and edible plants, which can exert pharmacological effects through multiple pathways and targets, it is extremely difficult to elucidate their molecular basis and mechanism of action using traditional pharmacological methods. With the development of bioinformatics and systems biology, network pharmacology has been proposed as a new method for drug discovery . NetworkIn this study, we first analyzed the possible key component of FS in the prevention and treatment of cholestasis via network pharmacology and molecular docking techniques. Then, a cholestasis mouse model was established to validate the effectiveness and molecular mechanisms of the key component to alleviate cholestasis. The aim of this study is to clarify the molecular basis of FS for the prevention and treatment of cholestasis and to lay a scientific foundation for the further development of functional foods and drugs for the treatment of cholestasis.https://pubchem.ncbi.nlm.nih.gov/ (accessed on 2 June 2022)). Then they were entered into the SwissTargetPrediction (http://www.swisstargetprediction.ch/ (accessed on 2 June 2022)) and SuperPred (https://prediction.charite.de/index.php (accessed on 2 June 2022)) online platforms [Four representative active ingredients (RAIs)  used for quality control under “FS” and “FS extract” in the Ch.P (2020) were selected for follow-up study. SMILES for RAIs were downloaded from the PubChem database ) and GeneCards database (http://www.genecards.org/ (accessed on 13 July 2022)) using the keyword “Cholestasis”, then integrating the data and de-duplicating them. Using the Venny_v2.1.0 platform (https://bioinfogp.cnb.csic.es/tools/venny/index.html (accessed on 13 July 2022)), Venn diagrams of the RAI targets and cholestasis targets were plotted, with the intersection being the target of the RAIs for cholestasis treatment. Subsequently, the relationships between the four RAIs and their targets for the treatment of cholestasis were collated and introduced into Cytoscape_v3.8.2 software to construct the “components-targets” network [Cholestasis targets were collected in the DisGeNET database ( network .https://string-db.org/ (accessed on 13 July 2022)), and the PPI relationships between the targets were obtained using “Homo sapiens” as the study species. The PPI network was established via Cytoscape_v3.8.2 software [The Gene Symbols of the targets of the RAIs for cholestasis treatment were imported into the STRING_v11.0 data platform ) [p-value from smallest to largest, we selected the top 25 results to draw an advanced bubble chart on the OmicShare website (https://www.omicshare.com/ (accessed on 13 July 2022)).The Gene Symbols of the targets of the RAIs for cholestasis treatment were imported into the DAVID_v6.8 database (y 2022)) ,32, and The top 25 signal pathways were selected from the outcomes of the KEGG pathway enrichment analysis, and the relationship between pathways and targets, as well as between targets and components, were sorted out and imported into Cytoscape_v3.8.2 software to construct a “components-targets-paths” network . MeanwhiNetwork pharmacological studies (as above) were conducted on the key component screened out above, including the construction of the PPI network, enrichment analysis of the KEGG pathway and GO biological process, and the main targets and pathways of key components for the treatment of cholestasis were screened out.https://www.rcsb.org/ (accessed on 28 September 2022)) and were used as receptors, which were prepared using OpenBabel_v2.4.1, PyMol_v2.4 and Autodock_v1.5.6 software and converted into qdbqt format [The 3D structure files of the RAIs and FTA downloaded from the PubChem database were used as ligands. At the same time, the structure files of targets were downloaded from the RCSB database (t format .https://www.uniprot.org/ (accessed on 28 September 2022)), with an exhaustiveness of 20, using Autodock Vina_v1.1.2 software for molecular docking [The center coordinates of the docking pocket of the receptor proteins with the original ligand were set with reference to the original ligand, and the receptor proteins without the original ligand were set with reference to the center coordinates of the active amino acid residues included in the UniProt database : control group, DDC group, FTA low-dose (FTA-L), FTA medium-dose (FTA-M), and FTA high-dose (FTA-H) groups. The experimental protocol has been approved by the Chengdu University of Traditional Chinese Medicine . Subsequently, in the DDC, FTA-L, FTA-M, and FTA-H groups, mice were fed a diet containing 0.1% DDC for four weeks, while the control group received a normal diet. After being fed the diet for one week, the mice in the FTA-L, FTA-M, and FTA-H groups were administrated with FTA , respectively, for three consecutive weeks. In the control group and the DDC group, mice received normal saline (10 mL/kg) for three weeks. After the experiment, the mice were euthanized by carbon dioxide, and blood and liver tissue were collected. The detailed experimental process is shown in The experimental subjects of this study were 30 8-week-old, male C57BL/6J mice (20 ± 2 g) purchased from Chengdu Dashuo Experimental Animal Co., Ltd. . Mice were housed in an SPF environment, and irradiated and sterilized feed and drinking water were freely available. A week later, the mice were fed adaptively and were randomly divided into five groups  staining. Collagen deposition in liver was evaluated via Masson staining. In addition, serum AST and ALT were tested by assay kit to assess the change in liver function. At the same time, serum TNF-α, IL-6, and IL-1β were tested by ELISA kit to estimate systemic inflammation.p-NF-κB expression. Briefly, paraffin sections were made using mice liver tissue. The sections were placed in PBS (PH 7.4) to perform antigen repair. The sections were placed in a 3% hydrogen peroxide solution to block endogenous peroxidase. The sections were sealed at room temperature for 30 min using 3% BSA. Subsequently, the sections were incubated with primary and secondary antibodies and colored with DAB, respectively. Finally, dyed pictures were observed and collected under a microscope.According to the previous experimental methods of our research group , we used−ΔΔCT.MMP-2, TNF-α, IL-1β, IL-6, and F4/80 mRNA expression levels were detected via qPCR. Briefly, a total RNA isolation kit was used to extract total RNA from liver tissues. Subsequently, the total RNA was then synthesized into stable cDNA using ChamQ SYBR qPCR Master Mix. Next, RT-qPCR was performed by adding ChamQ SYBR qPCR Master Mix according to the instructions of the reagent manufacturer. p- NF-κB p65 were tested via Western blot. Briefly, total protein was extracted from liver tissues and inactivated at 100 °C. Subsequently, electrophoresis, membrane transfer, and other operations were carried out to transfer the protein to the stable PVDF membrane. Then, the membrane was incubated in diluted MMP-2, α-SMA, TLR4, MYD88, NF-κB p65, and p-NF-κB p65 primary antibody (1:1000) and diluted secondary antibody (1:5000). Finally, the developer was dripped onto the membrane and the chemiluminescence was collected.According to the previous experimental methods of our research group , MMP-2, p < 0.05.The data were compared among multiple groups by one-way ANOVA using SPSS 26.0 statistical software, and all results were expressed by mean ± SD. Histograms and heat maps were drawn using GraphPad Prism 9.0 software. Results were considered statistically significant when Basic information about the RAIs can be found in A total of 63 proteins were collected through the STRING_v11.0 database with 221 complex interactions between them b. Cyto-sp ≤ 0.05) were obtained, involving the pathway in cancer, HIF-1, PI3K-Akt, Prolactin, Relaxin, and Toll-like receptor signaling pathway, etc. Subsequently, the top 25 pathways were visualized and analyzed with advanced bubble plots (p ≤ 0.05), involving protein phosphorylation, inflammatory response, response to hypoxia, negative regulation of the apoptotic process, signal transduction, etc. The top 25 entries were visualized and analyzed with advanced bubble plots  were obtained, involving the HIF-1 signaling pathway, PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, relaxin signaling pathway, etc. Subsequently, the top 20 pathways were visualized and analyzed with advanced bubble plots (p ≤ 0.05), involving positive regulation of cell migration, positive regulation of polymerase II promoter transcription, positive regulation of tumor necrosis factor production, positive regulation of inflammatory response, and positive regulation of ERK1 and ERK2 cascades. The top 20 entries were visualized and analyzed with advanced bubble plots , but also alleviate systemic inflammation . More importantly, FTA intervention could also block the conduction of the TLR4/MYD88/NF-κB pathway, thus effectively preventing the liver from suffering from inflammatory damage.In addition, cholestasis often accompanies the development of liver fibrosis, mainly due to impaired or interrupted bile production resulting in intracellular retention of toxic bile components, an excess of which will cause liver damage and fibrosis ,52,53. LEven though the TLR4/NF-κB pathway may be more important, network pharmacology results suggested that the role of other signaling pathways in the prevention and treatment of CLD by FS was also worth investigating. For example, as reported, the modulation of the PI3K-Akt signaling pathway has been shown to have a positive effect on the treatment of cholestasis ,60,61,62We revealed the potential molecular mechanisms of FS and its key components to prevent and treat cholestasis through network pharmacological methods and molecular docking techniques. Meanwhile, in vivo experiments showed that the key ingredient FTA could play an anti-inflammatory, hepatoprotective, and anti-fibrosis role by modulating the TLR4/NF-κB pathway, repressing HSC activation, ECM accumulation, and inflammatory factor release . All in