Taken together, these results indicate that YME1L1 is essential for regulating mitochondrial fission, function, and apoptosis during porcine embryo preimplantation development.YME1L1, a mitochondrial metalloproteinase, is an Adenosine triphosphate (ATP)-dependent metalloproteinase and locates in the mitochondrial inner membrane. The protease domain of YME1L1 is oriented towards the mitochondrial intermembrane space, which modulates the mitochondrial GTPase optic atrophy type 1 (OPA1) processing. However, during embryonic development, there is no report yet about the role of YME1L1 on mitochondrial biogenesis and function in pigs. In the current study, the mRNA level of YME1L1 was knocked down by double strand RNA microinjection to the 1-cell stage embryos. The expression patterns of YME1L1 and its related proteins were performed by immunofluorescence and western blotting. To access the biological function of YME1L1, we first counted the preimplantation development rate, diameter, and total cell number of blastocyst on day-7. First, the localization of endogenous YME1L1 was found in the punctate structures of the mitochondria, and the expression level of YME1L1 is highly expressed from the 4-cell stage. Following significant knock-down of YME1L1, blastocyst rate and quality were decreased, and mitochondrial fragmentation was induced. YME1L1 knockdown induced excessive ROS production, lower mitochondrial membrane potential, and lower ATP levels. The OPA1 cleavage induced by YME1L1 knockdown was prevented by double knock-down of YME1L1 and OMA1. Moreover, cytochrome  Mitochondria are energy-supplying organelles that participate in diverse cellular processes and are important determinants for the oocyte maturation and embryo development in mammals . In oocyThe mitochondrial metabolism is related to fusion and fission morphology transitions . MitochoHowever, the effects of YME1L1, which regulates mitochondrial function and morphology during porcine embryo preimplantation development, have not been reported. In the present research, we hypothesized that YME1L1 is crucial for the porcine embryo preimplantation development by maintaining the mitochondrial morphology and the balance of fission and fusion. To evaluate this hypothesis, YME1L1 was knocked down using double-stranded RNA (dsRNA) at the zygote stage and cultured until the blastocyst stage for further analysis.All reagents were purchased from Millipore Sigma , and all manipulations of live embryos were performed on a heating plate at 38.5°C, unless otherwise indicated.2.Porcine ovaries were collected and stored at 37 °C in saline containing 75 mg/mL penicillin G and 50 mg/mL streptomycin sulfate and delivered from a local slaughterhouse  to the laboratory. The diameter of follicles around 3–6 mm was aspirated using a 10 mL disposable syringe with an 18-gauge needle. The porcine COCs with more than two layers cumulus cells were collected and washed with HEPES briefly. Approximately 100 COCs were transported to each well of 4-well plates with IVM medium  and incubated for 44–48 h at 38.5°C and 5% CO2, 0.05 mM MgS04, 0.01% polyvinyl alcohol , and 0.5 mM 4-(2-hydroxyethyl) piperazine-l-ethanesulfonic acid]. To prevent the pseudo-second polar body extrusion, the activated oocytes were treated with 7.5 μg/mL cytochalasin B for 3–6 h. Subsequently, approximately 50 embryos were washed and cultured in one of 4-well plates with IVC medium for microinjection. The embryos were then cultured for 7 days at 38.5°C and 5% CO2. The blastocysts on the 7th day were randomly collected for further experiments. The total cell number of blastocyst was counted under the epifluorescence microscope (Nikon).The COCs with extended cumulus cells were pipetting for around 40–50 times in 1 mg/mL hyaluronidase to remove cumulus cells. MII stage oocytes with first polar body were then selected and parthenogenetically activated by 2 direct-current pulses of 120 V for 60 µs in activation buffer [297 mM mannitol (pH 7.2) supplemented with 0.1 mM CaClYME1L1 and OMA1were amplified from cDNA of porcine COCs using the primers containing the T7 promoter sequence  to synthesize dsRNA according to the manufacturer’s instructions. After 10 h in vitro transcription, dsRNA mixture was treated with DNase I for 15 min to degrade the DNA template, and then purified using Riboclear™ Plus . The purified dsRNA was dissolved in RNase-free water and stored at −80 °C until use.The fragments of sequence . In vitrYME1L1 or OMA1 was microinjected into 1-cell stage embryos after CB treatment using a Nikon Diaphot Eclipse TE300 inverted microscope  connecting an Eppendorf Femto-Jet  and equipped with a Narishige MM0-202N hydraulic 3-D micromanipulator . After microinjection, the embryos were continually cultured in IVC medium for 7 days. The dsRNA of green fluorescent protein (GFP) was microinjected as the control group.For the knock-down experiments, the dsRNA of 2O, real-time qPCR was performed using a pair of primers for ND1  were used for measurement, the standard reaction solution was prepared and placed on ice avoiding the light before use. Next, 5 µL lysate were transported to 96-well plates and equilibrated for 10 s, and 150 µL of the standard reaction solution was mixed with lysate in each well. ATP levels were measured using a lurninometer  for 10 s after 2 s delay. The signal intensity of YME1L1 knock-down group was relative to the control group which was arbitrarily set to one.Mitochondrial ROS were detected using MitoSOX Mitochondrial Superoxide Indicator (Thermo Fisher Scientific), as previously described . Brieflyth day blastocysts were treated with 2.5 µM 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraemyl-imidacarbocyanine iodide (JC-1)  in the IVC medium for 30 min at 38.5°C in 5% CO2 and visualized by the epifluorescence microscope (Nikon). The ratio of intensity of red fluorescence of the activated mitochondria (J-aggregates), to green fluorescence of less-activated mitochondria (J-monomers) was calculated as the membrane potential   and visualized by using a confocal microscope . The number of TUNEL-positive blastomeres was counted under the epifluorescence microscope (Nikon). The ratio of TUNEL-positive to the total blastomeres was indicated as apoptosis index.Following the instructions of the zerland) , ten blaThe BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 (Beyotime) was used for the cell proliferation assay. Briefly, the morula stage embryos were incubated with 10 μM EdU for 10 h at 38.5 °C. Next, the blastocysts were fixed with 3.7% paraformaldehyde at room temperature for 30 min and permeabilized with 1% Triton X-100 for 30 min. After washing thrice with PBS/PVA, the blastocysts were incubated in 100 μL of click reaction cocktail and 5 μg/mL of Hoechst 33342 in the dark for 30 min. Images were captured after mounting in PBS/PVA using a confocal microscope . The number of EdU-positive blastomeres was counted under the epifluorescence microscope (Nikon). The ratio of EdU-positive blastomeres to the total number of blastomeres was calculated as the proliferation rate.c was stained following the instruction in the immunofluorescence and confocal microscopy subsection.Ten blastocysts per group were treated with 500 nM MitoTracker Red CMXRos  in IVC medium at 38.5°C for 30 min. After washing thrice with PBS/PVA, blastocysts were fixed, and cytochrome st antibody at 4 °C overnight including anti-YME1L1 , anti- TOM20 , anti-cytochrome c  and anti-cleaved caspase-3  diluted in blocking solution (3% BSA and 0.1% Triton X-100). After washing more than thrice with PBS/PVA, the embryos were incubated with 2nd antibody such as Donkey anti-Mouse IgG, Alexa Fluor™ 488 , Goat anti-Rabbit IgG, Alexa Fluor™ 488  or Donkey anti-Rabbit IgG, Alexa Fluor™ 546  for 1 h at room temperature. After washing thrice with PBS/PVA, the embryos were then mounted onto slides using VECTASHIELD® Antifade Mounting Medium with DAPI  and captured using the confocal microscope (Zeiss LSM 710 Meta). Images were processed using the Zen software .Approximately ten embryos in each group were fixed in 3.7% paraformaldehyde at room temperature for 30 min, permeabilized with 1% Triton X-100 in the PBS/PVA for 30 min, and then blocked in 3% BSA containing 0.1% Triton X-100 in the PBS/PVA for 1 h at room temperature. Subsequently, the embryos were incubated with 1YME1L1 mRNA and 18S rRNA (reference gene) was performed as follows: initial denaturation at 95°C for 3 min, followed by 40 repeats of cycle for the denaturation at 95°C for 15 s, annealing at 60°C for 25 s, and extension at 72°C for 15 s, and a final extension at 72°C for 5 min. The data were quantified using the 2−ΔΔCT method . Reverse transcription was performed using the LeGene Express 1st Strand cDNA Synthesis System . Real-time RT-PCR for the T method .Approximately one hundred embryos in each group were lysis with 10 μL RIPA buffer and 10 μL 2X loading buffer on ice. After boiling the samples for 10–15 min, protein samples were separated by the different concentration of SDS-PAGE gel depends on the size of target protein and transferred onto polyvinylidene fluoride membranes for 1 h under 250 mA. Subsequently, the membranes were blocked with 5% skim milk in TBST buffer at room temperature for 1 h and then incubated with first antibody at 4°C overnight such as anti - YME1L1 , anti - DRP1 , anti - LC3B , anti - OPA1 , anti - OMA1 , anti-cleaved caspase-3  or anti - GAPDH . After washing with TBST buffer for three times, the membranes were treated with secondary antibody for 1 h at room temperature such as horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG . The membranes were then exposed using a UviSoft software  with SuperSignal™ West Femto Maximum Sensitivity Substrate .According to the manufacturer’s protocol, cytosolic and mitochondrial proteins of 500 blastocyst stage embryos were extracted using the Mitochondria/Cytosol Fractionation Kit . The expression of glyceraldehyde 3-phosphate dehydrogenase (GADPH) was measured as the purity of mitochondria fraction by western blot analysis.p < 0.05 was considered statistical significance. SPSS software v.19  was used for all calculations. All of experiments were replicated at least three times.The statistical data analysis was carried out by one-way analysis of variance (ANOVA) or Student’s t-test. All data were presented as the mean ± SEM. To evaluate the subcellular localization of YME1L1 during porcine embryo preimplantation development, YME1L1 and TOM20 were detected using immunofluorescence. The expression of YME1L1 was lower and colocalized with TOM20 in 1–2-cell stage embryos. However, YME1L1 was partially colocalized with TOM20 after the 4-cell stage at a higher level . The mRNYME1L1 knock-down was analyzed at blastocyst stages (p < 0.001). YME1L1 knock-down was evaluated by western blotting and immunofluorescence, which showed that the protein level of YME1L1 was also significantly reduced in YME1L1 KD blastocysts (p < 0.05). The ability of development to blastocyst stage was significantly decreased after YME1L1 knock-down  . These data suggest that defective YME1L1 may disrupt early embryo development.To investigate the role of YME1L1 in porcine embryo preimplantation development, YME1L1 dsRNA (YME1L1 KD) was injected into 1-cell stage embryos, and then cultured in IVC medium for 7 days. The efficiency of t stages . Compare< 0.005) . Additio< 0.005) . YME1L1 p < 0.01, p < 0.05, To evaluate the effect of YME1L1 on the porcine embryo mitochondrial morphology after knock-down, the blastocysts were stained with TOM20 as total mitochondria and MitoTracker Red CMORos as active mitochondria. The intensity of MitoTracker and fragmentation of mitochondrial clusters were analyzed using ImageJ, the MitoTracker signal were highly distributed and colocalized with TOM20 in all control blastomeres. However, the active mitochondria were observed in partial blastomeres with lower intensity in YME1L1 KD group (Additionally, YME1L1 KD induced more OMA1 due to the reduced degradation of OMA1 . YME1L1 p < 0.05, p < 0.001, Based on the changes in the morphology of mitochondria, we next investigated if YME1L1 knock-down can promote the mitochondrial dysfunction in porcine embryos, which can be induced by ROS over generation, low mitochondrial membrane potential, and low ATP levels. Accordingly, mitochondrial ROS were detected at the blastocyst stage. As expected, compared to control blastocysts, mitochondrial ROS levels in YME1L1 KD blastocysts were significantly increased (c is released from the mitochondria (c and the level of cytochrome c in mitochondria fraction at blastocysts stage was evaluated using immunofluorescence and western blotting after YME1L1 knock-down (c and MitoTracker was less in YME1L1 KD blastocysts, as detected by Mander’s overlap coefficient (c by western blot. The cytochrome c in the cytosol remained relatively unchanged between the control and YME1L1 KD groups; however, mitochondrial cytochrome c decreased in the YME1L1 KD groups compared with the control.Previous research has demonstrated that cytochrome chondria . The colock-down . Immunoffficient . As showp < 0.05, As YME1L1 KD induced mitochondrial dysfunction and blastocyst total cell number reduction, the TUNEL assay was performed to evaluate whether apoptosis can be induced by the knock-down of YME1L1. The ratio of TUNEL-positive signal number to total nuclear number was significantly higher in YME1L1 KD blastocysts than in the control group (Because of the poor developmental ability and poor quality of embryos after YME1L1 KD, we investigated whether YME1L1 could regulate embryo proliferation. As shown in Previous work reported that mitochondria are important for the apoptosis, calcium homeostasis, cellular signaling pathways, gene expression, organic compound biosynthesis and thermogenesis . ApproxiLow oocyte mtDNA content has been reported to result in abnormal embryo development in pigs . Therefoc (c promotes the caspase 3 activation, resulting in apoptosis (c release and the level of active caspase 3. We found that YME1L1 knock-down modulated the cytochrome c release (The dynamic organization of mitochondria with a high membrane potential is crucial for embryo development . Therefoc . The relpoptosis . In this release  and indu release . Moreove release . We foun release .c release in porcine blastocysts. Moreover, the TUNEL and activated caspase-3 were induced by the knock-down of YME1L1 during porcine embryo preimplantation development. These results indicate that YME1L1 plays a crucial role in porcine embryo preimplantation development by modulating the oxidative stress, mitochondrial morphology and functions, and apoptosis.Overall, YME1L1 knock-down significantly reduced the blastocyst quality during porcine embryo preimplantation development . YME1L1


The in vitro experiments were performed to validate the effect of AHI on HCC cell and explore the molecular mechanism by western blotting analysis.Methods: The TCMSP platform was used for drug ingredient screening. The GeneCards database and DisGeNET database are used to collect liver cancer targets. PPI network construction of active component-target intersection target was completed with string database. The GO and KEGG pathway analyses were performed via bioinformatics analysis. The molecular docking was used to confirm AHI’s target proteins. The In vitro experiments reveled that AHI could suppress tumor proliferation, invasion and metastasis of HCC cells. Further analysis showed that AHI inhibited tumor growth by PI3K/AKT signal pathway in HCC.Results: Through the intersection, 155 intersection targets are finally obtained. The top 15 active ingredients were quercetin, kaempferol, beta-sitosterol, luteolin, beta-carotene, Stigmasterol, naringenin, formononetin, baicalein, Anhydroicaritin, isorhamnetin, licochalcone, 7-O-methylisomucronulatol, aloe-emodin and 8-O-Methylreyusi. The molecular mocking analysis showed that the four active components  and targets had a good binding activity . Conclusions: The Bushen Huayu Decoction and its active ingredient AHI could fight HCC. The potential mechanism may be associated with inhibiting the activation of PI3K/AKT signal pathway, which may serve as a potential treatment for HCC therapy. At present, the cancer-related mortality rate is the second in the world, second only to lung cancer –3. The land vivo . Wu et aand vivo . Liu et and vivo .The active ingredients of Bushen Yangxue Huayu Decoction included Radix Rehmanniae, Eucommia ulmoides, Radix Paeoniae Alba, Radix Achyranthis, Radix Astragali, Herba Epimedii, Radix Angelicae Sinensis, Radix Carthamus, Caulis spatholobi, Cistanche deserticola, Rhizoma dogleg, and Radix Aucklandiae. Icariin (ICA) is the main active ingredient of Epimedium . ICA, asin vitro experiments. Network pharmacology and colony formation experiments demonstrated the anti-HCC potential of AHI. Western blot analysis confirmed that AHI suppressed tumor progression via the PI3K/AKT signaling pathway in HCC.In the present study, the anti-hepatoma mechanism of AHI was deeply studied through bioinformatics analysis, network pharmacology, molecular docking and Through TCMSP platform and Chinese herbal medicine platform, OB ≥ 30%, DL ≥ 0 18 was acted as the filter condition. The active components of Radix Rehmanniae, Eucommia ulmoides, Radix Paeoniae Alba, Achyranthes bidentata, Radix Astragali, Epimedium, Angelica sinensis, safflower, Caulis spatholobi, Cistanche deserticola, Rhizoma dogleg and Radix Aucklandiae were retrieved respectively. There were 145 active components in the soup of invigorating the kidney, activating blood circulation and removing stasis, and a total of 3944 targets were collected. After deleting the duplicates, 273 target genes were obtained by gene name transformation of the target protein of the active ingredient using Uniprot database.We searched with “multiple myeloma” as the key word, collected the relevant targets of MM, summarized the results of the disease database, and finally obtained 1671 disease genes. The disease target and the active ingredient target were intersected to obtain 155 intersection targets .Cytascape was used to draw the “active component-target” network diagram of 155 intersection targets . NetworkNext, we imported 155 intersection targets into the string database and adjusted the minimum required interaction score to high confidence (0.7) to obtain protein interaction network and corresponding network data. The data were imported into Cytoscape for analysis, and 148 nodes and 1490 edges are obtained, with an average degree of 12.79. The CytoNCA plug-in was used for protein clustering of gene groups, and the centrality analysis was carried out by Betweenness (BC), Closeness (CC) and Degree (DC) analysis conditions. TP53, AKT1, STAT3, JUN and MAPK3 are the core targets in the network, as shown in The R package was used for GO enrichment analysis of the intersection targets. A total of 629 biological process (BP) entries were obtained, involving cell response to chemical stress and oxidative stress, response to oxygen level and metal ions, regulation of active oxygen metabolism, regulation of apoptosis signal pathway, proliferation of muscle cells, aging and other processes. A total of 36 cell component (CC) entries involved transcriptional regulatory complex, nuclear chromatin, cyclin kinase complex, mitochondrial outer membrane, cytoplasmic membrane and other components and 80 molecular function (MF) entries, involving RNA polymerase II specific DNA-binding transcription factor binding, DNA-binding transcription factor binding, nuclear receptor activity, phosphatase binding, ubiquitin-like protein ligase binding, protein serine/threonine kinase activity and other functions. According to the number and diversity of genes, the top 20 items of three cluster analysis are drawn, as shown in The KEGG enrichment analysis of the obtained intersection targets was carried out using R package. The results showed that there were 173 pathways with p value<0.05, and the results of the top 20 entries were sorted according to the corrected p value, as shown in The four active components  with high intermediate value in the core network of the intersection target were linked with the three core proteins  in the PPI network by using Autodock software. The lower the binding energy of the two, the more stable the binding of ligand and receptor. The results show that the binding energy of the four active components and three core proteins is less than -5kcal·mol-1, as shown in Quercetin forms hydrogen bonds with ASP292, LYS18 and THR291 in the amino acid residues of AKT1, and hydrophobic forces with MET281, ALA177 and VAL164. Kaempferol forms hydrogen bonds with GLU191 and GLY162 in the amino acid residues of AKT1, and generates hydrophobic forces with LYS179, LEU181 and ASP292. Luteolin forms hydrogen bonds with GLU198, GLY162, LYS179 and THR195 in the amino acid residues of AKT1, and generates hydrophobic forces with LEU181 and VAL164. Anhydroicartin forms hydrogen bonds with LYS159, ASP292 and ASP439 in the amino acid residues of AKT1, and hydrophobic forces with GLU234, LYS179, PHE442, LEU156, PHE438 and MET281.The HepG2 cell was used to validate the effects of AHI on cell growth. In the colony formation assay, HepG2 cells were treated with different concentrations of AHI for 7 days. The result showed that the number and size of cell clones decreased with the increase of AHI concentration . SimilarWestern blot analysis was performed to compare the expression of Bax, Bcl2, p-PI3K, PI3K, p-AKT and AKT in HepG2 cells with AHI treatment. Bax/Bcl2 are key proteins related to apoptosis. As shown in TCM resources are an important source of chemical entities supporting drug discovery . There aLiver cancer is one of the common malignant tumors, and the global incidence rate and mortality are increasing year by year . Cancer The active ingredients of Bushen Yangxue Huayu Decoction included Radix Rehmanniae, Eucommia ulmoides, Radix Paeoniae Alba, Radix Achyranthis, Radix Astragali, Herba Epimedii, Radix Angelicae Sinensis, Radix Carthamus, Caulis spatholobi, Cistanche deserticola, Rhizoma dogleg, and Radix Aucklandiae. The present study aimed to classify the molecular mechanism of Bushen Yangxue Huayu Decoction in treating HCC. A total of 145 core active ingredients were identified. Moreover, using Uniprot database, 273 target genes were obtained by gene name transformation of target protein of active ingredient. The results of active component-target network analysis showed that the top 15 active ingredients were quercetin, kaempferol, beta-sitosterol, luteolin, beta-carotene, Stigmasterol, naringenin, formononetin, baicalein, Anhydroicaritin, isorhamnetin, licochalcone a, 7-O-methylisomucronulatol, aloe-emodin and 8-O-Methylreyusi, respectively. In addition, PPI analysis of drug active components-hepatoma intersection target found that TP53, AKT1, STAT3, JUN and MAPK3 were the core targets in the network.in vitro of active component, AHI. Our findings revealed that AHI treating was associated with repression of the abilities of proliferation, migration and invasion in HCC cells. Next, the changes in key proteins of the PI3K/AKT pathway were analyzed in detail. It was found that AHI could suppress the tumor growth, invasion by inhibiting PI3K/AKT signal pathway.Next, the four active components  with high degree in the core network of the intersection target were linked with the three core proteins  in the PPI network by using Autodock software. It was found that almost the four active components and targets had a good binding activity . Anhydroicartin, a prenylated flavonoid naturally occurring in several species of Epimedium, is recognised as one of the active compounds in the famous traditional Chinese herb Epimedium. Anhydroicartin exhibits a variety of biological activities such as activating apoptosis and inhibiting the growth of cancer cells, protecting against beta-amyloid-induced neurotoxicity and promoting neuronal and cardiac cell differentiation , 31. HowThrough the validation of bioinformatics and experimental research, this study analyzed the potential mechanism of Bushen Huayu Decoction and its active ingredient AHI against HCC. AHI plays an anti-HCC effect by suppressing tumor proliferation and metastasis. The potential mechanism may be associated with inhibiting the activation of PI3K/AKT signal pathway, which may serve as a potential treatment for HCC therapy, providing a new direction for the clinical management of HCC.https://tcmspw.com/tcmsp.php), the set conditions were oral bioavailability (OB) ≥ 30%, and drug-likeness (DL) ≥ 0.18 to screened out drug gradients. The active ingredients and corresponding targets of 12 traditional Chinese medicines, including Radix Rehmanniae, Eucommia ulmoides, Radix Paeoniae Alba, Radix Achyranthis, Radix Astragali, Herba Epimedii, Radix Angelicae Sinensis, Radix Carthamus, Caulis spatholobi, Cistanche deserticola, Rhizoma dogleg, and Radix Aucklandiae, were searched. Finally, the collected active ingredient targets will be converted into corresponding gene names and ID with the help of Uniprot database (https://www.uniprot.org).Through the TCMSP platform (https://www.genecards.org/) and DisGeNET database (https://www.Disgenet.org) were used for collecting disease targets. Then, we used “liver cancer” as the key word to search, filter and collect relevant targets, and filter by the median, and finally delete and merge the results of the two databases.GeneCards database  was used to construct the PPI network for the active component-target intersection target, and the Cytoscape and CytoNCA plug-ins were used to analyze the centrality of the protein network to obtain the core protein with a higher degree.The string database  was used to obtain the structure of the compound. The PDB database (https://www.rcsb.org/) was applied to get protein structure. Moreover, Autodock Vina software was used for molecular docking, and Discovery Studio software was used to optimize the output of the results.The components quercetin, kaempferol, luteolin and Anhydroicartin were linked with the core proteins  with higher degree in PPI network. The PubChem database . The HepG2 cell was cultured in complete medium (RPMI 1640 plus 10% FBS and 1% penicillin - streptomycin). Cell was cultured in a 37° C incubator with a humidified 5% COAnhydroicaritin (AHI) was purchased from Shanghai Weiao Medical Technology Co. Ltd. . Colony formation assays were used to further validate the inhibitory effect of AHI on the tumorigenicity of liver cancer cell. A total of 2000 HepG2 cells/well were seeded in 6-well plates. Then, the different concentrations of AHI were performed to treat cells for 7 days. After fixing with 5% methanol for 30 minutes, they were dyed with crystal violet solution for 10 minutes, and counted the colonies containing more than 50 single cells under the microscope.2 low-adhesion culture flask. 10ml of the above medium was added to regulate the number of cells to 1 × 105. They were incubated in a constant temperature of 37° C and 5% CO2 incubator, shaken gently once every 24 hours, and changed the liquid by half volume every 3 days. After two weeks, we can basically see the globular cells.HepG2 cells were digested with trypsin and made into single-cell suspension. After washing with PBS, they were inoculated into serum-free DMEM/F12 medium containing basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF) and other factors, and then cultured in 25cm4 cells suspended in 150 μl FBS-free medium were seeded onto top chambers for the migration assay, and 700 μl RPMI-1640 medium with 20% FBS was added to the lower chambers. After 36 h of incubation, cells that had invaded were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet for counting. All assays were repeated three times.HepG2 cell treated with different concentrations of AHI for wound healing assay was cultured in 6-well plates and grown to full confluence. Next day, we made artificial homogeneous wounds using a 200 μl pipette tip. Cells were serum-starved overnight after washing twice with PBS to remove debris. At 0 h and 48 h in the cell incubator, artificial scratch wound areas were photographed under microscopy. Migrate rate was calculated as follows: migrate rate % = /original scratch area×100%. Transwell assay was performed to assess cell migration. 8.0-μm transwell chambers in 24-well plates were used. A total of 10× 10Total proteins of HepG2 cell were extracted with RIPA buffer which added protease inhibitors and phosphatase inhibitors (200:1:1), and then was quantified by BCA Assays. The lysates were separated on SDS-PAGE gels under constant voltage and transferred onto 0.22μm polyvinylidene fluoride (PVDF) membranes. 5% non-fat dry milk was applied for blocking the blots for 2 h at room temperature. And the blots were incubated with primary antibodies at 4° C overnight. Next day, HRP-conjugated secondary antibodies were incubated 1 h at room temperature after washing with TBST for three times. All blots were examined by ECL reagent and visualized using a Tanon system. β-actin expression was used to quantify the data. The assay was repeated three times.Each group of experiments was repeated three times and data were analysed using GraphPad Prism 8.0 and SPSS 10.0, with results expressed as mean ± standard deviation All supporting experiment data are available from the corresponding author upon request.


Spatholobi caulis (SC) is a herbal medicine with potential hepatoprotective effects; however, its active compounds and underlying mechanisms have not been fully explored. In this study, we combined a multiscale network-level approach with experimental validation to investigate SC’s antioxidant properties and their impact on NAFLD. Data collection and network construction were performed, and active compounds and key mechanisms were identified through multi-scale network analysis. Validation was conducted using in vitro steatotic hepatocyte models and in vivo high-fat diet-induced NAFLD models. Our findings revealed that SC treatment improved NAFLD by modulating multiple proteins and signaling pathways, including AMPK signaling pathways. Subsequent experiments showed that SC treatment reduced lipid accumulation and oxidative stress. We also validated SC’s effects on AMPK and its crosstalk pathways, emphasizing their role in hepatoprotection. We predicted procyanidin B2 to be an active compound of SC and validated it using a lipogenesis in vitro model. Histological and biochemical analyses confirmed that SC ameliorated liver steatosis and inflammation in mice. This study presents SC’s potential use in NAFLD treatment and introduces a novel approach for identifying and validating active compounds in herbal medicine.Nonalcoholic fatty liver disease (NAFLD) is a global health problem that is closely associated with obesity and metabolic syndrome.  Non-alcoholic fatty liver disease (NAFLD) refers to a condition marked by the accumulation of fat (mainly over 5%) in the liver tissue of patients who do not consume large quantities of alcohol. NAFLD is a typical liver metabolic disorder and spans various conditions, such as simple steatosis, steatohepatitis, liver fibrosis, and hepatocellular carcinoma . Recent Spatholobi caulis (SC), which is the dried vine of Spatholobus suberectus Dunn, belonging to the Leguminosae family, has been used as a traditional herbal medicine [medicine . SC has medicine ,10,11. Imedicine . Additiomedicine . The accmedicine . OxidatiNetwork pharmacology, inspired by a systems biology approach, has emerged as a potent tool for recognizing rational drug targets and repurposing drugs . This apIn this study, we investigated the antioxidant properties of SC and its impact on NAFLD by combining a multiscale network-level approach with experimental validation. An integrated approach that combines computational and experimental methods was employed to evaluate the hepatoprotective effects of SC, as well as the key mechanisms and active ingredients underlying them. To this end, we first collected information on the detectable ingredients of SC and their experimentally validated targets. By analyzing the collected dataset, we identified key targets of SC and signaling pathways that were significantly associated with these key targets. The signaling pathways associated with this proposed therapeutic effect were comprehensively validated using an in vitro oxidative stress model. Furthermore, in vivo experiments confirmed that the hepatoprotective effect of SC could be achieved through oral administration. To identify the active ingredients, we utilized protein overlap and multiscale-level approaches to identify the association between SC ingredients and the disease. The mechanisms of the candidates were proposed by analyzing diffusion profiles and were further validated by subsequent experiments. Through this approach, we sought to contribute to the development of more effective therapeutic strategies for NAFLD and provided valuable insights into the potential applications of herbal medicines.https://oasis.kiom.re.kr. accessed date: 26 February 2023) database [A compound-target network was constructed by identifying compounds and their protein targets. These compounds were retrieved from the Oriental Advanced Searching Integrated System  or HFD , for 4 weeks, depending on the group. Subsequently, mice were orally injected with a vehicle (40% PEG) or SC  three times a week for 5 weeks. All mice were fasted from the last administration until sacrifice.The animal experimentation procedures of this study were approved and monitored by the Institutional Animal Care and Use Committee of Dongguk University. Male C57BL/6 mice  obtained from Charles River Orient Bio  were randomly divided into 3 groups: Normal diet (ND) + vehicle, HFD + vehicle, HFD + SC 30 mg/kg. Mice were fed either ND (PicoLabAspartate aminotransferase (AST), triglyceride, and LDL cholesterol levels were analyzed at Chaon Research Lab  using Diagnostics Reagents for AST and triglyceride  and Diagnostics Reagents for LDL cholesterol  on an AU680 chemistry analyzer .For haematoxylin and eosin (H&E) staining, liver tissue samples isolated from the left lateral lobe were fixed in 4% paraformaldehyde in PBS at room temperature for 24 h. The fixed liver tissue samples were then dehydrated in a sequential gradient of alcohol and xylene before it was embedded in paraffin. Subsequently, the paraffin-embedded tissues were sectioned into 5 μm thick slices. Hematoxylin and eosin (H&E) staining was performed on the sectioned liver tissues. For Oil Red O staining, the left lateral lobe of the liver was frozen-sectioned and fixed in 4% paraformaldehyde for 30 min. After washing with tap water, the tissue was rinsed in 60% isopropanol and then stained with fresh Oil Red O solution for 10 min. The tissue was then rinsed in 60% isopropanol again, followed by the counterstaining of cell nuclei with hematoxylin. Finally, the stained tissues were observed using light microscopy .Anti-SREBP-1c, anti-phospho-AMPKα, anti-phospho-acetyl-CoA carboxylase (ACC), anti-phospho- liver kinase B1 (LKB1), anti-phospho-YAP, anti-YAP, anti-phospho-large tumor suppressor kinase 1 (LATS1), anti-heme oxygenase 1 (HO-1), anti-Lamin A/C, and anti-β-actin were purchased from Cell Signaling Technology . Anti-Nrf2 was purchased from Santa Cruz Biotechnology . HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were purchased from Enzo Life Sciences . Trizol reagent was purchased from Invitrogen . Compound C (Com C), verteporfin (VP), T0901317 (T090), Harris hematoxylin and eosin, and Oil Red O solution were purchased from Sigma-Aldrich . Polyethylene glycol (PEG) 400 was obtained from Yakury Pure Chemical Co., Ltd. . SC is a medicinal-standard herb manufactured at a GMP facility  certified by the Korean FDA and was prepared and standardized as previously described [HepG2 and Huh-7 cells  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) at high glucose with 10% fetal bovine serum (FBS), 50 μg/mL streptomycin, and 50 units/mL penicillin and RPMI 1640 with 10% FBS, 50 μg/mL streptomycin, and 50 units/mL penicillin, respectively.The total RNA was extracted from liver tissue using the Trizol reagent. cDNA was reverse transcribed using the cDNA Synthesis Kit . The reaction mixture, including cDNA, forward and reverse primers, and the Premier qPCR Kit , was amplified using Light Cycler 1.5 . The sequences of primers were then as follows: mouse NOX-4(Entrez Gene ID: 50490)-F: 5′-GATCACAGAAGGTCCCTAGCA-3′, NOX-4-R: 5′-GTTGAGGGCATTCACCAAGT-3′, GAPDH (Entrez Gene ID: 14433)-F: 5′-AACTTTGGCATTGTGGAAGG-3′, GAPDH-R: 5′-ACACATTGGGGGTAGGAACA-3′.6 and then treated with SC at the concentrations and times indicated in each figure. For cell lysate preparation, the RIPA buffer was used at 4 °C, and the protein quantification of lysates was performed using a BCA assay kit . The quantified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane for subsequent antibody conjugate probing. The membrane was then incubated with primary and secondary antibodies, and chemiluminescence was detected using a Chemidoc image analyzer  to visualize the protein-bound membrane.HepG2 cells were seeded in a 60Ø dish at a density of 1.6 × 10p < 0.05 or p < 0.01.Statistical analysis was conducted using Student’s t-test or ANOVA to determine significant differences between the control and treatment groups. Blood chemistry data from animal experiments, including AST, triglyceride, and LDL cholesterol values, were presented as the mean ± standard deviation (SD). The criterion for statistical significance was set at We first performed a network pharmacological analysis to investigate the complex relationships between the compounds, targets, and biological effects of SC on NAFLD. This initial analysis provided a comprehensive overview of the possible interactions between SC ingredients and their protein targets in the context of NAFLD, setting the stage for further investigations into the specific mechanisms involved. A compound-target network of SC was constructed by collecting ingredients and their target information from various databases. We initially found 589 CPIs between 21 SC ingredients and 321 protein targets and 91 liver injury-related proteins associated with oxidative stress. Among the protein targets of SC, we found that CYP19A1, NFKB1, CASP3, NOS2, CA12, ESR2, and PTGS2 had the highest degree . On the other hand, we found that around two-thirds of the protein targets (210/321) interacted with only one SC target. To focus on the protein targets associated with a sufficient number of SC ingredients, we considered 111 targets that interacted with two or more ingredients as the key targets and conducted subsequent analyses focusing on them.p-value < 10−30). This result indicated that the key targets of SC were closely associated with oxidative stress, highlighting the potential of SC as a therapeutic agent for NAFLD.We then investigated the association between key targets in SC and NAFLD and oxidative stress-related proteins and found that approximately 20% of the proteins (27/111) overlapped with oxidative liver injury-associated targets. A hypergeometric test was conducted to check whether the observed number of overlapping targets was higher than random expectations. The values of random expectation were obtained by randomly selecting targets that were equal to the number of key targets from the assembled dataset and then repeatedly calculating the number of overlapping targets between the disease-related proteins and the selected targets. We found that the observed value was significantly higher (22-fold) than the random expectations  increased the expression of p-LKB1, p-AMPK, and p-ACC. In particular, AMPK phosphorylation was significantly highest at 10 min, and ACC phosphorylation was significantly highest at 1–3 h A. To detWe further identified the Hippo-YAP signaling pathway, which is associated with the AMPK signaling pathway and was recently reported to be involved in lipogenesis. SC increased the expression of p-LATS1 and p-YAP and decreased the expression of YAP. In particular, p-YAP was significantly highest at 10 min, and YAP was significantly lowest at 6 h C. To invTo investigate the influence of AMPK regulation on the Hippo-YAP signaling pathway, pre-treatment with Com C resulted in the decreased phosphorylation of ACC, a downstream kinase of AMPK, and the decreased phosphorylation of LATS1: an upstream kinase of YAP E.HFD-fed mice showed a marked formation of micro and macrovacuolation in hepatocytes, as observed in H&E staining, whereas 30 mg/kg SC treatment improved these phenomena A. HFD inIn HepG2 cells, SC treatment increased the expression of Nrf2, a key transcription factor of the antioxidant system that regulates the induction of antioxidant genes, with the highest expression observed at 3 h D. SC treWe subsequently predicted the active compounds of SC by considering protein overlap and multiscale network analysis . The proWe further investigated the hepatoprotective effects of SC ingredients. Among the prioritized active compounds, we focused on Procyanidin B2, as this is not only a potential quality marker for SC but was also easily detectable by chromatography technique ,34. We fElevated oxidative stress plays a significant role in liver pathogenesis, contributing to various stages such as cellular damage, inflammation, fibrosis, and cirrhosis. While mitigating oxidative stress may be advantageous for NAFLD management, pinpointing potential candidates and understanding their underlying mechanisms remain challenging tasks. In this study, we successfully investigated the antioxidant properties of SC and its impact on NAFLD using a multiscale network-level approach combined with experimental validation. The integrated approach allowed us to elucidate the hepatoprotective effects of SC, as well as the key mechanisms and active ingredients underlying these effects. Our findings provided the identification of key targets and significantly associated the signaling pathways of SC, which were further validated through in vitro and in vivo experiments. Additionally, we identified the active ingredient procyanidin B2 as a potential therapeutic agent for NAFLD. We believe that these findings provided valuable insights into the potential applications of herbal medicines, such as SC, and in the development of more effective therapeutic strategies for NAFLD.Unlike previous studies, our study presents an integrated framework that combines network pharmacology analysis and experimental validation, making an important contribution to the exploration of system-level mechanisms in herbal medicine. Prior investigations have often relied on protein overlap or protein–protein interactions (PPIs) between drug targets and disease proteins to understand therapeutic effects. Our study, however, expanded on this approach by incorporating not only PPIs but also the relationships between biological functions, enabling a more comprehensive elucidation of key components and their underlying mechanisms. In addition, some early studies only presented hypotheses based on network pharmacology analysis without experimental validation. In our research, we comprehensively performed experimental validation for both the therapeutic effects and the identified active ingredients, enhancing the reliability of our findings. Collectively, our results introduce a novel methodology for a more comprehensive understanding of the mechanisms of herbal medicine, which hold the potential to make a significant impact in the field.p-value < 10−30), emphasizing the potential of SC as a therapeutic agent for NAFLD. Through an over-representation analysis based on the KEGG database, we discovered that the majority of the signaling pathways were significantly associated with the main targets of SC. These findings have guided in vitro and in vivo experimental trials while also highlighting the complex and multifaceted nature of the potential for treating NAFLD. Although our research showcased integrated approaches, we primarily focused on an HFD model to evaluate the efficacy of SC via oral administration. Future interesting studies may involve investigating the effects of SC on various models, such as different species, including rats, with a HF diet or genetically fatty liver. Utilizing these different models to explore the capacity of SC may lead to a better understanding of the therapeutic potential of SC in different contexts and conditions.The results of the network pharmacological analysis investigated the complex relationships among the compounds, targets, and biological effects of SC on NAFLD. This comprehensive analysis provided insights into the potential interactions between SC ingredients and their protein targets concerning NAFLD. By constructing a compound-target network of SC using various databases, we initially identified 589 CPIs involving 21 SC ingredients and 321 protein targets, with related proteins associated with oxidative stress. Focusing on 111 targets that interacted with two or more ingredients, our subsequent analysis revealed a significant 22-fold overlap between the key targets of SC and oxidative stress (Our results confirm that the protective effects of SC against NAFLD are mainly mediated by the AMPK and YAP signaling pathways. AMPK, a crucial sensor of cellular energy levels, is activated in response to various conditions that have a falling cellular energy status, including nutrient deprivation, such as glucose, hypoxia, physical exercise, and exposure to toxicants . AMPK, wThe Hippo–YAP pathway is a signaling pathway that regulates organ size and growth and plays essential roles in cell proliferation, differentiation, and apoptosis . Yap hasOur study proposed an integrated approach to uncover the antioxidant properties of SC on NAFFLD. We identified its putative mechanism and the key components of SC through analysis on a multiscale network. In vivo, we confirmed that SC alleviated HFD-induced hepatic fat accumulation, diabetes, hyperlipidemia, and liver tissue damage. In vitro, we validated the regulation of the key signaling pathway, the AMPK-YAP pathway, and identified Procyanidin B2 to be the major active compound. These results suggest that SC and its major compound, Procyanidin B2, have potential as antioxidants and lipogenesis inhibitors.


Wedescribe herein a robust strategy to address the current situation, usingvarious isotope-aided NMR technologies. The feasibility of our approach isdemonstrated for the normal residues with protonated Although both the  Given the lack of generallyapplicable strategies to overcome this obstacle, only a few NMR studies haveprobed the structural aspects of stereospecifically assigned Lys residues.The ionization states of the Lys hydrophobic methylene chain and the hydrophilicdual nature of the Lys side chain, we have applied variousisotope-aided NMR technologies, including the stereo-array isotope labeling(SAIL) method .Detailed studies on the structures and dynamics of the Lys residues in aprotein have been severely hampered by the difficulty in gatheringcomprehensive NMR information on their side-chain moieties. It is especiallychallenging to establish The Lys We used a variant of staphylococcal nuclease, Although the SNase variant contains 21 Lys residues , including theengineered Lys-66, the 22.1E. coli BL21 (DE3) straintransformed with a pET3 vector (Novagen), encoding the E. coli cells werecultured at 37 The 2.2The 600 MHz 2D The Lys The 125.7 MHz 1D 33.1stereospecifically deuterated, i.e., [U-Although the chemical shifts with sequential assignments for the backbone3.2not corrected for the variousisotope-induced shifts caused by the complicated isotope-labeling pattern ofthe SAIL-Lys structure . Based on comprehensive NMR data, weshould be able to elucidate the dual role of the Lys side chains in terms of theconformational dynamics and functional properties of a protein in furtherdetail, using various solution NMR methods. In this section, we brieflyinterpret the chemical shift data to characterize the local conformationalfeatures by the fully SAIL-labeled SNase variant , it was obviousthat the chemical shift data with exclusive and unambiguous assignments forthe Lys residues contain an abundance of information on the side-chainconformations and ionization states of the electrostatic interaction between the positively charged Note that the chemical shifts in Table 1 for the 21 Lys residues in theSAIL-Lys-labeled SNase variant are The upfield shifts of the side-chain methylenes, induced by the neighboringaromatic rings, were also detected for Lys-28, Lys-84, Lys-110, and Lys-133.Considering the local structures of Lys-28 and Lys-84 in the crystal , the relative orientations between Lys-28 and Tyr-27 and between Lys-84 andTyr-85 seem to be similar to those in the crystal and are responsible forthe large upfield shifts for only their On the other hand, the unusual chemical shifts observed for the Lys-66residue, which is trapped within the hydrophobic environment engineered inthe engineered SNase variant , clearly reveal the strong influenceof the ionization state of the deprotonation shifts, in particular, those of In comparison with charged Lys side chains, deprotonation of the 3.3exceptionally slow hydrogenexchange rates . For example, in a protein selectivelylabeled with [In our previous studies investigating the effects of the deuterium-inducedisotope shifts on the Although the idea of estimating the hydrogen exchange rates by thedeuterium-induced isotope shifts on the We first examined the 1D free [Although the We also measured the 1D 4In this article, we have shown that comprehensive NMR information can beobtained using cutting-edge isotope-aided NMR technologies for the Lys side-chain moieties, comprising a long hydrophobic methylene chain and a hydrophilic


Due to various unpleasant side effects and general ineffectiveness of current treatments for prostate cancer (PCa), more and more people with PCa try to look for complementary and alternative medicine such as herbal medicine. However, since herbal medicine has multi-components, multi-targets and multi-pathways features, its underlying molecular mechanism of action is not yet known and still needs to be systematically explored. Presently, a comprehensive approach consisting of bibliometric analysis, pharmacokinetic assessment, target prediction and network construction is firstly performed to obtain PCa-related herbal medicines and their corresponding candidate compounds and potential targets. Subsequently, a total of 20 overlapping genes between DEGs in PCa patients and the target genes of the PCa-related herbs, as well as five hub genes, i.e., CCNA2, CDK2, CTH, DPP4 and SRC were determined employing bioinformatics analysis. Further, the roles of these hub genes in PCa were also investigated through survival analysis and tumour immunity analysis. Moreover, to validate the reliability of the C-T interactions and to further explore the binding modes between ingredients and their targets, the molecular dynamics (MD) simulations were carried out. Finally, based on the modularization of the biological network, four signaling pathways, i.e., PI3K-Akt, MAPK, p53 and cell cycle were integrated to further analyze the therapeutic mechanism of PCa-related herbal medicine. All the results show the mechanism of action of herbal medicines on treating PCa from the molecular to systematic levels, providing a reference for the treatment of complex diseases using TCM. Prostate cancer (PCa) is a widespread adenocarcinoma of the urinary system, ranking the second causes of cancer-related deaths in American males . Due to Traditional herbal medicine is a whole medical system originated from several thousand years of clinical experience , 5. Nowain silico pharmacokinetic model system was employed to screen out the potential active compounds of anti-PCa herbs and then the targets of these potential active ingredients were fished. Thirdly, the overlapping genes among differentially expressed genes (DEGs) in PCa patients and the target genes of the PCa-related herbal medicines were obtained. Subsequently, five hub genes were applied to perform survival and tumour immunity analysis, revealing their critical roles in PCa.Presently, to explore the mechanism of herbal medicines on treating PCa from a systematic level, a comprehensive approach combining knowledge mapping, pharmacokinetic evaluation, multi-targets fishing, bioinformatics analysis and computer experiments validation was proposed. Firstly, using the bibliometric analysis and text statistics mining, 10 most frequently used herbs of PCa and their corresponding constituents were obtained. Secondly, an Additionally, computer experiments validation including the simulations of molecular dynamics (MD) and molecular docking were carried out to verify the reliability of interactions between the key drugs and the hub target genes. In final, through integrated analysis, four signaling pathways including PI3K-Akt, MAPK, p53 and cell cycle pathways were determined, demonstrating that herbal medicines show the therapeutic effects on treating PCa by acting on the target genes in these pathways. To extract the relevant herbal medicine for prostate cancer, Web of Science (WoS), a crucial global database containing large amounts of academic literatures, was employed to perform bibliometric analysis. Using the keywords “prostate cancer” and “herbal medicine”, the related literatures were collected in WoS from January 2001 to December 2021. Presently, VOSviewer was used as the bibliometric analysis tool to extract the hot herbal medicine against PCa.https://old.tcmsp-e.com/tcmsp.php), PubMed and the clinical trial database . Then, a statistical index  as a ratio was used to assess the relationship between herbal medicine and PCa. Herein, P value is used to evaluate the relationship between herbs reported in literature and PCa and when P-value < 0.01, this herb is considered to have strong association with PCa [Further, to obtain the herbal medicines associated with PCa, a data mining approach based on the keywords “prostate cancer” and “herbal medicine” was carried out from TCMSP  and PreDL (predict drug-likeness).in silico model PreOB [For the potential compounds, OB, an essential pharmacokinetic parameter, is a good indicator to reflect the efficiency of oral drugs entering the systemic circulation . Herein,el PreOB  was empli and αi* are the Lagrange multipliers, b is the regression parameter K and  is the kernel function, which uses the radial basis function, as shown in formula (3).Where αWhere γ is the nuclear parameter.Drug-likeness (DL) represents the similarity of a compound’s structure to that of a known drug . A compoWhere x is the descriptors of the herbal ingredients associated with PCa, and y denotes the mean of the total molecular descriptors of all drugs in the Drugbank database.To sum up, the screening thresholds for the two models are set as OB ≥ 30% and DL ≥ 0.18, respectively. The compounds in herbal medicines can be identified as potential active ingredients if they meet these two screening conditions at the same time.Generally, drug molecules can exert their biological effects only by interacting with their targets. Therefore, in order to accurately and comprehensively predict the targets of candidate compounds in herbal medicines associated with PCa, SysDT model , which iIn final, the obtained target proteins are further subjected to TTD database to fish the PCa-related targets.Biological network can provide an intuitive display of the interaction between nodes. Presently, three nets, i.e., compound-target (C-T), target-function (T-F) and compound-target-pathway (C-T-P) networks are established and analyzed by using Cytoscape 3.3.8 . In thesTo identify the differentially expressed genes (DEGs) in the prostate tumor, gene expression profile of GSE134073 was downloaded from Gene Expression Omnibus (GEO) database . This afTo reveal significantly enriched biological processes and molecular function of the obtained genes, Gene Ontology (GO) analysis was carried out through the R package clusterprofiler . Only thhttp://gepia.cancer-pku.cn/) was applied based on TCGA samples. Hub genes were subjected to survival analysis in GEPIA and Genes with |log2(FC) | > 1 and P < 0.01 were considered significant. In addition, differential analysis for these hub genes was carried out by using the Human Protein Atlas online tool.To filter the hub genes in human PCa tumors, the Gene Expression Profiling Interactive Analysis (GEPIA) (https://cistrome.shinyapps.io/timer/), a website dedicated to analyzing tumor immune correlation, was employed. Through TIMER, the mRNA expression data for the obtained hub genes in TCGA PCa tumor samples and their correlations with tumor infiltration of 6 immune cell types including B cells, CD4+T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells was analysed.In order to explore the correlation between hub gene expression and immune cells, TIMER  and ΔGSA (non-electrostatic solvent component), respectively.where ΔKi was calculated from the MM-PBSA. The equation of Ki is listed as follow [The inhibitory constant s follow , 22:–3 kcal/K-mol) and absolute temperature of drug-target complex, respectively. ΔGbind denotes the binding free energy.Where R and T (298.15 K) represent gas constant , the statistical analysis was carried out. Additionally, t-test was used to assess the statistical significance. When P value is less than 0.05, the result is considered statistically significant.Hedysarum Multijugum Maxim., Licorice, Poria Cocos (Schw.) Wolf., Hedyotis Diffusae Herba, Lobeliae Chinensis Herba, Scutellariae Barbatae Herba, Coicis Semen, Atractylodes Macrocephala Koidz., Cornus Officinalis Sieb. Et Zucc. and Curcumae Rhizoma, which are significantly correlated with PCa, were obtained , accounting for a large proportion in all herbs  . Then, aTo filter the hub target genes that are associated with human PCa prognosis, the GEPIA online tools was employed. As a result, five hub genes including CCNA2, CDK2, CTH, DPP4 and SRC were obtained, which are used for further analysis. The expression analysis of these hub genes in PCa and normal tissues reveals that CCNA2, CDK2, CTH and DPP4 are unregulated in PCa tissue compared with normal tissue, while SRC is down-regulated in PCa tissue compared with normal tissue –5F. In aTo investigate the relationship between CCNA2, CDK2, CTH, DPP4, SRC and tumor purity as well as immunity, TIMER database was used to analyze the relationship between these hub genes and immune cell infiltration in PCa. As shown in Molecular docking is a key method for dealing with C-T interactions, which generates posture and dimensionless fitness scores in structural molecular biology and computer-aided drug design. In the present study, in order to verify the C-T interaction between all the active ingredients of PCa-related herbs and their corresponding hub targets, the advanced molecular docking was carried out to generate the set of docking conformations. The active components from the ten herbs were docked into the five hub targets with the default settings through the Lamarckian genetic algorithm program GOLD 5.1 Score fitness function to its prediction target . The heaTo verify the reliability of the C-T interactions and to further clarify the possible binding modes and binding stability of active compound at the protein binding site, we selected two proteins from the five hub genes following the rules of randomness and performed 100 ns MD simulations for the ligand-protein complex.The Root Mean Square Deviation (RMSD) for the proteins with their ligands is depicted in Additionally, in order to demonstrate the flexibility of binding residues in the target protein, we also calculate the root mean square fluctuation (RMSF) of each residue. Obviously, some important residues in the hydrophobic region have the lowest RMSF values, indicating that the rearrangement, variation, deviation and internal movement of the structure reduce the activity complexity of these residues and the flexibility of the binding site , 9F. FurKi by hitting the corresponding target (In order to further validate the activities of ingredients, the binding free energy between the active ingredients and their predicted targets from the C-T interactions is performed by using MM-PBSA method. It can be clearly observed that the two active compounds show low binding free energy of -153.058 and -161.812 KJ/mol and low g target , indicatAs a result, a conclusion can be drawn that van der Waals force and nonpolar solvation energy mainly contribute to the free energy of binding.Taken together, MD simulation provides indirect information about the biological efficacy of active compounds, which may be achieved through corresponding targets for cancer treatment, thus further validating our C-T network.As the most common reproductive system cancer in men, the incidence of prostate cancer is higher in Europe and the United States than that in Asia. However, the treatment of PCa has so far remained unsatisfactory, and more and more people use herbal medicines to treat PCa. Presently, a comprehensive approach combining knowledge mapping, pharmacokinetic evaluation, multi-targets fishing, network analysis and validation was proposed to explore the therapeutic mechanism of PCa-related herbal medicines.As a result, a total of 109 potential ingredients  and 139 Additionally, to find the intersection of DEGs in the prostate tumor and the target genes of the PCa-related herbs, we firstly identified 1880 DEGs in GSE134073  and thenBesides, to further explore the major signaling pathways implicated in treatment of PCa by ten herbals, 4 PCa-related biological pathways were extracted from KEGG analysis. Using the targets and their corresponding pathways, the C-T-P network was established . The yelAs displayed in All in all, these findings reveal the importance of PI3K-Akt, cell cycle, MAPK and p53 signaling pathways in PCa induction. Because these four pathways have a variety of functions, such as cell growth, cell proliferation, apoptosis regulation and immune activation, we speculate that these ten herbs may disrupt these pathways, thus showing direct anticancer therapy, immunotherapy and anti-inflammatory strategies. Candidate compounds may mediate the interaction and crosstalk between different pathways through target proteins, indicating that ten herbs may play synergistic roles in different pathways . At lastPresently, a comprehensive approach combining bibliometric analysis, pharmacokinetic screening, target fishing, network and bioinformatics analysis was employed to elucidate the mechanisms of the anti-PCa related herbal medicines. The predicted results were also computationally validated through molecular docking as well as MD simulations. The main findings of this paper are listed in the following.A total of 109 potential ingredients and 139 corresponding targets are obtained from 10 anti-PCa related herbal medicine through bibliometric analysis, ADME screening and target fishing.GO and T-F network analysis show that the biological processes of these targets are mainly associated with the inflammation response, modulating the immune system and direct anti-cancer effects, suggesting that these 10 herbs have therapeutic effects on PCa from these three aspects.Bioinformatics analysis reveal five hub genes including CCNA2, CDK2, CTH, DPP4 and SRC, which are validated by the MD simulations, are expected to be potential prognostic markers to improve PCa survival and prognostic accuracy.The C-T-P network analysis of anti-PCa herbs showed that herbal medicines may simultaneously target PI3K-Akt, MAPK, p53 and cell cycle signaling pathways to achieve the effect of treating diseases, providing useful clues to make more effective therapeutics against PCa.To sum up, this work offers an integrative analysis to study the herbal drugs and understand the action mechanism of herbal medicines for treating PCa from molecular level to pathway level, which provides new ideas for exploring new drug treatments for complex diseases.Supplementary Table 1


Osteosarcoma is a rare disorder among cancer, but the most frequently occurring among sarcomas in children and adolescents. It has been reported to possess the relapsing capability as well as accompanying collateral adverse effects which hinder the development process of an effective treatment plan. Using networks of omics data to identify cancer biomarkers could revolutionize the field in understanding the cancer. Cancer biomarkers and the molecular mechanisms behind it can both be understood by studying the biological networks underpinning the etiology of the disease.In our study, we aimed to highlight the hub genes involved in gene-gene interaction network to understand their interaction and how they affect the various biological processes and signaling pathways involved in Osteosarcoma. Gene interaction network provides a comprehensive overview of functional gene analysis by providing insight into how genes cooperatively interact to elicit a response. Because gene interaction networks serve as a nexus to many biological problems, their employment of it to identify the hub genes that can serve as potential biomarkers remain widely unexplored. A dynamic framework provides a clear understanding of biological complexity and a pathway from the gene level to interaction networks.Our study revealed various hub genes viz. TP53, CCND1, CDK4, STAT3, and VEGFA by analyzing various topological parameters of the network, such as highest number of interactions, average shortest path length, high cluster density, etc. Their involvement in key signaling pathways, such as the FOXM1 transcription factor network, FAK-mediated signaling events, and the ATM pathway, makes them significant candidates for studying the disease. The study also highlighted significant enrichment in GO terms , such as cell cycle signal transduction, cell communication, kinase binding, transcription factor activity, nucleoplasm, PML body, nuclear body, etc.To develop better therapeutics, a specific approach toward the disease targeting the hub genes involved in various signaling pathways must have opted to unravel the complexity of the disease. Our study has highlighted the candidate hub genes viz. TP53, CCND1 CDK4, STAT3, VEGFA. Their involvement in the major signaling pathways of Osteosarcoma makes them potential candidates to be targeted for drug development. The highly enriched signaling pathways include FOXM1 transcription pathway, ATM signal-ling pathway, FAK mediated signaling events, Arf6 signaling events, mTOR signaling pathway, and Integrin family cell surface interactions. Targeting the hub genes and their associated functional partners which we have reported in our studies may be efficacious in developing novel therapeutic targets. The prominence of Osteosarcoma dates back to the early nineteenth century when the French surgeon Alexis Boyer first coined the term and William Enneking described the disease. A recent study by the American Cancer Society found that 186.6 per 100,000 children and adolescents were diagnosed with cancer each year from birth to age 19 . The mosAlthough it is seen in both young and adults, it has been observed that the tumor spreads rapidly when the bone undergoes the stages of its growth. It has a bimodal age distribution with an adolescence and elderly peak in incidence. The incidence often peaks between the ages of 10 and 14 years, after which it starts to subside. Adults over 65 see the second peak in Osteosarcoma incidence, more likely to be a second malignancy commonly linked to Paget disease . NumerouMDM2, p14ART, and CDK4, may increase a person's risk of acquiring Osteosarcoma  is an online, publicly accessible database harboring information on protein-protein interaction. The interactions include direct as well as indirect connections. It provides a versatile way for analyzing and visualizing the data, such as setting confidence scores that reflect the level of interaction, no of interactors, network type, display mode, etc. In order to categorize the interactions, String uses the confidence scores: highest (above 0.90), high (0.7–0.89), medium (0.4–0.69), and low (0.15–0.39). The STRING database accepts the input in various forms, such as protein by name, protein by sequence, multiple proteins, protein families (COG), etc. The outcome of the network can be saved in a variety of formats such as bitmap image, vector graphic, TSV format, tab-delimited file, etc.  . It provides a user-friendly interface that allows the user to seamlessly operate the software. It supports various plugins, which serve various purposes such as clustering of genes, enrichment analysis, annotation, determining topological properties of a network, etc. Output from STRING was used as an input for the Cytoscape.Gene interaction networks can be visualized and analyzed using Cytoscape . Genes are clustered by MCODE based on their connectivity, in which the same cluster contains more interconnected genes with the optimal neighborhood density. Genes that are associated with MCODE scores are clustered together. .http://www.funrich.org/) is a tool used for functional enrichment and network analysis. It can be utilized to conduct functional enrichment analysis on background databases incorporating diverse genomic and proteomic resources. The outcomes of the enrichment studies may be depicted using a wide range of graphical layouts, such as column graphs, bar graphs, pie charts, Venn diagrams, heat maps, and doughnut charts. Users can download information from the UniProt and standard human-specific FunRich databases. Additionally, users can create their custom datasets and carry out enrichment analyses regardless of the organism  which gave an interaction for 71 genes and their functional partners. Gene networks were constructed and further analyzed based on STRING interaction data .TP53, CCND1, CDK4, and STAT3 with no interactors 45, 33, 28, and 27, respectively. The network analysis of 71 genes was carried out using NetworkAnalyzer. To study the gene interaction network, it analyzed different topological parameters such as degree, no of nodes and edges, characteristic path length, clustering coefficient, closeness centrality, and betweenness centrality. The top genes with the highest degree values are viz. C1, C2, and C3 . Commonly, significance levels of 0.05 are considered significant. The genes observed in Osteosarcoma revealed various contributions in Gene Ontology terms such as Biological Processes (BP), Molecular Function (MF), and Cellular Compartment (CC). The significantly enriched terms in BP included regulation of cell cycle signal transduction, cell communication, regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism , limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis, along with the emerging hallmarks of cancer which includes deregulating cellular energetics and avoiding immune destruction , 25. AttCCND1, CDK4, VEGFA, CDKN2A, SRC, CHEK2, ERBB2, CD44, CCNE1, PLK1, CDC6, AURKA, CCNB2, and TNFSF11. It has been reported that patients suffering from Osteosarcoma cells develop resistance toward the kinase inhibitor drug, Sorafenib due to the mTOR signaling pathway. The mammalian target of rapamycin (mTOR) facilitates all cell proliferation, apoptosis, and autophagy. There is evidence showing that the mTOR signaling pathway plays a significant role in a number of diseases, including osteosarcoma.  via the extracellular domain, supplying anchoring . IntegriOur study has also revealed various highly enriched pathways, such as the FOXM1 transcription factor network, ATM pathway, signaling event mediated by FAK, Arf6 signaling events, and Class 1 PI3K signaling events. FOXM1, a Forkhead Box Transcription Factor, is known for maintaining the homoeostatic environment and other cellular functions, such as cell proliferation, cell cycle progression, DNA damage repair, angiogenesis, etc. Being associated with a large number of cellular processes, it has also manifested its role in several diseases as well as cancer. It has been studied that FOXM1 plays a role in tumor growth and progression . ForkheaTP53, CCND1, CDK4, STAT3, and VEGFA, can be considered potential biomarkers because they are involved in the major biological pathways of Osteosarcoma.To identify possible drug targets for Osteosarcoma, which plays an essential role in various biological pathways, we used NetworkAnalyzer, which is a built plugin in Cytoscape. NetworkAnalyzer uses various parameters such as degree, average shortest path length, closeness centrality, and betweenness centrality. Degree refers to the no. of direct interactors, and more no. of degrees will indicate more no. of gene interactors which will help us to understand the progression of a pathway. The significance of the gene in gene-to-gene communication increases with decreasing average shortest path length and increasing closeness centrality. Based on the parameters mentioned above, our study has revealed the top five genes viz. TP53 gene is a potential biomarker with the most no. of direct interactors of 45 with the shortest average path length of 1.343 and the highest closeness centrality of 0.744. TP53 is seen to be involved in various biological pathways. In our study, such as the FOXM1 transcription factor network, ATM pathway, Class 1 PI3K signaling events, and mTOR signaling pathway. In normal conditions, TP53 is a tumor suppressor gene that initiates numerous stress-induced pathways, including DNA damage, senescence, cellular death, and reprogramming. It stimulates numerous genes encoding proteins responsible for apoptosis (TP53 is mutated, which loses its ability to suppress the tumor, thereby promoting uncontrolled cell proliferation. Over 50% of human neoplasms have somatic mutations in the TP53 gene. About 10% of the changes are nonsense mutations, resulting in shortened p53 proteins, while most variants are missense mutations. Sixty percent of neoplasms with missense TP53 mutations have their second TP53 allele deleted (TP53 have revealed that reverse regulation of TP53 through the mTOR pathway also modifies the synchronization of growth signals and stressors (TP53 gene is considered a hallmark in cancer studies and serves maximum potential for developing therapeutic targets for treating Osteosarcoma.poptosis . In a ca deleted . Earliertressors . The TP5CCND1 gene can serve as a drug target with 33 direct interactors having a path length of 1.582 and closeness centrality of 0.632. CCND1 or Cyclin D1 gene synthesizes a protein that governs cyclin-dependent kinases in the cell cycle. It is well recognized as a regulator of cell cycle progression in the nucleus, modifying the transition from the G1 to the S phase. Although Cyclin D1 is well recognized for its function in the nucleus, current clinical investigations link it to tumor invasion and metastasis when it is present in the cytoplasmic membrane (membrane . It is amembrane .CCND1 gene mutations that cause nuclear retention and constitutive CDK4/6 kinase activation are oncogenic (CCND1 is also seen to be involved in biological pathways such as the FOMX1 transcription pathway, mTOR signaling pathway, etc. CCND1 is seen to be involved in response to leptin, which is a peptide hormone produced by adipocytes. Leptin helps in the maintenance of normal cellular homeostasis. Downregulation of the apoptotic reaction and upregulation of the cell cycle is due to the pro-carcinogenic impact of leptin (CCND1 gene may aid in halting Osteosarcoma development.Additionally, emerging evidence reveals that ncogenic . CCND1 if leptin . TherefoCDK4 gene plays a significant role in the completion of the cell cycle and are often hyperactive in cancer. CDKs are serine/threonine kinases that are activated in association with a cyclin partner. It has a no. of direct interactors of 28 with an average shortest path length of 1.626 and closeness centrality of 0.614. During the G1-S transition, retinoblastoma protein acts as a negative cell cycle regulator by binding to the transcription factor E2F and suppressing transcriptional activity during the early G1 phase. D-type cyclins express themselves more often in response to mitogenic stimuli, and they join forces with CDK4/6 to phosphorylate RB. The E2F transcription factor family's inhibitory control on RB is partially relieved by hypo phosphorylated RB, which encourages the expression of E2F target genes like cyclin E and speeds up the G1 phase transition (CDK4 is involved in the regulation of the mTOR pathway activated, thus making it a potential drug target (ansition . Studiesg target .VEGFA gene is considered a hallmark in cancer-related studies because of its role in angiogenesis, accomplished periodically from pre-existing vascular networks (the FOXM1 transcription factor regulates VEGFA to promote tumor angiogenesis (VEGFA gene had a degree value of 27 and an average shortest path length of 1.701.The networks . The tumnetworks . Studiesogenesis . VEGFA gSTAT3, plays a vital role in DNA replication. Being an essential STAT protein family member, it plays a crucial part in various vital cellular functions, including proliferation, differentiation, survival, immunosuppression, angiogenesis, and cancer (STAT3-activated genes enhance angiogenesis and metastasis, prevent apoptosis, promote cell proliferation and survival, and suppress antitumor immune responses (STAT3 regulates mitochondrion functions (STAT gene has been revealed to have direct interactors of 27 with an average shortest path length of 1.716 and closeness centrality of 0.587.The signal transducer and activator of transcription, d cancer . STAT3-aesponses . In addiunctions . STAT geTP53, CCND1 CDK4, STAT3, and VEGFA have the highest no. of interactions and showed a high clustering density. Their involvement in the major signaling pathways of Osteosarcoma makes them potential candidates to be targeted for drug development. The highly enriched signaling pathways include the FOXM1 transcription pathway, ATM signaling pathway, FAK mediated signaling events, Arf6 signaling events, Class 1 PI3K signaling events, mTOR signaling pathway, and Integrin family cell surface interactions. Targeting the hub genes and their associated functional partners, which we have reported in our studies, may be efficacious in developing novel therapeutic targets.Osteosarcoma is one of the most frequently occurring sarcomas with a high potency of tumorigenesis. Although chemotherapy and radiotherapy are available as treatment options that have improved patients' lives, there is still some gray area regarding the etiology of the disease. To develop better therapeutics, a specific approach toward the disease targeting the hub genes involved in various signaling pathways must be opted to unravel the complexity of the disease. Our study has mentioned hub genes viz. The original contributions presented in the study are included in the article/Conceptualization: HD, KV, GD, and AE. Methodology: HD and KV. Formal analysis: HD, SK, and AA. Investigation: HD, KV, GD, and HZ. Data curation: HD and SK. Writing—original draft preparation: HD, KV, and AE. Writing—review and editing: KV, AE, GD, and HZ. Supervision: KV and AE. All authors have read and agreed to the published version of the manuscript.


Juvenile spondyloarthropathies (JSpA) are defined as a heterogeneous group of diseases that start before the age of 16. The study aimed to identify key genes and pathways that are influenced by circRNAs and to screen potential therapeutic agents for JSpA. The study involved the analysis of circRNA expression profiles, identification of circRNA-miRNA-mRNA regulatory networks, and functional annotation of differentially expressed genes. The results of the study may have provided insights into the molecular mechanisms underlying JSpA and potential therapeutic targets for this disease.In this study, sequencing data of circRNA, miRNA, and mRNA were obtained from the GEO datasets. The data were then analyzed to identify candidates for constructing a circRNA-miRNA-mRNA network based on circRNA-miRNA interactions and miRNA-mRNA interactions. Functional enrichments of genes were performed using the DAVID database. A PPI network was constructed using the STRING database and visualized using Cytoscape software. The MCODE plugin app was used to explore hub genes in the PPI network. The expression changes in immune cells were assessed using the online CIBERSORT algorithm to obtain the proportion of various types of immune cells. Finally, the Connectivity Map L1000 platform was used to identify potential agents for JSpA treatment. Overall, this study aimed to provide a comprehensive understanding of the molecular mechanisms underlying JSpA and to identify potential therapeutic agents for this disease.A total of 225 differentially expressed circRNAs (DEcircRNAs), 23 differentially expressed miRNAs (DEmiRNAs) and 1324 differentially expressed mRNAs (DEmRNAs) were identified. We integrated 5 overlapped circRNAs, 7 miRNAs and 299 target mRNAs into a circRNA–miRNA–mRNA network. We next identified 10 hub genes based on the PPI network. KEGG pathway analysis revealed that the DEGs were mainly associated with JAK-STAT signal pathway. We found that neutrophils accounted for the majority of all enriched cells. In addition, we discovered several chemicals as potential treatment options for JSpA.Through this bioinformatics analysis, we suggest a regulatory role for circRNAs in the pathogenesis and treatment of JSpA from the view of a competitive endogenous RNA (ceRNA) network.The online version contains supplementary material available at 10.1186/s12969-023-00855-2. Juvenile spondyloarthropathies (JSpA) are a complex and heterogeneous group of diseases that typically manifest before the age of 16. JSpA is characterized by peripheral joint involvement, axial skeletal involvement, enthesitis, and HLA-B27 positivity. The different subtypes of JSpA include juvenile ankylosing spondylitis, psoriatic arthritis, reactive arthritis, inflammatory bowel disease-associated arthritis, seronegative enthesopathy arthropathy syndrome, and enthesitis-associated arthritis [Circular RNA (circRNA) is a type of endogenous noncoding RNA that was only recently discovered. These molecules have a covalently closed loop structure and are formed by back splicing. CircRNAs are highly abundant, stable, and exhibit tissue/stage specificity, as well as species conservation . Recent Integrative ceRNA regulatory networks, which incorporate data on circRNA, miRNA, and mRNA, can provide valuable insights into complex gene interactions and help identify potential biomarkers for the diagnosis and treatment of diseases such as JSpA. The Gene Expression Omnibus (GEO) database provides a rich source of RNA sequencing data on circRNA, miRNA, and mRNA, making it an excellent resource for data mining and biological discovery. Constructing integrative ceRNA regulatory networks based on GEO database can lead to the identification of more accurate prognostic markers for JSpA and other diseases. However, few studies have explored the use of GEO database to construct integrative ceRNA regulatory networks, highlighting the need for further research in this area.In this current research, the GEO database was utilized to identify DEcircRNAs, DEmiRNAs, and DEmRNAs in peripheral blood mononuclear cells (PBMCs) of JSpA patients. Interactions between miRNA-circRNA and miRNA-mRNA were determined using website tools, and a ceRNA network was constructed. Functional enrichment analyses were conducted to assess the biological functions of DEmRNAs, and hub genes were identified through a PPI network. The expression changes in immune cells were assessed using the online CIBERSORTx algorithm, and the proportion of various types of immune cells was determined. Finally, a connectivity map (CMap) analysis was performed to identify potential compounds that could be useful in the treatment of JSpA. The use of GEO databases in this research helped ensure the accuracy and repeatability of the analysis results.The NCBI GEO is a public functional genomics data repository that contains high-throughput gene expression data, chips, and microarrays . We condWe utilized the GEO2R web-based tool  to detecTo predict the target miRNAs of DEcircRNAs, we referred to the Circbank database . The tarWe established the circRNA-miRNA-mRNA regulatory network by integrating the potential circRNA-miRNA pairs and miRNA-mRNA pairs. The Cytoscape 3.8.2 software  was emplDAVID is an internet-based biological information database utilized for annotation, visualization, and integrated discovery . KEGG isUsing the online search tool, Search Tool for the Retrieval of Interacting Genes (STRING), a protein-protein interaction (PPI) network of the differentially expressed mRNAs (DEmRNAs) was constructed. The PPI network was then presented visually through the utilization of Cytoscape (version 3.8.2). Furthermore, the identification of hub genes was performed through the use of the Molecular Complex Detection (MCODE) application in Cytoscape.In order to evaluate alterations in immune cell expression and to obtain the distribution of different immune cell types, we applied the CIBERSORTx algorithm online . To condTo explore potential therapeutic agents for JSpA, we employed the CMap tool . SpecifiWe performed microarray analysis to determine the expression levels of circRNA, miRNA, and mRNA, as summarized in Table In our analysis, we identified 10 interactions between circRNA and miRNA, which consisted of 5 circRNAs  and 7 miRNAs . We merged the predicted mRNAs of these 7 miRNAs from miRDB with the DEmRNAs retrieved from the GEO database, resulting in a total of 299 target mRNAs. Finally, we integrated the 5 circRNAs, 7 miRNAs, and 299 target mRNAs into a circRNA-miRNA-mRNA network for further investigation, as depicted in Fig. Functional and pathway enrichment analyses were conducted using DAVID to obtain a better understanding of the biological roles of the 299 DEmRNAs. Figure The PPI network analysis of the 299 DEmRNAs resulted in a complex network consisting of 299 nodes and 307 edges, as depicted in Fig. We can conclude that neutrophils accounted for the majority of all enriched cells, followed by monocytes, NK cells and T cells. Low percentage of B cell and plasma cell infiltration. While, no differential expression proportion of immune- enriched cells in the JSpA and normal groups is shown in Fig. It is important to note that while these compounds Table  may showThe expression patterns of circRNAs have become increasingly noteworthy in various diseases , 22. A sThis study aimed to investigate the impact of circRNAs on the pathogenesis and treatment of JSpA. By conducting a microarray data analysis, we established a circRNA-miRNA-mRNA network and identified mRNAs that were significantly altered, enabling us to identify relevant GO terms and the KEGG pathway. Subsequently, we identified hub genes through the use of the PPI network. Additionally, we discovered several effective compounds, which could offer a novel approach to treating JSpA.Based on the results of our functional enrichment analysis, we propose that in JSpA, the aberrantly expressed circRNAs utilize both protein and RNA binding to affect the regulation of transcription from the RNA polymerase II promoter, transcription and DNA-templated processes, signal transduction, and cell proliferation. Our KEGG pathway analysis indicates that the differentially expressed genes (DEGs) are primarily associated with the JAK-STAT signaling pathway. The JAK/STAT signaling pathway is a widely expressed intracellular signal transduction pathway that plays a critical role in numerous biological processes, such as cell proliferation, differentiation, apoptosis, and immune regulation. It provides a direct mechanism for extracellular factors to regulate gene expression. Recent studies have focused on the inflammatory effects of this pathway , as the Previous studies have shown the significance of circRNAs in the pathogenesis of AS. For example, Wang T et al. identified two down-regulated circRNAs (circPTPN22 and circFCHSD2) and constructed a circRNA-miRNA-mRNA regulatory network based on these two circRNAs . AdditioIn our study, we identified five circRNAs  in the final circRNA-miRNA-mRNA network. To our knowledge, there have been no relevant studies on the potential functions of the top four circRNAs in JSpA or other diseases. However, a study on lung adenocarcinoma (LUAD) identified hsa_circ_0102537 to be downregulated in LUAD plasma exosomes and tissues, suggesting that it could be involved in LUAD progression . RegardiThrough CMap analysis, we identified ten drugs  as potential treatment options for JSpA. Previous studies have shown that the PI3K/AKT pathway plays a significant role in the pathogenesis of AS, and the PI3K pathway is highly active in this disease –36. A-66The construction of a circRNA-miRNA-mRNA network in this study has illuminated the regulatory mechanisms of juvenile spondyloarthropathies (JSpA) and has yielded valuable insights into the underlying molecular mechanisms and potential therapeutic targets for this disease. Nevertheless, it is important to acknowledge the limitations inherent in this study. Due to the absence of our own sequencing data, this study relied on existing datasets for analysis, which may have introduced certain limitations and constraints. For example, patients from GSE178408 were not JspA, and three datasets were obtained from different biological tissues or sources. However, this study serves as a springboard for future exploration. It has highlighted the need for subsequent experiments to be conducted in future studies, where independent sequencing data can be generated to further validate and expand upon the findings presented here.To summarize, the results of our study provide valuable information regarding the regulatory mechanisms of JSpA through the establishment of a circRNA-miRNA-mRNA network using microarray data and comprehensive bioinformatics analyses. This study sheds light on potential therapeutic targets for JSpA, and may facilitate the development of new treatment strategies.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2