Dry eye disease (DED) is a multifactorial disease of the ocular surface, which affects the quality of life and work efficiency of affected patients. The traditional Chinese medicine formula Qiju Dihuang Pill (QJDHP) has a good therapeutic effect on DED. However, the pharmacological mechanism is not clear.  To explore the mechanism of QJDHP in the treatment of DED based on network pharmacology.  The active components in QJDHP were screened in Traditional Chinese Medicine Systems Pharmacology (TCMSP), and putative molecular targets of QJDHP were identified using the SwissTargetPrediction database. DED-related targets were screened by GeneCards and OMIM. We established protein-protein interaction (PPI) and core targets and corresponding active compound network by Cytoscape to identify the core targets and main compounds of QJDHP against DED. DAVID database was utilized for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Molecular docking was used to evaluate the binding activity between key active compounds and core targets.  The results of network pharmacology showed that 253 targets of QJDHP were related to DED. PPI network analysis showed the 18 core targets. The binding affinity of docking results ranged from -5.7 to -9.3 kcal/mol, indicating a good docking effect. The results of GO enrichment analysis showed that the mechanism of QJDHP in the treatment of DED mainly involved biological processes such as apoptosis, oxidative stress, response to estrogen, angiogenesis, and the regulation of transcription factors. KEGG analysis showed that QJDHP may be regulated by the TNF signaling pathway, Toll-like receptor signaling pathway, MAPK signaling pathway, and estrogen signaling pathway in the treatment of DED.  In this study, we demonstrated the multicomponent, multitarget, and multichannel action mechanism of QJDHP in the treatment of DED and provided a foundation for further drug development research. Dry eye disease (DED) is a multifactorial ocular surface disease. It is primarily characterized by destabilization of the ocular surface environment and is associated with ocular discomfort. Tear film instability, high tear osmolarity, inflammatory damage to the ocular surface, and neurosensory abnormalities are the main pathological manifestations of DED . The worThe pathological mechanism of DED is complex and involves multiple aspects such as inflammation, apoptosis, oxidative stress, and endocrine dysregulation. Most of the current drugs for DED target a single pathological mechanism and have limited efficacy. Chinese medicine prescriptions are characterized by the ability to exert therapeutic effects through multiple components and targets. Qiju Dihuang Pill (QJDHP) is a traditional Chinese medicine prescription for the treatment of DED. According to TCM theory, DED is a complex syndrome, and QJDHP is mainly used to treat liver and kidney yin deficiency type DED. Multiple meta-analyses have shown that QJDHP significantly improves tear film stability, increases tear production, prolongs tear film rupture time, and promotes repair of damaged corneal epithelium in patients with DED –11. QJDHNetwork pharmacology is an emerging discipline that provides a comprehensive explanation of disease mechanisms and drug action mechanisms from the perspective of biological networks. Drug targets and disease-related molecules are mapped to biomolecular networks. By analyzing the complex and multilevel various networks, it is possible to reveal the multicomponent and multitarget synergies of drugs and identify the potential mechanisms of drugs in disease treatment . This reIn this study, the active components, potential targets, pathways, and biological processes of QJDHP for the treatment of DED were systematically analyzed. And the affinity between the main active components of QJDHP and disease targets was predicted by the molecular docking method to explore the potential molecular mechanism of QJDHP for the treatment of DED. The whole research process involved in the study is shown in http://tcmspw.com/tcmsp.php, TCMSP, Version 2.3) [QJDHP has eight herbs, including Gouqizi (GQZ), Juhua (JH), Fuling (FL), Zexie (ZX), Mudanpi (MDP), Shudihuang (SDH), Shanzhuyu (SZY), and Shanyao (SY). Compounds of the eight herbs were searched in Traditional Chinese Medicine Systems Pharmacology (ion 2.3) . Traditiion 2.3) , 19. Thehttp://www.swisstargetprediction.ch/). If there was no standard SDF format, the Open Babel GUI 3.1.1(http://openbabel.org/wiki/) would be used to convert mol2 format to SDF format [Import the standard SDF format of compounds into SwissTargetPrediction, respectively (F format . PotentiF format .https://www.genecards.org/) and OMIM (https://omim.org/). We searched with the keyword “dry eye disease” in the two platforms and selected the organism as “Homo sapiens.” We merged the results of two databases and eliminated duplicated genes. Finally, standardized names were implemented via the UniProt database (https://www.uniprot.org/).The target genes of DED were derived from the following two databases. The GeneCards database (https://bioinfogp.cnb.csic.es/tools/venny/). To explore the interaction between target proteins, overlapping target proteins between QJDHP and DED were uploaded to the STRING database (https://string-db.org/) to obtain information on protein-protein interaction (PPI). The confidence scores were limited to ≥0.4. Set species as “Homo sapiens” and then exported PPI data. The visual network graphs were constructed by Cytoscape 3.8.2 (https://cytoscape.org/). We used the analyzer plugin to analyze the PPI network and built the preliminary hub network by taking twice the median of the degree. Then, we used the CytoNCA plugin to analyze the preliminary hub network and took the excess median of BC, CC, DC, and NC to obtain the core network [The common targets of drug and disease were charted by Venny 2.1.0 ( network .https://david.ncifcrf.gov/). The filtering of searched results was with a threshold value of FDR < 0.05. The value of FDR was the adjusted P value, which reflected the importance of protein biological function. The bar and bubble charts were drawn by R language.To reveal the potential biological function of QJDHP in the treatment of DED. Functional enrichment analyses of the screened core genes were performed by the DAVID database (https://www.rcsb.org). The mol2 format of key compounds was obtained from TCMSP data. The molecular docking was conducted by AutoDock Vina software [Molecular docking was used to evaluate the binding activity between key active compounds and core targets. The crystal structures of core proteins were downloaded from the PDB database  were simulated with 18 core targets, respectively. The binding affinity of docking results ranged from -5.7 to -9.3 kcal/mol see . In addiThe core pathogenesis of DED is tear film instability, with major pathological changes including tear hyperpermeability, inflammatory response, squamous metaplasia, and apoptosis , 26. Excα and has significant therapeutic effects in rabbit dry eye models [κB signaling pathways, respectively [κB and MAPK signaling pathways [QJDHP is an appropriate prescription for the treatment of DED. In this study, 105 active ingredients and 765 potential targets of QJDHP were systematically screened based on network pharmacology. Through further screening, 83 key compounds and 18 core target genes were obtained. Network pharmacological analysis reflects the holistic nature of the combined action of multiple components of Chinese medicine. Based on the core targets and corresponding active compound network analysis, the top six key compounds of the QJDHP were alisol B, luteolin, kaempferol, chryseriol, diosmetin, and alisol B 23-acetate. All six compounds have been shown to have anti-inflammatory effects in previous studies. Kaempferol can promote tear production and corneal repair by inhibiting proinflammatory factors such as IL-1B, IL-6, IL-8, and TNFe models . Chryserectively , 30. Dioectively , 32. Alipathways .α and IL-1, and CXCL8 is highly expressed in the eyes of patients with chronic dry syndrome [α, and IL-1 [The results of the PPI network analysis showed that the main targets of QJDHP were AKT1, VEGFA, TNF, MAPK3, EGFR, CXCL8, MAPK1, CASP3, JUN, PTGS2, MMP9, IL1B, MAPK8, CCND1, ESR1, APP, PI3K3C, and NOS3. Six major compounds were mapped to these 18 key targets, and the predictions were visualized using PyMOL software. The binding affinity range for the docking results was -5.7 to -9.3 kcal/mol, indicating that the binding was stable. AKT1 is involved in the regulation of cell growth, proliferation, and survival and plays an important role in maintaining epithelial cell function in the lid gland . VEGFA, syndrome , 38. MAPsyndrome . Both kaand IL-1 . Disruptand IL-1 . The reland IL-1 . Chryserand IL-1 . CASP3 iand IL-1 . Kaempfeand IL-1 . EGFR isand IL-1 . The ocuand IL-1 . Thus, sand IL-1 . As an oand IL-1 .κB, leading to the release of the proinflammatory cytokines TNFα, IL-1β, IL-6, and the protease MMP9 at the ocular surface [β, TNFα, and IL-8 and the protease MMP9 on the ocular surface. Inflammatory mediators can induce cupped cell and corneal epithelial cell damage by apoptotic and nonapoptotic means [κB p65 nuclear translocation to express proinflammatory factors [α and IL-6 levels in patients with DED, while downregulating the expression of inflammatory factors IL-1β, IL-8, and the protease MMP9 in the tears of patients with DED [GO analysis results show the multifaceted and complex role of QJDHP. QJDHP exerts its therapeutic effects on DED mainly by participating in various biological processes such as apoptosis, oxidative stress, estrogen response, angiogenesis, and regulation of transcription factors. KEGG enrichment analysis showed that the key targets of QJDHP for DED were mainly enriched in TNF signaling pathway, Toll-like receptor signaling pathway, MAPK signaling pathway, and estrogen receptor signaling pathway. TNF signaling pathway, Toll-like receptor signaling pathway, and MAPK signaling pathway are important inflammatory regulatory pathways that are involved in the regulation of many inflammatory processes. Exposure of ocular surface cells to hyperosmolarity activates the MAPK signaling pathway and subsequently activates the downstream transcription factor NF- surface . Proinfl surface . Multiplic means , 53. In ic means . Kaempfe factors . Luteoli factors . All sixwith DED , 58. Thewith DED , 60. Topwith DED . In a clwith DED .In summary, this study revealed the main active components, key targets, and signaling pathways of DED treated by QJDHP through network pharmacology and molecular docking approach. However, there are still some limitations in this study. First of all, network pharmacology and molecular docking are based on the results of database screening and computer simulation prediction, and further improvement of the database of targets of Chinese medicine components is needed to improve the reliability of network pharmacology analysis. Secondly, the effect of a Chinese medicine compound is the result of the interaction of various drugs, not the simple addition of a single Chinese medicine. Therefore, not only the independent analysis of QJDHP active ingredients is needed but also a comprehensive study of the synergistic relationship between drugs. Third, our study is based on database screening, computer simulation prediction, and analysis of existing literature studies, so further experimental validation is still needed in the future.In this study, we used a network pharmacology approach to identify the bioactive compounds of QJDHP and their potential targets in DED. It is concluded that QJDHP may exert anti-inflammatory, antiapoptotic, and endocrine regulatory effects in the treatment of DED through a synergistic multitarget and multipathway action.


Among them, map3k2, csnk1e and gtf2h were expressed in growing tips, mapk8(1) was expressed in static tips and prkcq was expressed in retracting tips. Venn diagrams revealed more than 40 components of MAPK signaling pathways, including jnk and p38 isoforms, which were differentially distributed in growing, static and/or retracting tips. Real-time q-PCR and immunohistochemistry verified the colocalization of map3k2 and csnk1e in growing axon tips. Thus, differentially regulated MAPK and circadian rhythm signaling pathways may be involved in activating either programs for axon regeneration or axon retraction and apoptosis.Axotomy in the CNS activates retrograde signals that can trigger regeneration or cell death. Whether these outcomes use different injury signals is not known. Local protein synthesis in axon tips plays an important role in axon retraction and regeneration. Microarray and RNA-seq studies on cultured mammalian embryonic or early postnatal peripheral neurons showed that axon growth cones contain hundreds to thousands of mRNAs. In the lamprey, identified reticulospinal neurons vary in the probability that their axons will regenerate after axotomy. The bad regenerators undergo early severe axon retraction and very delayed apoptosis. We micro-aspirated axoplasms from 10 growing, 9 static and 5 retracting axon tips of spinal cord transected lampreys and performed single-cell RNA-seq, analyzing the results bioinformatically. Genes were identified that were upregulated selectively in growing ( When an axon is severed, e.g., due to spinal cord injury (SCI), its cell body responds in one of two ways. In some neurons, a regenerative response is activated, which allows the axon to regrow. In other cases, the neuron undergoes retrograde death or atrophy. Several retrograde signals have been implicated in these injury responses, including members of the mitogen-activated protein kinase (MAPK) family ,2,3. So Here, we have combined micro-aspiration and the isolation of total RNA from individual axon tips with deep sequencing to gain insight into the role of local protein synthesis in selecting regenerative vs. degenerative responses to axotomy in the lamprey spinal cord. The results support differential roles for different MAPK signals in axon regeneration and retraction.Petromyzon marinus), 10–13 cm in length , were used for the current research. They were either purchased from Lamprey Services  in Michigan or gifted (100) by Dr. Nicholas Johnson of the USGS Great Lakes Science Center  and maintained with light cycles in freshwater tanks at 15 °C until use. Among them, 200 animals were used for the micro-aspiration in practice and in formal experiments. The rest were used for histological studies. Lampreys were anesthetized by immersion in saturated aqueous benzocaine for 5 min and were pinned to a Sylgard  plate filled with ice-cold lamprey Ringer . The spinal cord was exposed via a dorsal incision and was transected under direct microscopic vision with iridectomy scissors at the level of the 7th gill. Spinal axons and their cell bodies in brainstems were retrogradely labeled with the fluorescent dye dextran-tetramethylrhodamine  at the time of SC-TX by inserting a 0.5 mm dye-soaked Gelfoam pledget into the TX site. The cut axons and their cell bodies became brightly labeled within several hours. The Gelfoam was no longer distinguishable from the overlying scar tissue by 10 days after insertion. Background fluorescence was largely cleared from the area of dye application by 1 week after application. Although a fluorescent label was picked up by glial cells and small neurons in the region adjacent to the application, the resulting low level of fluorescence did not interfere with the observation of the large RAs and their cut tips. Animals were covered with a lamprey Ringer-moistened paper towel and were kept on ice for 3 h to facilitate clot formation. Animals were returned to fresh water and were allowed to recover at 4 °C overnight, and then they were housed at room temperature.A total of 260 larval lampreys  with a CCD camera  and software  without lamprey Ringer. Fiduciary landmarks such as surface capillaries and local neuronal cell bodies were noted and used for later alignment. Then, the spinal cord was covered with a Gelfoam pledget pre-soaked with lamprey serum, and its head and body were covered with Kimwipes pre-soaked with lamprey Ringer. The animals were kept in a cold, high humid environment (a Coleman Cooler with ice) for 3–4 h, and their spinal cords were reimaged after 2–4 h. The growth status of a given axon tip  during that interval was determined by measuring the change in the location of the axon tip relative to the fiduciary landmarks. To avoid bias in finding the front edge, images were converted to gray mode and were processed using the tool “Find Edges” . This allowed us to discriminate distances as short as 2–3 µm between observations. A tip was deemed “growing” or “retracting” if it had moved more than 10 µm towards or away from the transection site. The rest were judged “static tips”. Axon tips that had moved between 3 and 10 µm were excluded to avoid ambiguity. Of 33 microaspirated axoplasms, 9 were excluded, either because of this ambiguity, because the leading edge of the tip could not be identified unambiguously in the fluorescence micrographs or because the tip had to be impaled a second time in order to microaspirate sufficient axoplasm, which may have introduced contaminating material from surrounding cells . Their rThe lamprey spinal cord is flat and translucent, allowing the visualization of cells and fluorescently labeled axon tips in living preparations ,28. The ® Single Cell Kit  and (2) Nextera XT DNA Library Prep Kit /IDT® for Illumina® DNA/RNA UD Indexes Set A, Tagmentation . Briefly, a 2 µL aliquot of axoplasm from an individual axon tip was incubated with RNase inhibitor in 1 × reaction buffer with 3′ SMART-Seq CDS Primer II A at 72° for 3 min, and it was reacted with RT Master Mix for 180 min at 42 °C to synthesize first-strand cDNA, which was amplified by LD-PCR in SeqAmp CB PCR buffer, containing PCR primer and SeqAmp DNA polymerase . The quality of the cDNA was checked by a 2100 Bioanalyzer . NGS libraries were then constructed with the Nextera XT DNA Library Prep kit per Illumina’s instructions. The quality of DNAs was rechecked, and the DNA was quantified and pooled to generate a final concentration of ~5 nM in 120 µL.Two kits were used in constructing the libraries: (1) SMART-Seqhttps://genealacart.genecards.org/, accessed on 26 July 2022) and were uploaded onto the DAVID internet platform. After “Fold Enrichment” and “FDR” options were checked, the “Functional Annotation Chart” module was re-calculated. Results were downloaded for further analysis in an excel file. Principal component analysis (PCA) and regression analysis were performed using the R/Bioconductor DESeq2 package. For protein interaction analysis, 2 original networks were generated by searching the PubMed database with “map2k3” and “csnk1e” in Cytoscape (v. 3.9.0) software, analyzed and trimmed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) [The fragment sizes of libraries were verified using the Agilent 2100 Bioanalyzer, and the concentration was determined using a Qubit instrument . The libraries were loaded onto Illumina Novaseq 6000 for 2 × 100 bp paired-end read sequencing. FASTQ files were generated using the bcl2fastq software for further analysis. Sequence reads were aligned to the curated Lamprey genome  using STAR . The num(STRING)  and DAVIThe length of the body containing the brain and spinal cord rostral to the TX site was obtained by cutting the frozen tissue in the olfactory sac in the head and 1 mm below the level of the 7th gill in the spinal cord. The head was separated from the spinal cord by a third cut at the 1st gill, and the spinal cord stump that contained the TX site was obtained by a 4th cut at the 3rd gill. The frozen stumps were placed in stainless steel molds  filled with Optimal Cutting Temperature (OCT) medium. The orientation of the frozen tissue was adjusted to obtain transverse or coronal  sections. The molds were placed directly on top of liquid nitrogen in a Dewar flask until the OCT became completely solid. Blocks were stored at −80 °C until sectioning. Cryosectioning was performed on a Leica CM1950 Cryostat with the chamber temperature at −17 °C . The frozen blocks were placed in a cryochamber for 30 min to achieve uniform temperature before sectioning. The OCT-embedded specimens were cut serially at 15 µm and were mounted on glass slides. The slides were kept temporarily in a Styrofoam box filled with dry ice during sectioning and were then stored at −80 °C until use.For IHC, slides were thawed and dried in a plastic box filled with Drierite desiccant  at 4 °C for 15 min and then at room temperature for 30 min. The tissue was fixed with ice-cold acetone for 2 min and was washed with phosphate-buffered saline (PBS) twice for 30 s to remove OCT and the fluorescent dye DTMR. Nonspecific binding was blocked with 4% fetal bovine serum  for 5 min. Slides were incubated with antibodies against map3k2  and csnk1e  overnight at 4 °C. The next day, samples were washed in Tris-buffered saline solution  and were blocked by normal animal serums depending on the origin of the secondary antibodies. For immunofluorescence, slides were incubated with either Alexa Fluor 488 donkey anti-mouse IgG polyclonal antibodies  or Alexa Fluor 594 donkey anti-rabbit IgG polyclonal antibodies  at 1:200 in blocking solution for 1 h at room temperature. Finally, the specimens were washed twice with TBS and were mounted with DAPI Fluoromount-G . Images were captured using a Nikon Eclipse 80i epifluorescence microscope with a Roper Scientific CoolSNAP digital camera , under the control of NIS Elements software. Negative controls employed the same protocol, omitting the primary antibody. For chromogenic IHC, protocols recommended by Cell Signaling Technology were followed. It generated brown to gray–black colors.® Single Cell Kit in the “Construction of libraries for scRNA-seq and sequencing” were used to perform the q-PCR with Power SYBR Green Master Mix  on a qPCR apparatus . Real-time q-PCR was performed in triplicate on 96-well reaction plates under the following conditions: activation (95 °C ×10 min), PCR 40 cycles  and data collection , followed by cooling to 40 °C. The primers used in q-PCR are presented in Pre-amplified tip cDNAs (20 cycles) by SMART-SeqPetromyzon marinus specimens were anesthetized in saturated aqueous benzocaine, and their brains and spinal cords were dissected. Tissues were homogenized in RIPA buffer  containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4 and 1 µg/mL leupeptin, by sonication . The homogenate was centrifuged at 10,000× g for 20 min. The supernatant was collected and used. Their protein concentrations were quantified by the Lowry method. Proteins (35 µg) were separated in a 10% SDS mini-gel and were electrophoretically transferred onto nitrocellulose membranes  using a Bio-Rad transblot apparatus. The membranes were blocked in an Odyssey Blocking Buffer  for 2 h and were incubated with antibodies against map3k2  and csnk1e  overnight at 4 °C, followed by washing with PBS containing 0.2% Tween 20 (PBST) and by incubation with the fluorescence-labeled secondary antibody IRDye 680RD  for map3k2 and IRDye 680RD  for csnk1e for 1 h at room temperature. The membranes were then rinsed in PBST and were scanned by the Odyssey infrared imaging system .A total of 10 p < 0.05. The statistical analysis was calculated through SigmaStat software for Windows (version 2.0). Sample numbers are indicated in the figure legends.All data are represented as the mean ± standard error for all performed repetitions. Statistically significant differences among three or more groups were analyzed by a one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc analysis. The significance level was set to n = 30). Representative examples of these three types of tips are shown in A total of 80 animals were used in sample collection by micro-aspiration, as described in the Thirty-three RNA-seq libraries were constructed with the SMART-Seq Single Cell Kit and Nextera XT DNA Library Prep kit. Due to the limited amount of total RNA (1–10 pg in 0.1 µL) in each aspiration, it was not practical to measure the quality of mRNAs before PCR amplification. Therefore, we postponed the quality assessment until the mRNAs were reverse-transcribed and amplified by the first round of PCR amplification  and until after tagmentation, followed by another around of PCR amplification . An amount of 2 µL of each PCR product was checked for the quality of synthesized transcriptomes. The top image in the right panel of FASTQ files (100+ Gb), generated using the bcl2fastq software, were analyzed to identify differentially expressed genes (DEGs). Data trimming and alignment were processed as described in the p-values  were calculated for each gene in three comparisons . Only genes with FC ≥ 1.5 and FDR ≤ 0.2 were included in subsequent hierarchical cluster analyses. This threshold resulted in three lists of genes that were differentially expressed between G tips and R tips , MAPK , sonic hedgehog (smo) and keratinocyte differentiation (prkcq) pathways. KEGG generated more than 40 pathways related to 26 genes, including the 5 genes mentioned above  and pckcq. These are presented in red, green, blue and purple, respectively, for their upregulation in G, S and R tips. As a result of the space limitation, and because the genes upregulated in G tips are of greater priority in the present context, we especially focused on map3k2, csnk1e and their related pathways, on MAPK and the circadian rhythm pathways. Although MAPK signaling pathways have been studied for many years with regard to their roles in axon regeneration, their true effects remain unclear due to the complexities associated with the co-existence of Erk, Jnk, p38 cascades and the cross-interactions among them. Circadian timing has been implicated in the regulation of several cell functions linked to tissue regeneration via the homeostasis of stem cell proliferation [map3k2 and csnk1e are found in the right side (upregulated) of the left (G vs. R) and right (G vs. S) panels of Based on similarities in gene expression patterns, the genes in these three groups were classified in expression clusters by the hierarchical algorithm . These cdatabase , presented above . It is w10FDR values , 1 is involved in protein synthesis (eif2s1a), 3 are involved in mitochondrial function , 1 is involved in chromosome regulation (gpatch11), 1 is involved in the heat shock protein of the Hsp70 family (dnaja4) and 1 is involved in phosphatidylinositol glycan biosynthesis (pigq). The biological functions of the 10 genes in the category R > G include involvement in lymphocyte signaling and differentiation , the inhibition of cAMP accumulation (rxfp3) and protein degradation (mul1a). In another category (G > S), genes were identified that are involved in the ras-related signaling pathway  or in the structural maintenance of chromosomes (smchd1). In contrast, genes involved in exocytosis (scamp5), the extracellular matrix (tectb) or insulin-like growth factor (IGF) binding (wisp2) were found in the S > G category. Interestingly, the Erk/MAPK-signaling-pathway-related gene raf1a was found among the top 10 of the S > R category. Therefore, a ras-raf-MAPK signaling pathway may be involved in static tips to maintain axon stability. In the R > S group, sdc2 and prkcq are genes related to apoptosis.From the three groups, A, B and C, we also identified 60 genes, the top 10 of each of the 6 possible categories  in FC and −logR values , with thp-value < 0.05). The most significant GO terms (FDR < 0.05) were found in group A genes  and are included in p-values.The DAVID Bioinformatics Resources program was used for the functional analysis of transcripts . Three gp-value = 0.057, fnta (protein farnesyltransferase) and prkcq (PKC-θ), are listed in one GO term: “cellular component disassembly involved in execution phase of apoptosis” .Compared with group A . GO terms with higher FDR values  was selected in order to increase the chances of identifying genes whose encoded proteins are related to epigenetic modifications, or that are involved in signaling pathways, because these genes and coded proteins are more likely to be differentially expressed in different growth states, even if their expression levels are low. This increased the number of genes that could be analyzed by gene ontology (GO) enrichment, whereas a higher stringency criterion (FDR < 0.05) was used in most cases in order to exclude noisy signals. The low-threshold FDR did not affect the identification of To assess the variation of whole samples, we performed PCA on all sequencing data from 24 transcriptomes  were obtained from paired counts from G1-G2, G1-G3, …, G1-G10, G2-G3, etc. Sums of R2s for each transcriptome  were calculated , representing the repertoires of RNA-seq in G, S and R axon tips. A Venn diagram was plotted (BioVenn) with areas proportional to the number of gene species . Since the numbers of tips in each group differed, which can result in bias toward the selection of more genes in the larger groups if all samples were used, we selected the five most related transcriptomes from each group  to participate in the Venn diagram analysis, based on a Pearson correlation analysis of counts within each group. For example, in group G, the coefficients of determination  are shared by all tips (v-GSR), but histone acetyltransferase (hat1) is present exclusively in the v-GS. There are two unique genes in the v-R: elp3 and usp22. Since they may play a role in axon retraction, they were selected for validation by real-time q-PCR.The results of the above analyses suggest that MAPK pathways may be involved in axon regeneration, as shown in erk, jnk1/2 and p38α/β), two genes limited to the v-R region of the Venn diagram (elp3 and usp22) and two genes differentially expressed in G tips , whereas elp3 (not shown) and usp22 were differentially expressed in R tips  were the most upregulated genes found in G tips and S tips, respectively. Enrichment analysis of the listed 32 genes indicated that they belong to the “MAPK signaling pathway” (FDR = 1.47× 10−12), or more specifically, the “JNK cascade” (FDR = 3.98 × 10−6). A pivotal node “jun” is missing from the RNA-sequencing list because it has not been annotated in the lamprey genome. The csnk1e network consists of 24 nodes, among which 9 genes (green color) were highly upregulated in one of the three tip types. GO enrichment analysis showed that they are related to “circadian rhythms” (FDR = 7.14 × 10−18), or “circadian regulation of gene expression” (FDR = 1.37 × 10−14). Data used for constituting 2 PPI networks are provided in As a result of their apparent prominence in differentiating growing tips from static or retracting ones, we performed network analyses of the proteins related to map3k2  and csnkAfter axotomy, the neuron responds by turning on genetic programs that either promote axon regeneration or signal neuronal death. Which response pattern predominates may depend on environmental factors, such as the presence of growth-inhibiting molecules . HoweverMany investigations have been performed to elucidate the mechanisms of axon regeneration by identifying mRNAs in neurites. Early techniques, such as conventional PCR methods or microarray analysis, have low efficiencies and detect only dozens or hundreds of transcripts ,47. With(1)map3k2 and csnk1e: From the G > S and R list in hierarchical cluster analysis Genes involved in ribosomal function: Approximately 300 of the genes found in axon tips are believed to be directly involved in RNA processing, ribosome biogenesis and protein synthesis .(3)eif2s1a and eif4b are translation initiation factors. The protein encoded by alas2 catalyzes the first step in the heme biosynthetic pathway.G > R genes: Among the top 10 genes upregulated more in growing tips than in retracting tips , eif2s1a(4)hadac1, hadac3 and hdac8, encode histone deacetylase isoforms 1, 3 and 8, respectively, found in all three types of tips  are thought to act in maintaining the stability of chromatin structures Genes related to RNA binding.casc3, lrpprc and slbp; genes related to ribonucleoprotein granule formation include dhx30, rpusd4 and tbrg4; and genes related to signal recognition particles include srpra and srp72.RNA-binding proteins (RBPs) bind to RNA in cells and participate in the formation of ribonucleoprotein complexes. RBPs contain structural motifs for binding to specific regions of RNA. They play major roles in the post-transcriptional control of RNAs, including RNA transport, splicing, polyadenylation, stabilization, localization and translation. GO enrichment analysis from DEGs in G vs. R tips  revealed(1)dcaf17 encodes a protein associated with cullin 4A/damaged DNA binding protein 1 (DDB1), which is involved in protein degradation. The gene prkcq encodes protein kinase C theta, which is involved in T cell activation and other functions.Genes found in the R > S and G list D: Among (2)cdk7, rxfp3, oprm1, grm7,and sdc2. Proteins encoded belong to the “Gα(i) signaling events” family . They inhibit the cAMP-dependent pathway through the inhibition of adenylate cyclase [prkcq and sdc2 encode protein kinase C theta (above) and syndecan 2, and both participate in apoptosis of osteoblasts [Genes found in the G < R or R > S lists: Among the top 10 genes expressed at higher levels in retracting than in growing tips G < R,  or in re cyclase . Genes peoblasts .(3)usp22  or oxidoreduction reactions . Cells are powered by two main energy sources: glycolysis and mitochondrial respiration. The energy for local protein synthesis in neurites is mostly from mitochondria. The synthesis of protein granules in the dendrites of rat primary hippocampal cultures was inhibited by the ATP synthase inhibitors, antimycin or oligomycin [Xenopus laevis embryos, approximately 35% of endosomes (a new translation platform) were found adjacent to mitochondria, and 80% of these were associated with RNA granules [Many mRNAs coding for ribosomal proteins were present in all three types of axon tip in this genome-wide analysis. There were 130, 121 and 87 transcripts that encode ribonucleoprotein and 66, 51 and 36 transcripts that were involved in “protein biosynthesis” expressed in G, S and R tips, respectively. Genes involved in mRNA , tRNA-  and rRNA-  processing also were present in the transcriptomes, suggesting that the maturation of mRNA, tRNA and rRNA occurs in axonal compartments. Interestingly, “mitochondrion” transcripts were the first and second significant GO terms in the G vs. R  and S vsigomycin . In Xenogranules . This idgranules .C. elegans through a p38/MAPK-like pathway [The basic assembly of MAPK pathways is a three-component module composed of MAPK kinase kinase (MKKK), MAPK kinase (MKK) and MAPK. In mammals, there are 14 MAPKs in 7 groups. Conventional MAPKs consist of the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino (N)-terminal kinases 1, 2 and 3 (JNK1/2/3), p38 isoforms  and ERK5 . The lam pathway ,66,67. H pathway . The sam pathway , neurite pathway  and cell pathway . The dur pathway . Moreove pathway ,71. The  pathway . For allwisp2, col19a1 and ptprk  that were annotated to participate a pathway in the BIOCARTA database  pathway . This pathway is inhibited by another 7-transmembrane protein Ptch1 and is released when it binds to ligand Shh. The regulation of gene transcription results from the translocation of Gli family proteins to the nucleus [prkcq is a member of the PKC family. It is a calcium-independent and phospholipid-dependent protein kinase [prkcq in the nervous system is unclear. It may function through the activation of the NF-κB transcription factors, as occurs in the immune system [prkcq in neural degeneration and regeneration has been evaluated recently [Gene  pathway . Hedgeho nucleus . The sti nucleus . The pron kinase , involvee system . NF-κB ie system ,85. A rorecently . Prkcq edcaf17, prkcq, cdk7, rxfp3, oprm1, grm7 and sdc2) may also be involved in signaling regenerative failure and delayed cell death. The present study also suggests possible cross-talk between the MAPK and circadian rhythm pathways, which previously seemed unrelated to the mechanisms of axon regeneration. We recognize that the descriptive results obtained in this study require validation by molecular manipulations. However, the study includes a great deal of valuable data that we and others can mine to make many more discoveries about the role of local protein synthesis in axon regeneration and about the roles of specific proteins in regeneration and degeneration. We used morpholino antisense oligonucleotides to conduct knockdown studies in the lamprey, and we may be able to do so with a very small number of genes selected on the basis of the uncovered data. However, with its very long life cycle, the lamprey may not be the best animal in which to test the functions of discovered local mRNAs. Our data may point the way to experimental manipulations in species, such as mice, that are more convenient for molecular manipulation, but in which it would be very difficult to obtain the information contained in the present report.Although they are far from an in-depth analysis of the roles of all kinases of the MAPK pathways in regulating axon dynamics, the results of this genome-wide gene expression analysis provide multiple targets for further studies aimed at understanding the mechanisms of post-injury axon dynamics. the selective upregulation of a subset of MAPK pathways, such as map3k2/map2k4/p38α, may be an effective strategy to improve axon regeneration. The present study provides the first overall picture of a complex signaling network involving different subsets and isoforms in axon elongation and the opposite process of axon retraction. In addition, in lamprey, there are close correlations among the distances of axon retraction, regeneration failure and delayed apoptosis ,25,26,87


Astragalus mongholicus Bunge [Fabaceae] (AMB), a traditional Chinese medicine (TCM), has been widely used to treat liver diseases in the clinic. However, the efficacy and mechanism of AMB in the treatment of nonalcoholic fatty liver disease (NAFLD) remain unclear. The purpose of this study was to systematically investigate the active components and mechanisms of AMB against NAFLD based on network pharmacology, molecular docking, and experimental verification.  First, the bioactive components and relevant targets of AMB were screened from the Traditional Chinese Medicine Systematic Pharmacology (TCMSP) database, and NAFLD-related targets were obtained from the GeneCards database. Then, the AMB-NAFLD protein target interaction network was built by the STRING database. GO and KEGG pathway enrichment analyses were performed using the DAVID database. The component targets were visualized using Cytoscape software. Finally, molecular docking and experiments were used to verify the results of network pharmacological prediction. α. In addition, we further discovered that quercetin could increase ACC phosphorylation and CPT1α expression in PA-induced HepG2 cells.  Network pharmacology predicted that quercetin may be the main active component in AMB, and the TNF and MAPK signaling pathways may be the key targets of AMB against NAFLD. Molecular docking validation results demonstrated that quercetin, as the main active component of AMB, had the highest binding affinity with TNF. Furthermore, quercetin played a distinct role in alleviating NAFLD through in vitro experiments. Quercetin upregulated the phosphorylation levels of AMPK and inhibited the expression of p-MAPK and TNF-α and AMPK/ACC/CPT1α signaling pathways to inhibit inflammation and alleviate lipid accumulation. Our results indicated that quercetin, as the main active component in AMB, exerts an anti-NAFLD effect by regulating the AMPK/MAPK/TNF- Nonalcoholic fatty liver disease (NAFLD) refers to excessive lipid deposition in hepatocytes that has become a leading cause of chronic liver disease worldwide, with a prevalence of 25% in adults , 2. NAFLAstragalus mongholicus Bunge [Fabaceae] (ABM) is one of the most widely used traditional Chinese medicines (TCMs) in clinics and is widely distributed in Northeast, North, and Northwest China as well as in Mongolia and Korea . The rooDue to the characteristics of multicomponents and multitargets, traditional experimental methods cannot systematically explain the pharmacological mechanism of TCM. Thus, we adopted network pharmacology analysis based on bioinformatics and systems biology to carry out the research . NetworkIn this study, we first predicted the bioactive compounds and mechanisms of ABM in ameliorating NAFLD based on network pharmacology. Then, molecular docking technology and experiments were utilized to verify the reliability and accuracy of the above results. To our knowledge, this study is the first to reveal the effect and mechanisms of AMB against NAFLD based on network pharmacology, molecular docking, and experimental validation. This research was carried out to provide a theoretical basis for AMB in the treatment of NAFLD. The workflow is shown in http://lsp.nwu.edu.cn/tcmsp.php). Then, oral bioavailability (OB) ≥ 30% and drug − like quality (DL) ≥ 0.18 were defined as important ADME-related pharmacokinetic parameters for identifying active ingredients in AMB [The related chemical components of AMB were obtained from the Chinese Medicine System Pharmacology Database , a comprehensive functional database that contains genomics, proteomics, and transcriptomics [Information on NAFLD-related targets were obtained from the GeneCards database . Its function is to visually present the direct or indirect interactions between proteins. First, the common component-disease targets were entered into the STRING database and selected within the scope of “Homo sapiens”. PPI information was exported in tab-separated value (TSV) format with confidence score set to 0.4. Then, the results in TSV format were imported into Cytoscape  software to visualize the protein interactions.The PPI network was constructed based on the STRING database . GO analysis was used for gene functional classification analysis, including biological process (BP), molecular function (MF), and cell component (CC). A P value < 0.05 was employed for further analysis.Gene Ontology (GO) Knowledgebase and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the common AMB-NAFLD targets were performed using the DAVID database (https://pubchem.ncbi.nlm.nih.gov). Then, the SDF format was transformed to PDB format by minimizing energy using ChemBio3D software. Finally, they were preprocessed and saved in PDBQT format as docking ligands in AutoDock Tools software. On the other hand, for protein receptors, their X-ray crystal structures were obtained from the Protein Data Bank (PDB) (https://www1.rcsb.org/), including AKT1 (PDB ID: 1unq), IL6 (PDB ID: 6 mg1), TNF (PDB ID: 6q00), TP53 (PDB ID: 4cz5), JUN (PDB ID: 6osn), PTGS2 (PDB ID: 1pxx), CXCL8 (PDB ID: 4xdx), MAPK8 (PDB ID: 2xrw), MMP9 (PDB ID: 6esm), and CASP3 (PDB ID: 2dko). Afterwards, solvent and organic protein receptors were removed using PyMOL software and converted to pdbqt format through AutoDock Tools [To validate the binding affinities of ingredient targets, molecular docking was performed using AutoDock Vina 1.5.6. . First, ck Tools . FinallyQuercetin  were purchased from Chengdu Must Biotechnology Co. Ltd. . The purity of quercetin was >98% and kept protected from light and refrigerated at 4°C. Palmitic acid (PA) was dissolved in ethanol and mixed with fatty acid-free bovine serum albumin (BSA) to prepare stock solutions. The AMPK inhibitor compound C (P5499) was purchased from Sigma-Aldrich.2 atmosphere. To mimic the NAFLD model in vitro, HepG2 cells were grown in medium containing palmitic acid (PA) at a concentration of 0.4 mM. The experiment was divided into three groups: the control group (BSA+DMSO), PA group (0.4 mM PA+DMSO), and PA+quercetin group (0.4 mM PA+25 μM quercetin). The overall experimental time was 24 hours. Moreover, HepG2 cells were cultured in the absence or presence of compound C (20 μM) to verify the specificity of quercetin in AMPK activation.HepG2 cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% 100 U/mL penicillin–streptomycin at 37°C in a 5% COHepatocyte TG content was quantified using a commercial kit . All experimental manipulations were performed in accordance with the manufacturer's instructions. In addition, oil red O staining was also used to assess lipid accumulation in cells. The cells were immobilized in 10% paraformaldehyde and then stained shielded from light. All pathological images were observed using a light microscope.-△△CT method, and the results are expressed as the fold change relative to the control. The PCR primers used are shown in Total RNA was isolated from cell cultures using RNAiso plus reagent (TaKaRa) according to the manufacturer's instructions. Reverse transcription was performed using the PrimeScript RT reagent kit (TaKaRa). Real-time PCR was performed on a Roche LightCycler 480  using SYBR Green . Relative gene expression levels were calculated by the 2α , and anti-GAPDH . The appropriate secondary antibodies conjugated to horseradish peroxidase (HRP)  were used at a 1 : 5000 dilution. An Alpha Q detection system was used to visualize the bound primary antibodies.Total proteins were extracted from HepG2 cells using protease inhibitors and phosphatase inhibitors . Protein concentrations were measured using a BCA Protein Quantitative Assay Kit. The target proteins were blotted with the following antibodies: anti-phospho-p38 , anti-p38 , anti-phospho-ERK , anti-ERK , anti-phospho-JNK , anti-JNK , anti-phospho-AMPK , anti-AMPK , anti-phospho-ACC , anti-ACC , anti-CPT1P value < 0.05 was considered statistically significant.The data are presented as the mean ± standard deviation (SD). Statistical analysis was performed in GraphPad Prism 8.0 software. One-way analysis of variance (ANOVA) was performed to examine differences between multiple groups. A Eighty-seven components of AMB were acquired from TCMSP. According to the screening standards of OB ≥ 30% and DL ≥ 0.18, 20 active components of AMB were finally obtained for subsequent research and analysis . Then, 4A total of 1674 NAFLD-related protein targets were downloaded from the GeneCards database . Among them, 383 GO terms were biological processes (BP)  in the presence or absence of PA (0.4 mM) for 24 h. PA-induced HepG2 cells were used as the NAFLD model. The cell TG content results showed that TG levels were markedly higher in the PA group than in the control group. However, quercetin treatment significantly reduced cell TG levels  is a direct substrate of AMPK, and its phosphorylation level was increased after quercetin treatment. Quercetin also significantly enhanced the level of CPT1α. However, the upregulation of p-ACC and CPT1α was abrogated by compound C , Jun N-terminal kinase (JNK), and p38 , 44. Thepathways . In addiα can be activated by MAPK signaling pathways [α signaling pathway to inhibit the inflammatory response.Furthermore, in the present study, we found that quercetin could increase AMPK phosphorylation levels and that the inhibitory effect of quercetin on the MAPK signaling pathway was abolished by the AMPK inhibitor compound C in PA-induced HepG2 cells. These results indicate that the AMPK signaling pathway regulates the expression of MAPK and that MAPK is a downstream protein of AMPK. In addition, previous studies have illustrated that TNF-pathways . In summβ-oxidation accelerates lipolysis, which contributes to reducing lipid deposition in hepatocytes. AMPK is regarded as a key factor that regulates energy metabolism homeostasis, and AMPK signaling pathway activation has been confirmed to have a protective effect on NAFLD [β-oxidation. In our current study, quercetin treatment significantly increased AMPK and ACC phosphorylation levels, which inhibited ACC activity, and ultimately, the expression of CPT1α was upregulated. However, this effect was abolished by the AMPK inhibitor compound C. Altogether, quercetin alleviated hepatic lipid accumulation by enhancing fatty acid β-oxidation through activating the AMPK/ACC/CPT1α signaling pathway in PA-induced HepG2 cells.Beyond this, we further explored the mechanism by which quercetin improves lipid metabolism because quercetin alleviates lipid accumulation in HepG2 cells, which was also consistent with literature reports , 48. Hepon NAFLD . ACC caton NAFLD . PhosphoHowever, there were several limitations in the present study. First, although quercetin was considered to be the main component of AMB proven to have an anti-NAFLD effect, it could not fully represent AMB. Therefore, to elucidate the mechanism of AMB in the treatment of NAFLD, further experiments are essential. Second, this study only confirmed the effect and mechanism of quercetin against NAFLD in vitro. Thus, further in vivo experiments and clinical trials are needed to verify our conclusions.β-oxidation, and alleviate hepatic lipid accumulation via the AMPK/MAPK/TNF-α and AMPK/ACC/CPT1α signaling pathways to exert its anti-NAFLD effect (In conclusion, this study adopted an integrated strategy that combined network pharmacology, molecular docking, and experimental verification to illustrate novel mechanisms of AMB in the treatment of NAFLD. Our findings revealed that quercetin, as the main active component of AMB, could inhibit the inflammatory response, enhance fatty acid D effect . This di


PI3K, AKT and mTOR-rpS6K. Therefore, the PI3K/AKT/mTOR pathway seems to have the capability to serve as research hotspot in the future. Currently, various inhibitors are being tested in cells, animals, and clinical trials, which targeting at different parts of this pathway. In this work, we reviewed the research progress on the PI3K/AKT/mTOR pathway how to influence biological behaviors in ESCC, and discussed the interaction between signals downstream of this pathway, especially eukaryotic translation initiation factors (eIFs) and the development and progression of ESCC, to provide reference for the identification of new therapeutic targets in ESCC.Esophageal squamous cell carcinoma (ESCC) is a malignant tumor developing from the esophageal squamous epithelium, and is the most common histological subtype of esophageal cancer (EC). EC ranks 10th in morbidity and sixth in mortality worldwide. The morbidity and mortality rates in China are both higher than the world average. Current treatments of ESCC are surgical treatment, radiotherapy, and chemotherapy. Neoadjuvant chemoradiotherapy plus surgical resection is recommended for advanced patients. However, it does not work in the significant promotion of overall survival (OS) after such therapy. Research on targeted therapy in ESCC mainly focus on EGFR and PD-1, but neither of the targeted drugs can significantly improve the 3-year and 5-year survival rates of disease. Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is an important survival pathway in tumor cells, associated with its aggressive growth and malignant progression. Specifically, proliferation, apoptosis, autophagy, and so on. Related genetic alterations of this pathway have been investigated in ESCC, such as  In 2020, the incidence and mortality of esophageal carcinoma (EC) ranked tenth and sixth in the world, with about 70% of the cases affection males. According to statistics, in 2020, 544,076 people died due to EC worldwide1]. The incidence in eastern Asia is the highest in the world , but theWith the improvement of various examination methods, the detection rate of esophageal squamous dysplasia is increasing, especially in the areas with high incidence of ESCC. Esophageal squamous dysplasia has the potential to develop into ESCC. The two can be regarded as a continuous pathological process . The symAccording to the degree of differentiation of ESCC, three grades can be differentiated: highly, moderately, and poorly   during surgery for patients with resectable EC was found to have an overall survival benefit . It was Currently, neoadjuvant chemoradiotherapy and esophagectomy are the mainly therapy for ESCC. In a guide to the management of EC, it is suggested that patients with locally advanced ESCC should receive neoadjuvant chemoradiotherapy . MultimoJapanese scholar Masayuki Watanabe and colleagues found that patients with ESCC received neoadjuvant chemoradiotherapy plus surgical resection, the 3-year survival rate was 29.8% and the 5-year survival rate was 15.0%  in ESCC . At the In the phase III AIO/EORTC clinical trial conducted by M. Moehler and colleagues, they found that the use of panitumumab (an anti-EGFR antibody) combined with cisplatin and 5-fluorouracil did not improve survival compared to unselected advanced ESCC patients who received 5-fluorouracil alone . This rein vitro (P=0.07) (Programmed cell death protein 1 (PD-1) is an inhibitory receptor expressed on activated lymphocytes, can connect with the ligands of PD-L1 and PD-L2. And they are favorable to regulating the balance of T cell activation, immune tolerance, and immune-mediated tissue damage , 22. Blon vitro , 26. It n vitro , 28. Nivn vitro . Toshihin vitro . ThroughP=0.07) . Some scP=0.07) .Therefore, the current targeted therapies for EGFR and PD-1 respectively have encountered bottlenecks. The solution is to dig deeper into the molecular mechanisms of ESCC and find other sensitive targets. In the future, combination therapy with multiple molecular-specific targeted drugs may be a good option for the treatment of ESCC.The causes of ESCC are complex. Environmental and genetic factors are contributors for ESCC formation. It is believed that pathogenic genes have an important influence on it.ADH1B and ALDH2 had the highest risk to develop cancer   and genes  .ERBB, HDAC, PI3K family, XPO1, FGFR1, TP53, JAK-STAT3, and mTOR-rpS6K were defined to be recurrent candidate druggable targets  and epigenetic process . It should be noted that TP53 plays a role in inhibiting carcinogenesis in organisms   and RTK-PI3K  . While the class IB is composed of p110γ  encoded by PIK3CG and activated by G protein-coupled receptors (GPCRs)  , 50. The (GPCRs) –54. Clas (GPCRs) . Class I (GPCRs) .Class I PI3Ks are the research hotspot of PI3K signal transduction. In addition, class IA PI3Ks are widely found in carcinogenic processes. RTK or GPCR activation enrolls class I PI3Ks into the plasma membrane, where p85 (regulatory subunit) -mediated inhibition of p110 is released and p110 directly phosphorylates PIP2  into PIP3  . This liPI3K, RAS can potentially activate AKT  could activate the ras homolog enriched in brain (RHEB), a small GTP enzyme. When AKT is activated, it can mediate TSC2 phosphorylation, inhibit TSC1/2 complex and activate mTORC1 signal , the mainly active component secreted by Garcinia hanburryi tree, on the ESCC cells , 75. It These experimental data suggest that proliferation and apoptosis are closely related to PI3K/AKT/mTOR pathway in ESCC.in vivo , an Akt inhibitor, significantly inhibited p-Akt, HIF-1α, and VEGF expression in vivo . ESCC hain vivo . The effin vivo . The undThe mTOR inhibitors are divided into three generations . Currentin vitro, temsirolimus significantly reduced the size of subcutaneous tumors in nude mice and effectively extended the survival of mice with esophageal carcinoma in situ (the cell used for this experiment was TE-8)  .At the same time, the first generation of mTOR inhibitors only had little effect on the phosphorylation of 4E-BP1 .These inClinical trials with rapamycin and its analogues related to ESCC have not been carried out. Still some researchers have also had limited success in treating a small number of rare cancers with monotherapy, including mantle cell lymphoma and pancreatic neuroendocrine tumors , 99.The second generation mTOR inhibitors are some small molecule ATP competitive inhibitors. They can target mTOR or both mTOR and PI3K. Several categories according to the chemical structure exist . The pyrYu Huang and colleagues examined the antitumor effect of PP242 in ESCC cell lines include Eca-109 and TE-1 . As expePP242 is not tested in currently ongoing clinical trials. TAK-228 (derived from PP242) was well tolerated as a single agent and showed initial therapeutic activity in hematological malignancies (NCT01118689) .Regarding second generation mTOR inhibitors, some compounds were found to target both PI3K and mTOR . BEZ235 Some natural polyphenols extracted from plants, such as curcumin and resveratrol, have been confirmed to inhibit mTOR signaling pathway directly or indirectly in certain tumors . ResearcCurcumin is a polyphenolic compound extracted from turmeric roots. It is safe, non-toxic, and has anti-tumor effects in the human body . Many stResveratrol is rich in grapes, red wine, and peanuts . It is aEpigallocatechin-3-gallate (EGCG), a primary tea polyphenol, has been shown to inhibit the growth of certain human cancer cells . The mecThese natural compounds can inhibit the growth of ESCC cells and are inseparable from mTOR signal.via mTOR. Therefore, mTOR plays a leading role in the regulation of eIF functions   were associated with the development of specific tumors –131. Forn (FISH) , 139. Inn (FISH) , 137.P <0.05)  . TherefoThese previous examples reveal eIFs may be a promising target for future tumor treatment. At present, there are some inhibitors of eIFs in preclinical and clinical trials Table 2.JingJin and colleagues found an overexpression of eIF4E in most ovarian cancer patients. In addition, the eIF4E function is critical for the growth and survival of tumors . eIF4E iSeveral clinical trials related to ribavirin are underway, such as oropharynx squamous cell cancer (NCT01721525), acute myeloid leukemia (NCT01056523), melanoma (NCT00897312).ESCC is a complex disease, the external predisposing factors and genetic mutations both have an important impact on oncogenesis and tumor progression. So, we should focus on the prevention, warning high-risk individuals away from alcohol, cigarettes, and so on. At the same time, we should pay attention to the screening of disease and improve the early diagnostic rate. Once ESCC patients could be diagnosed in the early stage, their prognosis and living quality will be improved significantly.From the global cancer report, the incidence and mortality rate of ESCC are currently ranked tenth and sixth . This daIn all results of ESCC gene testing conducted by several research groups, the abnormal expression of PI3K/AKT/mTOR and its related pathways have been found. In this article, we discussed how the PI3K/AKT/mTOR pathway affects the growth, proliferation, and autophagy of ESCC. It is also discussed that inhibitors in different parts of PI3K/AKT/mTOR pathway can affect the growth and biological behavior of ESCC Table 1.Still, it should not be ignored that there are only few relevant studies on the application of PI3K/AKT/mTOR pathway inhibitors in ESCC. Most studies have used a single inhibitor and with limited efficacy. At present, there are few studies on the combination of multiple inhibitors with different targets in ESCC ongoing. It may be that researchers have not yet paid attention to the potential therapeutic effects of PI3K/AKT/mTOR pathway and its regulated eIFs in ESCC. It also could be that new drugs are being developed slowly and in fewer varieties. But why the effectiveness of a single inhibitor is limited?The researchers found that the activation of multiple pathways is the main cause of drug resistance in tumor cells . Such asThe PI3K/AKT/mTOR pathway and its regulated eIFs have been proved to be a key pathway involved in growth. And many other pathways in the body are inseparable from it. Therefore, the future treatment of ESCC must be related tightly with the PI3K/AKT/mTOR pathway and its regulated eIFs. Simultaneous suppression of multiple targets of this pathway may be a future research focus. One hypothesis: it may be focused more on the co-inhibition of PI3K/AKT/mTOR pathway and eIFs.In this context, multiple components of several oncogenic signaling pathways and eIFs participated in mRNA translation have been identified as biomarkers with potential diagnostic, therapeutic and prognostic value. Therefore, anti-tumor agents targeting the core elements of protein synthesis and related signaling pathways can get over intratumor heterogeneity and represent as novel promising anticancer drugs.Conceptualization, RH, JH, and YD. Writing—original draft preparation, RH, QD, RY, and QZ. Writing—review, revising and editing, RH, JH, and ZY. All authors have read and agreed to the published version of the manuscript.The project of Department of Science and Technology, Sichuan province, Grant/Award, Number: 2018JY0398.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Multi-omics approaches provide an integrated perspective to empower the development of personalised therapies through a better understanding of functional biology underlying the disease and molecular-driven selection of the best-targeted individualised therapy. In this study, we applied integrative metabolomic and transcriptomic analyses to elucidate tumour biochemical processes affected by imatinib treatment.A GIST xenograft mouse model was used in the study, including 10 mice treated with imatinib and 10 non-treated controls. Metabolites in tumour extracts were analysed using gas chromatography coupled with mass spectrometry (GC-MS). RNA sequencing was also performed on the samples subset (n=6).Metabolomic analysis revealed 21 differentiating metabolites, whereas next-generation RNA sequencing data analysis resulted in 531 differentially expressed genes. Imatinib significantly changed the profile of metabolites associated mainly with purine and pyrimidine metabolism, butanoate metabolism, as well as alanine, aspartate, and glutamate metabolism. The related changes in transcriptomic profiles included genes involved in kinase activity and immune responses, as well as supported its impact on the purine biosynthesis pathway.Our multi-omics study confirmed previously known pathways involved in imatinib anticancer activity as well as correlated imatinib-relevant downregulation of expression of purine biosynthesis pathway genes with the reduction of respectful metabolites. Furthermore, considering the importance of the purine biosynthesis pathway for cancer proliferation, we identified a potentially novel mechanism for the anti-tumour activity of imatinib. Based on the results, we hypothesise metabolic modulations aiming at the reduction in purine and pyrimidine pool may ensure higher imatinib efficacy or re-sensitise imatinib-resistant tumours. KIT or PDGFRA (Platelet Derived Growth Factor Receptor Alpha) genes. These genes encode receptor tyrosine kinase proteins, which following activation by a ligand, promote cell growth and proliferation through such signalling pathways as the PI3K/AKT pathway, the STAT pathway, and the MAP kinase pathway. Mutation of KIT or PDGFRA leads to constitutive kinase activation independent of the ligand, and consequent unrestrained induction of signalling cascades leading to tumour proliferation at an abnormally high rate KIT exon 11 or in PDGFRA gene (except exon 18 p.D842V) is associated with a higher response rate and longer recurrence-free survival than GISTs with a KIT exon 9 mutation  and by the project POWR.03.02.00-00-I026/17-00 co-financed by the European Union through the European Social Fund under the Operational Programme Knowledge Education Development 2014–2020.This work was supported by the Szymon Macioszek: Conceptualization, Investigation, Formal analysis, Writing – original draft. Danuta Dudzik: Formal analysis, Data curation, Visualization, Writing – review & editing. Rafał Bartoszewski: Formal analysis, Writing – original draft. Tomasz Stokowy: Software, Data curation. Diether Lambrechts: Investigation, Data curation. Bram Boeckx: Software, Data curation. Agnieszka Wozniak: Conceptualization, Resources, Writing – review & editing. Patrick Schöffski: Supervision, Writing – review & editing. Michał J. Markuszewski: Conceptualization, Supervision, Writing – review & editing.The authors declare no conflict of interest associated with this publication.


We aimed to explore the mechanism of the linoleic acid metabolism in metabolic syndrome (MetS). RNA-seq data for 16 samples with or without MetS from the GSE145412 dataset were collected. Gene set variation analysis (GSVA), gene set enrichment analysis (GSEA), and gene differential expression analysis were performed. Expression data of differentially expressed genes (DEGs) involved in the linoleic acid metabolism pathway were mapped to the pathway by using Pathview for visualization. There were 19 and 10 differentially expressed biological processes in the disease group and healthy group, respectively. 9 KEGG pathways were differentially expressed in the disease group. Linoleic acid metabolism was the only differentially expressed pathway in the healthy group. The GSVA enrichment score of the linoleic acid metabolism pathway in the disease group was markedly lower than that in the healthy group. The GSEA result showed that the linoleic acid metabolism pathway was significantly downregulated in the disease group. JMJD7-PLA2G4B, PLA2G1B, PLA2G2D, CYP2C8, and CYP2J2 involved in the pathway were significantly downregulated in the disease group. This study may provide novel insight into MetS from the point of linoleic acid metabolism dysregulation. Metabolic syndrome (MetS) is a common complex entity characterized by a set of metabolic abnormalities that serve as risk factors for the development of cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) . MetS doAs an essential nutrient, linoleic acid is the most abundant n-6 polyunsaturated fatty acid (PUFA) in the human diet . TherefoGene set enrichment methods focus on gene sets in the analysis of gene expression data. Gene set variation analysis (GSVA) calculates gene set enrichment scores for each sample and assesses the variation of gene set enrichment over the samples. It identifies the differential pathway activity in an unsupervised manner . Gene seHomo sapiens, GRCh38, version 23). Single-mapped reads were utilized to calculate the read count and TPM for each gene. TPM normalization was performed by the normalizeBetweenArrays function of the limma package [Raw data from the GSE145412 dataset  were dowHomo sapiens grouped by KEGG pathways and gene ontology BPs were downloaded from the Molecular Signatures Database (MSigDB) [2FC| > 0.5 and p < 0.05, while those of differentially expressed pathways were set as |log2FC| > 0.1 and p < 0.05. Significantly enriched pathways were identified by the GSEA method with the cutoff values of NOM p value <0.05 and FDR q value <0.25.Gene sets in (MSigDB)  by usingp < 0.05. Expression data of DEGs were mapped to the linoleic acid metabolism pathway for visualization by using Pathview [The gene set related to the linoleic acid metabolism pathway was extracted from MSigDB to construct a protein-protein interaction (PPI) network by using STRING, in which genes with the top 10 highest degrees were regarded as hub genes . DEGs bePathview , and prop < 0.05 was statistically significant.All the statistical analyses were performed by R Statistical Software (version 4.1.2). The Wilcoxon rank sum test was applied to compare the differences between groups. 16 samples were divided into four groups, including MetS lean, MetS obese, healthy obese, and healthy lean. Differentially expressed gene sets are shown in We next examined the differences in the GSVA enrichment score of the linoleic acid metabolism pathway between obese and lean samples in the disease group and the healthy group. As depicted in 29 genes involved in the linoleic acid metabolism pathway were obtained, and the PPI network is shown in MetS has become a global health concern currently. Except for heart disease and diabetes, MetS also has a link with cancer . The comIn the present study, we applied GSVA and GSEA to the RNA-seq data from patients with or without MetS and found that the enrichment score of the linoleic acid metabolism pathway was significantly lower in MetS patients, suggesting the dysregulation of the linoleic acid metabolism pathway in MetS. Through integrated proteomics and metabolomics analysis, Chen et al. also revealed that the linoleic acid metabolism pathway was significantly perturbed in the MetS rat model . SzczukoWe also found that several genes involved in the linoleic acid metabolism pathway were significantly downregulated in MetS patients, including JMJD7-PLA2G4B, PLA2G1B, PLA2G2D, CYP2C8, and CYP2J2. PLA2G4B, PLA2G1B, and PLA2G2D encode members of the phospholipase A2 family that generally hydrolyze the sn-2 acyl bond in phospholipids to release fatty acids and lysophospholipids. In the linoleic acid metabolism pathway, lecithin in the cell membrane is converted by phospholipase A2 enzymes to linoleate. PLA2G4B was associated with age-related changes in phospholipids and decreased energy metabolism in monocytes . PLA2G2DμM concentration by activating NF-κB and AP-1 transcription factors that mediate inflammation [CYP2C8 and CYP2J2 encode members of the cytochrome P450 (CYP) superfamily of enzymes. CYP monooxygenases participate in the metabolism of various endogenous substrates, including PUFAs. In a CYP-dependent manner, linoleic acid is metabolized to produce 9,10-epoxyoctadecenoic acid  and 12,13-epoxyoctadecenoic acid  . CYP2C8 ammation . EpOMEs ammation , 36, whiammation –39. In aammation .The study has several limitations. First, the sample size was small. Second, the bioinformatic analysis was restricted to the RNA-seq data of MetS, while proteomics and metabonomics may provide novel insight into the underlying mechanisms of MetS. Third, there is a lack of experiments to detect the linoleic acid metabolism pathway's activity. Therefore, future studies should be conducted with a larger sample size and in a more comprehensive way, combining multidimensional omics data analysis and experimental verification to enhance the understanding of MetS.In conclusion, we found the dysregulation of the linoleic acid metabolism pathway in MetS, and JMJD7-PLA2G4B, PLA2G1B, PLA2G2D, CYP2C8, and CYP2J2 involved in the linoleic acid metabolism pathway were significantly downregulated.


Objective: To study the potential targets and molecular mechanisms of Fritiliariae Irrhosae Bulbus (FIB) in the treatment of ischemic strokes based on a network pharmacology strategy, with a combination of molecular docking and animal experiments.Methods: The active components and targets of FIB were screened by TCMSP database and TCMIP database, and the related targets of ischemic strokes were screened by GeneCards, OMIM, CTD, and DrugBank, then the intersection targets of the two were taken. The protein interaction network was constructed by STRING, the PPI network diagram was drawn by using Cytoscape software, and the key targets of FIB treatment of ischemic strokes were analyzed by MCODE. The DAVID database was used for GO and KEGG enrichment analysis, and the potential pathway of FIB against ischemic strokes was obtained. Molecular docking was performed by using AutoDock Tools 1.5.6 software. Finally, a mouse model of ischemic stroke was established, and the results of network pharmacology were verified by in vivo experiments. Realtime Polymerase Chain Reaction was used to detect the expression levels of relevant mRNAs in the mouse brain tissue. Western blot was used to detect the expression levels of related proteins in the mouse brain tissue.Results: 13 kinds of active components of FIB were screened, 31 targets were found in the intersection of FIB and ischemic strokes, 10 key targets were obtained by MCODE analysis, 236 biological processes were involved in GO enrichment analysis, and key targets of KEGG enrichment analysis were mainly concentrated in Neuroactive light receptor interaction, Calcium signaling pathway, Cholinergic synapse, Hepatitis B, Apoptosis—multiple specifications, Pathways in cancer and other significantly related pathways. There was good binding activity between the screened main active components and target proteins when molecular docking was performed. Animal experiments showed that the infarct volume of brain tissue in the FIB treatment group was considerably reduced. RT-qPCR and the results of Western Blot showed that FIB could inhibit the expression of active-Caspase3, HSP90AA1, phosphorylated C-JUN, and COX2.Conclusion: Based on network pharmacology, the effect of FIB in the treatment of ischemic strokes was discussed through the multi-component-multi-target-multi-pathway. The therapeutic effect and potential mechanisms of FIB on ischemic strokes were preliminarily explored, which provided a ground work for further researches on the pharmacodynamic material basis, mechanism of action and clinical application. The Lancet in 2019 and 2021 presented that between 1990 and 2019, stroke is the second leading cause of death and the third leading cause of disability in the global population; meanwhile, the first cause of death in China  and Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine . According to the oral bioavailability (OB) of the drug ≥30% and the drug similarity (DS) ≥0.18, the active ingredients of FIB were further screened out (https://www.uniprot.org), then made a conversion of the targets into a unified gene name, defining the source of the gene to “Homo sapiens”.The active chemical components of FIB were collected through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform , OMIM (http://omim.org/), CTD (https://ctd.mdibl.org/) and DrugBank (https://go.drugbank.com/) database, taken “Ischemic Strokes” as retrieval termto search and collect the targets of ischemic strokes, and then, finally obtained the relevant targets of ischemic strokes. The targets obtained from the GeneCards database were classified by the correlation scores of the results, and filtered by using the median score>2-fold as the screening condition. After combining the genes of the four databases and removing the duplicate values, the result of relevant target of ischemic strokes will be showed.We have utilized disease databases GeneCards (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to map mutually the action target of FIB and the target of ischemic strokes related genes and acquire the intersection target of the two namely, the potential target of FIB treatment of ischemic strokes.The online tool Venny2.1.0 , the more nodes in the network related to the node directly, and then the more important the node in the network .https://string-db.org) online database platform; the species was set to “Homo sapiens”, and the confidence level was set to ≥0.4 to construct a network diagram of protein interaction between drugs and diseases. Then, the data information obtained from STRING platform was imported into the Cytoscape 3.9.1 software for analysis, sorted according to the degree value (degree), and filtered according to the degree ≥ median to obtain the core target of FIB treatment of ischemic strokes. Molecular complex detection (MCODE) is applied to identify the characteristics of closely connected networks and further screened the core target community. The setting parameters are as follows: find clusters = in the whole network, degree cutoff = 2, node score cutoff = 0.2, K-score = 0.2, max depth = 100 , gene ontology (GO) function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out for the intersection targets to obtain the biological process, cell components, molecular functions and related signaling pathway relationships of FIB in the treatment of ischemic strokes. The screening standard was set as p < 0.05, and the species were limited only to Homo sapiens. GO and KEGG enrichment analysis were made by R language software for visualization.Based on DAVID 6.8 database (http://www.rcsb.org) to retrieve receptor proteins Caspase3 (PDB: 7RNB), PTGS2 (PDB: 3NT1), JUN (PDB: 6Y3V), and HSP90AA1 (PDB: 4BQG) which those receptor proteins will be performed to remove water and ligands by using PYMOL 2.3.4 software The PubChem database (https://pubchem.ncbi.nlm.nih.gov) is used to obtain the SDF structure file of the compound, and AutoDockTools 1.5.6 software is used for hydrogenation, electronation and other operations. AutoDock software is used for molecular docking, and the PyMOL software for data visualization.Molecular docking is performed between the top 4 active pharmaceutical ingredients and the core targets in the degree value obtained after the analysis. Utilizing the PDB database (FIB (NO: DZYC-C-022) was purchased from Chengdu Herbpurify Co., Ltd.  and dissolved in Carboxymethyl cellulose . 2,3,5-Triphenyl Tetrazolium Hydrochloride (TTC) staining solution and BCA protein concentration determination kit were purchased from Beijing Soleibo Technology Co., Ltd. . The monofilaments were purchased from Beijing Cinontech Co., Ltd. . RNAeasy™ Animal RNA Isolation Kit with Spin Column and Reverse Transcription Kit were purchased from Beyotime Biotechnology Co., Ltd. . Specific primary antibodies  and HRP Goat Anti-Rabbit IgG (H + L) were purchased from Wuhan ABclonal Technology Co., Ltd. .The animal experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Chongqing Medical University. Male C57BL/6J mice weighing 20–22 g were provided by the Animal Research Center of Chongqing Medical University, and placed in an environment free of specific pathogens, temperature (22 ± 2)°C, and relative humidity of 50%–70% and 12 h light/12 h dark cycle with free access to food and water. Experiments were carried out after 1 week of adaptive feeding. Before surgery, the fasted mice for 12 h, had free access to drink water, and were anesthetized with 3% pentobarbital. Establishment of middle cerebral artery occlusion or reperfusion model by wire embolization . After dn = 6): sham operation group, MCAO group, FIB treatment group. FIB was dissolved in 0.5% carboxymethyl cellulose, and 3 h after reperfusion, 20 mg/kg was administered by gastric perfusion for 5 consecutive days. The sham operation group and the MCAO group were given the same volume of normal saline by gastric perfusion for 5 days.Eighteen experimental animals were randomly divided into the following groups  was used for analysis . The micAfter 5 days of reperfusion in mice, mice were killed by the overdose anesthesia, brain tissue was taken out and placed at - 20°C. After 30 min, take out the brain tissue and cut it into 6 pieces, and incubate it in TCC solution for 30 min at 37°C. The infarct size was calculated by ImageJ .−△△Ct method was performed to calculate the relative expression of genes  were incubated overnight at 4°C, washed 3 times with TBST, and incubated with corresponding secondary antibodies at 37°C for 1 h. Finally, the protein bands were imaged and analyzed using the Gel imaging analyzer system , and the value of t-test is used for comparison between two groups, and One-Way ANOVA test is used for comparison among multiple groups. All data were from at least 3 independent experiments. p < 0.05 were considered statistically significant.Statistical analysis was performed with SPSS 24.0 software. The results have been represented as After retrieving through TCMSP and TCMIP databases, screening according to OB and DL conditions, and deleting duplicate values, a total of 13 active ingredients were obtained, as shown in The FIB active ingredients and its potential action targets were introduced into the Cytoscape 3.9.1 software to build a “drug-active ingredient-core target” network diagram, as shown in We have analyzed the protein-protein interaction of common targets of FIB and ischemic strokes through STRING database, hidden 2 connectionless nodes  which involved a total of 31 nodes and 78 edges. The network owned an average node degree of 5.03 and an average local clustering coefficient of 0.612. By importing the obtained data into Cytoscape 3.9.1 software, analyzing the networkto obtain the degree centrality (DC), betweenness centrality (BC) and closeness centrality (CC) of each node in the network. Selecting the nodes with DC > 5, BC > 8.893939, CC >0.500159485, and obtain 10 core targets for FIB to treat ischemic strokes . CASP3, etc.The potential role is further clarified by enrichment analysis of intersection targets. GO function enrichment analysis of 31 differential genes through DAVID database . There wThe 31 intersecting targets of FIB and ischemic strokes were imported into the DAVID database for KEGG pathway enrichment analysis. A total of 72 pathways were acquired from the KEGG pathway enrichment analysis, which were sorted according to the -log10(p) value, and the top 10 were selected to draw a bubble map  and a CiCytoscape 3.9.1 software was used to construct the top 10 KEGG pathways and their related target-drug-active ingredient-disease network . The bluAutoDock software was used to dock the screened active ingredients (bis[(2R)-2-ethylhexyl] benzene-1,2-dicarboxylate、beta-sitosterol, Korseveriline, verticinone) with the core target proteins Caspase3 (PDB: 7RNB), PTGS2 (PDB: 3NT1), JUN (PDB: 6Y3V), and HSP90AA1 (PDB: 4BQG). The lower the binding energy value is, the better the binding effect between the two. Generally, the binding energy ≤ - 5.0 kJ/mol is taken as the standard. The docking results showed that all docking energies are less than - 5 kcal · mol-1, indicating that the docking is stable . The visp < 0.01). Compared with MCAO group, the infarct volume of brain tissue in FIB group was notably reduced (p < 0.01). In order to explore the effect of FIB on neural function defect of MCAO, we performed mNSS score on mice after the last administration (p < 0.01), but the brain mNSS of mice in the MCAO group was significantly improved after FIB treatment (p < 0.01).In order to evaluate the effect of FIB on brain nerve injury, we performed TCC staining of brain tissue to measure the infarct volume of brain tissue. No obvious lesions were found in the sham group. Compared with the Sham operation group, the infarct volume of brain tissue in the MCAO group increased significantly ( reduced  (p < 0.0stration . As showp < 0.01). Compared with the MCAO group, the levels of Caspase3, HSP90AA1, C-JUN and COX2 in the FIB group were significantly decreased (p < 0.01) (p < 0.01). Compared with MCAO group, the levels of active-Caspase3, HSP90AA1, phosphorylated C-JUN and COX2 in the FIB group were significantly decreased (p < 0.01). In conclusion, FIB has a significant protective effect on ischemic stroke through the regulation of relevant core targets, which further verifies the prediction of network pharmacology.We found that compared with the sham operation group, the levels of Caspase3, HSP90AA1, C-JUN, and COX2 in the model group were significantly increased by RT-qPCR ( < 0.01) . Then we < 0.01) . The resAt present, the overall prevalence of ischemic stroke is increasing year by year, and it emerges a development trend of rejuvenation. The current basic treatment strategy for stroke is to facilitate cerebral blood perfusion and neuro-protection. Nevertheless, the limited time window and high surgical cost of thrombolytic therapy restrict its clinical application . Furtherin vitro cell experiments. The effective components and action targets of FIB were screened by network pharmacological methods, and the intersection of FIB and Ischemic Strokes related targets were visually analyzed to build a drug component disease target network. Through the analysis of protein interactions in the network, the core target community was screened. The main pathways of core targets involved in the treatment of Ischemic Strokes were studied through bioconcentration analysis, and a target pathway network diagram was constructed. Finally, the four core target proteins of FIB for the treatment of Ischemic Strokes were analyzed by molecular docking. Thus, the main pathway, target protein and core components of FIB in the treatment of Ischemic Strokes can be obtained. Subsequently, an animal model of transient middle cerebral artery occlusion was established to verify the analysis of the therapeutic effect of FIB by network pharmacology.Traditional Chinese medicine emphasizes individualized treatment under the guidance of a “holistic view.” Meanwhile, the stroke prevention and treatment drugs represented by traditional Chinese medicine have the advantages of “multi-component, multi-target, and multi-effect.” In addition, various components in traditional Chinese medicine can act individually or synergistically to enhance the therapeutic effect, which possess unique advantages and promising application prospects in the prevention and treatment of ischemic stroke. The application of network pharmacology to the research of traditional Chinese medicine can explore the material basis of pharmacodynamics and the mechanism of action in the treatment of diseases from the perspective of molecular network regulation, which plays an essential role in promoting and guiding the research of traditional Chinese medicine . The pot2O2  were finally determined. Recent studies have shown that the activation of caspase-3 is a key link in neuronal apoptosis after the Ischemic Strokes, which will induce the formation of apoptotic bodies and the occurrence of apoptosis by cutting off contact with surrounding cells . Inhibit2O2 . FIB mig2+ was not only a crucial chemical involved in neurotransmitters, but also a key chemical in the release of intercellular signals in the nervous system which was also involved in neuronal activity and the immune system (The results of GO enrichment analysis showed that the protection of FIB on ischemic strokes were realized by simultaneously activating multiple biological processes, cell components and molecular functions. The targets and the signal pathways often play a role in coordination with each other. Through the enrichment analysis of the KEGG pathway of the intersection target, 72 signal pathways were obtained, most of which exist in the nerve, inflammation, circulation and other aspects. Neuroactive ligand receptor interaction signal pathway is the collection of all receptors and ligands related to intracellular and extracellular signal pathways on the plasma membrane . Ca2+ wae system . Americae system . Alpha7 e system . These rBased on the analysis method of network pharmacology, and combined with the prediction of active ingredients and targets, GO and KEGG enrichment analysis, molecular docking, animal experiments, etc., the molecular mechanism of FIB in the treatment of ischemic strokes were predicted and verified from a systematic perspective. FIB has a favorable therapeutic effect on Ischemic Strokes by influencing multiple targets and multiple related pathways. This also provides quite a few references for our subsequent exploration of the mechanism. In the future research, we will conduct more in-depth cell and animal experiments to further study the potential mechanism of FIB in the treatment of ischemic strokes, so as to ensure the reliability of the research results. In terms of clinical research, we will design a multi-center large-sample randomized controlled study to further observe the therapeutic effect and safety of FIB on Ischemic Strokes. In conclusion, FIB is expected to become a new drug for reducing cerebral infarction area, improving neurobehavioral capability, and anti-oxidative stress after ischemic stroke.