Clinical studies and basic science experiments have widely demonstrated the antidepressant and anxiolytic effects of the herbal formula Xiao-Yao-San (XYS). However, the system mechanism of these effects has not been fully characterized. The present study conducted a comprehensive network pharmacological analysis of XYS and sorted all pharmacologically active components (149) through the TCMSP webserver. Then, all potential molecular targets (449) were predicted, of which there were 99 genes clearly related to depression. To further investigate the mechanism of antidepressant effects of XYS, a compound-depression targets (C-DTs) network was constructed, and Gene Ontology (GO) functional and KEGG pathway enrichment analyses were performed for the 99 targets. Enrichment results revealed that XYS could regulate multiple aspects of depression through these targets, related to metabolism, neuroendocrine function, and neuroimmunity. Prediction and analysis of protein–protein interactions resulted in selection of three hub genes . In addition, a total of seven ingredients from XYS could act on these hub genes and they were identified through ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS), including paeoniflorin, quercetin, luteolin, acacetin, aloe-emodin, Glyasperin C, kaempferol. Hereafter, we investigated the effects of paeoniflorin and its predicted target, the results suggest that it can reverse the neurotoxicity produced by CORT and could be a neuroprotective effect by promoting the phosphorylation of Akt. Overall, our research revealed the complicated antidepressant mechanism of XYS, and also provided a rational strategy for revealing the complex composition and function of Chinese herbal formula. Depression is a psychological disorder with widespread prevalence, affecting more than 350 million individuals worldwide, and the prevalence is gradually increasing  Diels ; Paeonia lactiflora Pallas ; Bupleurum Chinense De Candolle ; Atractylodes macrocephala Koidzumi ; Poria cocos (Schweinitz) Wolf ; Mentha canadensis Linnaeus ; Glycyrrhiza uralensis Fischer ; Zingiber officinale Roscoe .Development of medicines that have antidepressant effects and produce less side effects than current antidepressants is a priority. Therefore, complementary and alternative medicine (CAM) and traditional Chinese medicine (TCM) have received attention as potential strategies for treatment and prevention of depression. Xiao-Yao-San (XYS) has been used in TCM clinics for more than a century to treat various diseases with characteristic features of liver stagnation and spleen deficiency syndrome (LSSDS) , which is a unique systems-level pharmacology server for TCM   and drug-likeness (DL) properties. Comprehensive analysis of bioavailability and structural descriptors for predicting OB values for each compound were previously determined P < 0.05 were determined by “limma” package in R studio software. Moreover, the connectivity map (cMap) database is a collection of genome-wide transcriptional expression data from cultured human cells treated with bioactive small molecules that uses simple pattern-matching algorithms allowing for elucidation of functional connections between drugs, genes, and diseases through transitory common gene expression changes  is a unique online database used as a bioinformatics and cheminformatics resource that compiles extensive drug-related information  for physicochemical-related parameters  though the prcomp function in R studio software. Therefore, known antidepressant drug/drug-like compounds from cMap, DrugBank, and active ingredients from XYS were also analyzed by PCA. Furthermore, the correlation of ADME properties of these compounds was also analyzed. Pearson r coefficients were calculated to evaluate the correlation between the three groups.http://www.uniprot.org)  were based on the pharmacophore model, and this server is a large, in-house collection of pharmacophore databases extracted from all targets in TargetBank, DrugBank, BindingDB, and PDTD  and depression-related targets, depression-related genes were retrieved from the OMIM database  KEGG pathway analysis: To explore potential biological mechanisms of predicted targets, the “clusterProfiler” R package was used to annotate Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of targets genes potentially regulated by active compounds contained in XYS. In addition, significantly enriched KEGG pathways should contain at least three genes, and have P < 0.05.To analyse targets groups of active compounds in XYS, we used enrichment analysis, as this method can effectively increase the reliability of the identification of biological phenomena, resulting in meaningful annotation information. (i) GO annotation: In this study, “clusterProfiler” package in R studio was used for GO (Gene Ontology) enrichment analysis. “ClusterProfiler” is based on multiple resources, and also serves as a user-friendly enrichment tool with integrated gene cluster analysis  were provided by Jiuzhitang Co., Ltd.  at 37°C. NGF  was used for PC12 differentiation.Differentiated PC12 cells were employed in the experiments. RPIM 1640 basic medium supplemented with 5% fetal bovine serum , 10% Horse serum , and 1% antibiotic mixture comprising penicillin and streptomycin . Cells were under the 5% CO3/100 μl, then incubated the cells under the conventional culture condition overnight. After 24 h treatment that with the mixture of 200μM CORT plus different concentrations of paeoniflorin , 10 μl MTT (0.5 mg/ml) was added to each well, followed by 4 h of incubation at 37°C. Then carefully remove the supernatant, and 100 μl DMSO was added to each well to dissolve the formazan crystal. The absorbance was recorded at 570 nm by microtiter plate reader. Cell viability was calculated as a percentage of the untreated or vehicle-treated control and averaged from three independent experiments.The effect of paeoniflorin reverse neurotoxicity from corticosterone, cell viability was determined by the MTT assay . The protein lysates separated by 10% SDS-PAGE gel were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes . The following specific primary antibodies were used for incubate membranes overnight at 4°C: anti-GAPDH , anti-AKT , anti-Phospho-AKT . After probed with a 1:5000 dilution of horseradish peroxidase (HRP) conjugated secondary antibody , the labeled proteins were detected by using enhanced chemiluminescence (ECL) detection.Furthermore, AutoDock 4.0 was used to evaluate the hub genes with active compounds, which is widely used for the study of bound conformation and binding free energy of ligand to the protein . Several of the herbal medicines in XYS contain the same active ingredients, such as quercetin, a natural flavonoid, which is present in BR and in GRH. In addition, stigmasterol is present in AS, RB and ZRR. In total, 149 active compounds were identified in XYS, including 12 in PN, 2 in AS, 18 in BR, 15 in PR, 7 in AMR, 91 in GRH, 5 in ZRR, and 10 in MH. Detailed information regarding these molecules is summarized in To determine the molecular diversity of the constituents of each herbal medicine, the constituents were evaluated based on four significant physicochemical properties , Table 1P < 0.05 using “limma” in R studio, a total of 29 significantly differentially expressed genes between depressed and normal patients were identified. Fourteen genes were down-regulated, and 15 genes were up-regulated  were identified. Of the twelve compounds, 2 had high positive connection scores, and the other ten compounds possess negative connectivity scores, indicating they can act with these dysregulated genes in depression. r value is 0.3404 (P < 0.0001) for MW, AlogP , Hdon , Hacc . These results indicated that bioactive compounds from XYS are highly correlated with known antidepressant compounds in molecular properties, and could as potential drug therapies for depression treatment.PCA was performed to characterize the physicochemical parameters  of these antidepressant drugs/compounds. PC1 and PC2, as shown in in silico target screening approach was performed to identify the putative targets of each bioactive component of XYS. Predictive tools were used, including the TCMSP server, STITCH, and PharmMapper data servers. In total, 446 targets were identified for 149 bioactive compounds (9 compounds did not receive predictions), and many compounds were predicted to target the same proteins, such as Monoamine Oxidase B (gene symbol: MAOB). Depression-related putative targets were obtained from Therapeutic Target, DrugBank, OMIM, and DisGeNET databases, and DEGs resulting from analysis of GSE12654. Finally, 121 bioactive compounds in XYS which associated with 99 depression-related targets were identified. Multiple therapeutic targets were mediated by active ingredients of XYS, such as IL2, IL4, IL6, IL10, and STAT3, which are involved in immune and inflammatory responses closely associated with depression. For example, STAT3 , a component of the JAK/STAT signaling pathway, influences the fate and function of brain cells.An A C-DT network was constructed  for furtTo investigate biological functions associated with this interaction network relative to treatment of depression, we performed GO and KEGG pathway enrichment analyses using the “clusterprofile” R package. Three GO categories were analyzed, which included CC, MF, and BP. The top 15 GO enrichment results for each category are listed in KEGG signaling pathway results are shown in To investigate the characteristics of protein targets in the C-DT network, protein–protein interactions were analyzed using the STRING online database, and the related PPI network was constructed and visualized using cytoscape software. Using confidence of 0.9 as the threshold, we found 90 nodes and 288 interactions in the PPI network. In addition, we used four different analysis methods  using the “cytoHubba” plugin to identify the top 10 hub nodes in the PPI network. Detailed results are shown in Paeonia lactiflora Pallas . Western blot analysis results showed that compared with the control cells, the expression of p-Akt in CORT stimulated cells was significantly decreased (P < 0.01). However, the down-regulated expression of p-Akt was significantly reversed by paeoniflorin treatment  is being more frequently chosen by patients to treat depressive and anxiety disorders. A meta-analysis reported that use of XYS as an adjuvant therapy with antidepressant drugs in 1,837 patients was more effective in improving symptoms than antidepressants alone  identified paeoniflorin, liquiritin, glycyrrhizic acid, ferulic acid, saikosaponins A and C, curcumin, and poria cocos alcohol were eight active compounds identified in XYS  network, and further enriched this network using GO KEGG pathways analyses of 99 depression-related targets. CC enrichment results indicated that a considerable number of genes were involved in formation of nerves and synapses. Synapses are the fundamental structures for information transmission between neurons, and they adapt to stimuli by continuously modifying neural connections and neurological circuits. Therefore, synaptic plasticity is the main manifestation of neuroplasticity, and depression and other psychological disorders are typically associated with decreased synaptic plasticity in the hippocampus  from XYS. And it is one of the quality identification standards for XYS in the Chinese Pharmacopeia. Thus, it should be a representative compound from this formula, so that the current research further analyzed its neuroprotective effect and validated its potential roles with predicted target Akt. Previous reports by several researchers had provided clues supporting the role for paeoniflorin in the protection of neuron cells and the therapeutic effects in neurological diseases (Mao et al., However, among these 7 ingredients, only paeoniflorin, a monoterpene glycoside compound, as main component of herb (in vitro, the pathogenesis of depression is complicated, which means that it is difficult to simulate whole pathogenic factors in vitro. Moreover, western blots suggested that paeoniflorin could regulate the phosphorylation of Akt, but it is only in protein expression level. The direct or indirect regulation between them would need to be further explored. Last but not least, hub genes were selected by PPI network in our study that they are usually as upstream molecules or central molecules in those important signaling pathways. However, there still exist multiple downstream proteins in pathogenesis of depression. And thus, it is important to discover and identify more compounds that interact with these downstream proteins through future works.Current research based on network pharmacology which could provide a novel and systematic analysis way for the research of Chinese herbal formula. However, there are still some limitations in our article. Firstly, as we all know, components of Chinese herbal are complicated, it's doubtful whether there are synergistic or side effects caused by the interaction between compounds that is hard to clarified in the prediction and composition identification of this study. Secondly, although CORT is one of the most common methods to simulate depression in vitro. The current findings provide a basis and new insights for XYS in the treatment of depression. Certainly, we will explore widely experiments of pharmaceutical ingredients to better understand the effects of XYS, such as component analyses, identification of drugs, and metabolic studies in animal models.Overall, this study systematically summarized key active compounds in XYS and comprehensively analyzed potential mechanisms of its action and pathways. We also evaluated for representative compound paeoniflorin https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12654.Publicly available datasets were analyzed in this study. This data can be found here: NY and JC main contributed research conception and design. NY, KT, LH, WH, XL, and QM collected and processed data. NY, LG, and KT wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the final submitted version. JC takes primary responsibility for communication with the journal and editorial office during the submission process, throughout peer review, and during publication.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.


Polygonum cuspidatum)–Shanzha (the fruit of Crataegus sp.), a classic herb pair, has been widely used for the treatment of CHD. In recent years, Huzhang–Shanzha herb pair (HSHP) was found to have a wide range of effects in CHD; however, its therapeutic specific mechanisms remain to be further explored. The aim of this study was to elucidate the molecular mechanism of HSHP in the treatment of CHD using a network pharmacology analysis approach.  Coronary heart disease (CHD) seriously affects human health, and its pathogenesis is closely related to atherosclerosis. The Huzhang  network was constructed using the STRING web platform and Cytoscape software. GO functional and KEGG pathway enrichment analyses were carried out on the Metascape web platform. Finally, molecular docking of the active components was assessed to verify the potential targets of HSHP to treat CHD by the AutoDock Vina and PyMOL software.  Totally, 243 active components and 2459 corresponding targets of LDP were screened out. Eighty-five common targets of HSHP and CHD were identified. The results of the network analysis showed that resveratrol, anthranone, emodin, and ursolic acid could be defined as four therapeutic components. TNF, ESR1, NFКB1, PPARG, INS, TP53, NFКBIA, AR, PIK3R1, PIK3CA, PTGS2, and NR3C1 might be the 12 key targets. These targets were mainly involved in the regulation of biological processes, such as inflammatory responses and lipid metabolism. Enrichment analysis showed that the identified genes were mainly involved in fluid shear force, insulin resistance (IR), inflammation, and lipid metabolism pathways to contribute to CHD. This suggests that resveratrol, anthranone, emodin, and ursolic acid from HSHP can be the main therapeutic components of atherosclerosis.  Using network pharmacology, we provide new clues on the potential mechanism of action of HSHP in the treatment of CHD, which may be closely related to the fluid shear force, lipid metabolism, and inflammatory response. Cardiovascular diseases, which seriously affect the quality of life of patients and bring a huge burden to the society, are the main cause of death worldwide –3. StudiPolygonum cuspidatum with detoxification effect and Hawthorn with activating blood effect can relieve the degree of angina pectoris to a certain extent. The cardiovascular activity and clinical significance of HSHP extracts have been extensively studied. Crataegus sp., commonly known as hawthorn, belongs to the family Rosaceae. Studies have showed that the main components of hawthorn extract, such as flavonoids, polyphenols, and oligo-procyanidins, have antiatherosclerotic effects [P. cuspidatum is often used in TCM to clear dampness and heat, and detoxifying, promoting blood circulation, and removing blood stasis. Moreover, P. cuspidatum is also used for its anti-inflammatory effects. In particular, resveratrol, which is main P. cuspidatum in component, was shown to play an important role in improving vasodilation and prevent thrombosis and atherosclerosis onset [The underlying mechanisms induced by the traditional Chinese medicine (TCM) are complex, and its components and targets are often multiple . Clinica effects . Howeveris onset . Our preis onset . At the is onset . HoweverIn addition, due to the diversity of TCM components and the complexity of their interactions with the human body, traditional single-agent or monomer studies cannot fully explain the specific therapeutic effects of TCM , 12. TheThis study uses network pharmacology and bioinformatics analysis to predict the candidate compounds and mechanisms of HSHP in the treatment of CHD. In addition, we also combined with literature analysis to clarify the relationship between the herbal active components and diseases, thereby identifying a new strategy for the prevention and treatment of CHD. The specific network pharmacology and bioinformatics approach employed in the study are shown in https://bionet.ncpsb.org/batman-tcm/) was specifically designed for the study of TCM molecular mechanisms, allowing to predict the TCM component targets and the complete component-target-pathway association network. The chemical constituents of P. cuspate and hawthorn were explored using the BATMAN-TCM database, as detailed in Supplementary P-value of 0.05 (as shown in Supplementary BATMAN-TCM database (https://www.un.org). GeneCard (https://www.genecards.org/), OMIM (https://www.omim.org/), PharmGkb (https://www.pharmgkb.org/), Therapeutic Targets Database (https://db.idrblab.net/ttd/), and DrugBank (https://www.drugbank.ca) were used to search CHD-related genes. Venny v2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) online tool was used to map the drug-diseases overlap genes. The common targets of HSHP and CHD could represent the potential therapeutic targets. Finally, Cytoscape software (Version 3.7.1) was used to map the disease network of drug components.UniProt identifiers of HSHP were obtained from the UniProt database (https://string-db.org/cgi/input.pl) was used to explore the PPI. The “organism” option was set to “Homo sapiens,” and PPI with a composite score greater than 0.5 was selected as the core gene.The “Herbal-compound-target” network was constructed using the network visualization software Cytoscape (Version 3.7.1). The network diagram of proteins and their interactome was also constructed. Topological parameters were used to screen the key active compounds and CHD-related targets of HSHP. The String database . The species parameter was set as “https://geneontology.org/), function annotation, and the Kyoto Encyclopedia of Genes and Genomes  pathway enrichment analysis were performed on the 85 common target genes of the identified drug-disease-target network using the R 3.6.1 software with the Bioconductor Package. The obtained target genes molecular function (MF), biological process (BP), and cellular component (CC) were analysed. P-value <0.05 was considered statistically significant. The top 30 items with the highest enrichment were selected for analysis. Lastly, the RGUI and pathview tools in the KEGG database were used to refine target list and identify the top 20 genes. The target pathway network was established through the connection between the target and KEGG pathway analysis data, and the pathway with a degree greater than or equal to the median, and related to CHD, was selected as the main pathway for further research.In order to further understand the underlying mechanisms of HSHP for the treatment of CHD, gene ontology  was used to find and download the drug molecular structure. The protein crystal structure of the CHD-related target was obtained from the RCSB Protein Data Bank database (https://www.rcsb.org/). The structure and chemical formula of the target and drug, respectively, were obtained from the RCSB Protein Data Bank structured and PubChem (https://pubchem.ncbi.nlm.nih.gov/). The AutoDock Tools 1.5.6 (https://autodock.scripps.edu) was used to perform hydrogenation and remove water molecules for protein processing and molecular docking and calculate the docking binding energy of each complex to evaluate the binding effect. PyMol 2.3.2 software (https://pymol.org/2/) was used to predict the protein molecular docking mechanism.The drug components of HSHP, and the targets of CHD, were obtained by analysing the drug-disease-target network. The chemical library database  by inhibiting the TLR4/NF-κB signalling pathway, thereby acting as an anti-inflammatory and antioxidant agent, protecting the vascular endothelium and preventing atherosclerosis [Active components of HSHP, and their corresponding disease targets, were acquired from the Batman-TCM database , OMIM (480 compounds), PharmGkb (115 compounds), TTD (2 compounds), and DrugBank (35 compounds) . Among tWe introduced 85 common targets into the STRING online service platform, and the date and figure of a PPI network were acquired . CytoscaP-value <0.05 as the threshold, the top 30 GO biological function analysis results were screened out  and their corresponding active compounds was performed. Discovery Studio software was used to observe the compounds entering the protein active pocket, and their affinity  was used to identify the degree of ligand binding to the receptor protein and verify the therapeutic mechanism of hawthorn in the treatment of CHD  in elderly patients with CHD landmark [Herein, NF-ytokines . Jin et ytokines  showed tg, China . In addi pathway . TNF-α ilandmark . Studieslandmark , 35. Morlandmark , 37. TheIn order to further understand the mechanism of HSHP against atherosclerosis, the GO biological function analysis and the KEGG functional enrichment analysis was performed. The GO biological function analysis revealed that the BP mainly involved were the regulation of inflammatory response, fat and steroid metabolic processes, blood pressure, as well as hormone-mediated and intracellular receptor signalling pathways. MF mainly focused on lipid- and inflammation-related aspects, such as steroid receptor activity, steroid binding, and nuclear receptor activity. CC was mainly concentrated in the transcription regulator complex, RNA polymerase II transcription regulator complex, nuclear chromatin, and other aspects. The KEGG analysis results showed that the therapeutic approaches of HSHP against atherosclerosis were mainly related to hemodynamic, metabolic disorders, and inflammatory signalling pathways, including fluid shear force and IR, as well as AMPK, cAMP, and mTOR signalling pathways. These findings are in agreement with our previous studies, which demonstrated that cAMP, mTOR, and AMPK pathways play key roles in the therapeutic mechanism of atherosclerosis by regulating inflammation, lipid metabolism, cell proliferation, and apoptosis.−/− mice, resveratrol could play an antiatherosclerosis role. Emodin is an anthraquinone compound, which have been shown to have anti-inflammatory and cardiovascular protective effects [in vivo. Other studies have shown that aloe emodin can promote retinal neovascularization by inhibiting the HIF-1/VEGF signalling pathway, possibly playing a role in the treatment of diabetic retinopathy [The results showed that resveratrol, anthranone, emodin, and ursolic acid were the most probable therapeutic components identified by the composition-to-target network and subsequent molecular docking affinity analysis . Modern  effects –42. Nemm effects  found th effects  found thinopathy . Ursolicinopathy . Studiesinopathy . TherefoκB, ESR1, and TNF signalling pathways and downregulate the expression of inflammatory factors IL-6, mainly by reducing serum lipid levels such as TG, TC, ApoB100, and Lp(a) [κB, ESR1, and TNF. TNF is an important proinflammatory factor that mediates inflammatory and immune responses. It can regulate lipid metabolism and stimulate the expression of adhesion molecules in itself, and IL-1 and IL-6 cells play a pro-inflammatory effect and accelerate the formation of atherosclerosis [α expression and lipid peroxidation levels [κB-p65 pathway and exerts an anti-inflammatory effect by blocking the expression of the main inflammatory cytokines TNF-α, IL-6, and IL-1β [α, MCP-1, IL-6, S100A8, and S100A9 triggered inflammation [Based on the previous related research of our team and other literature, we further studied the prediction results of network pharmacology. Our previous research results showed that HSHP may inhibit the activation of NF-clerosis . Some stclerosis . Resveran levels . In a mind IL-1β . In addiammation . In thisκB in straight arteries under normal endothelial shear stress would play an important biological role in improving eNOS expression, as well as the expression of its target genes, such as MCP-1, ICAM-1, and VCAM-1, which will in turn promote white blood cell aggregation and the inflammatory response.However, contrasting with previously reported data, these results also suggested that shear force and IR could be involved in the antiatherosclerotic mechanisms induced by HSHP. The turbulence and low shear stress generated at the bifurcation, branching exit, and bending of blood vessels may promote cell proliferation and apoptosis, promoting the inflammatory response, lipid uptake, and synthesis by the endothelium, as well as the subcutaneous aggregation of monocytes and lipids, thereby increasing the risk of plaque and thrombus formation . In addiɑ [ɑ and IL-6 can affect the insulin signal transduction and increase the release of free fatty acids (FFA), activates the MAPK pathway, endothelial DAG and PKC signal pathways, and intimal smooth muscle cells from middle to vascular endothelial migration and activation, as well as the synthesis and secretion of extracellular matrix proteins and fibrinolytic enzyme activator inhibitor 1 that will promote thrombosis [IR is the main line and central link of various metabolic abnormalities and CHD  and is cɑ . In turnrombosis .κB, TNF, and ESR1. Therefore, it is possible that HSHP plays an important role in the treatment of atherosclerosis, and their therapeutic mechanism is mainly mediated by the modulation of hub targets involved in blood flow shear force, IR, inflammation, and metabolism-related signalling pathways. However, the study also has some limitations, including the investigation of biologically active ingredients and further experimental verification to verify the impact of HSHP on CHD. Therefore, further research is needed to demonstrate HSHP potential therapeutic mechanisms.This study predicted the targets, mechanism, and related signal pathways of HSHP in the treatment of CHD through network pharmacology, verified the binding abilities of herb active ingredients and their targets. Through molecular docking, this study found that the docking effect of the common compounds of HSHP, including resveratrol, emodin, and ursolic acid, was similar to that of simvastatin. And these active components bind well with the key targets NF-


Alzheimer's disease (AD) and Huntington's disease (HD) are destructive worldwide diseases. Efforts have been made to elucidate the process of these two diseases, yet the pathogenesis remains elusive as it involves a combination of multiple factors, including genetic and environmental ones. To explore the potential role of forkhead box O1 (FOXO1) in the development of AD and HD, we identified 1,853 differentially expressed genes (DEGs) from 19,414 background genes in both the AD&HD/control and FOXO1-low/high groups. Four coexpression modules were predicted by the weighted gene coexpression network analysis (WGCNA), among which blue and turquoise modules had the strongest correlation with AD&HD and high expression of FOXO1. Functional enrichment analysis showed that DEGs in these modules were enriched in phagosome, cytokine-cytokine receptor interaction, cellular senescence, FOXO signaling pathway, pathways of neurodegeneration, GABAergic synapse, and AGE-RAGE signaling pathway in diabetic complications. Furthermore, the cross-talking pathways of FOXO1 in AD and HD were jointly determined in a global regulatory network, such as the FOXO signaling pathway, cellular senescence, and AGE-RAGE signaling pathway in diabetic complications. Based on the performance evaluation of the area under the curve of 85.6%, FOXO1 could accurately predict the onset of AD and HD. We then identified the cross-talking pathways of FOXO1 in AD and HD, respectively. More specifically, FOXO1 was involved in the FOXO signaling pathway and cellular senescence in AD; correspondingly, FOXO1 participated in insulin resistance, insulin, and the FOXO signaling pathways in HD. Next, we use GSEA to validate the biological processes in AD&HD and FOXO1 expression. In GSEA analysis, regulation of protein maturation and regulation of protein processing were both enriched in the AD&HD and FOXO1-high groups, suggesting that FOXO1 may have implications in onset and progression of these two diseases through protein synthesis. Consequently, a high expression of FOXO1 is a potential pathogenic factor in both AD and HD involving mechanisms of the FOXO signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and cellular senescence. Our findings provide a comprehensive perspective on the molecular function of FOXO1 in the pathogenesis of AD and HD. The funnormalizeBetweenArrays function in the limma package of R software (version 3.6.2) was used to normalize the gene expression profile [We downloaded the RNA gene expression profiles of GSE33000 from the Gene Expression Omnibus (GEO) database, which included 467 patients with neurodegenerative diseases (310 AD and 157 HD) and 157 nondementia controls . We then profile . Based o profile , 24. ThelmFit and eBayes functions of limma package in R software. The fold changes (FC) in the expression of individual genes were calculated, with ∣logFC | >0.15 and false discovery rate- (FDR-) adjusted P < 0.05 considered thresholds [DEGs were generated in the AD&HD/control and FOXO1-high/low groups using the resholds .WGCNA package in R software, the gene coexpression network analysis was constructed by clustering overlapping DEGs between the AD&HD/control and FOXO1-high/low groups into multiple functional modules. Weight gene correlation network analysis (WGCNA), an advanced data exploration technique, not only allows the construction of interconnected node modules that represent network-based data volumes and alleviate the problem of multiple testing but also screens for modules that are relevant to clinical traits [hclust function was used to eliminate outliers from the samples. A pickSoftThreshold function was used to determine the appropriate power value of 16 when the degree of independence was set to 0.8. To ensure high reliability of the results, each module contained at least 30 genes. From thousands of genes, interesting modules of DEGs were identified by WGCNA, and then, the Kyoto Encyclopedia of Genes Genomes (KEGG) pathway analysis was performed on the genes in each module using the ClusterProfiler package in R software. FDR adjusted P < 0.05 was used as the threshold to define the significant differences in biological functions and enrichment pathways enriched in each module.Using the l traits . The hclverboseScatterplot function to represent the correlation between intramodular connectivity and clinical trait. In modules related to the trait of interest, genes with higher module membership tend to have higher genetic significance and biological importance [http://STRING-db.org) online database [Correlations between intramodular connectivity and clinical phenotype were used to estimate module-pathway associations, so that highly phenotypically related expression modules could be readily identified. Gene significance (GS) was calculated as the absolute value of the correlation between the gene expression profile and each trait; module membership (MM) was defined as the correlation between the gene expression profiles in each module. Scatterplot of GS vs. MM in each module was plotted using the portance . The glodatabase , in whicdatabase .pROC package, AUC analysis was performed to predict the diagnostic performance of FOXO1 in differentiation between AD&HD and controls. Bilateral P value of less than 0.05 was considered statistically significant.Adopting the P value less than 0.05) applying default weighting statistic for each parameter. Enriched data in GSEA analysis was visualized using packages of ClusterProfler, ggplot2, enrichplot, and GSEABase.According to the median expression of FOXO1, samples were divided into the FOXO1-high and low expression groups. After normalization of the gene expression profile, GSEA was performed to screen for the biological process of Gene Ontology (GO) terms in the AD&HD and FOXO1-high groups . The thrTo investigate the functions of FOXO1 in the pathogenesis of AD and HD, we conducted systematic analysis following the steps in P < 0.0001). After removal of unannotated or duplicated genes, 19,414 background genes were summarized for further differential expression analysis. A total of 2,103 genes were differentially expressed in AD&HD compared with nondementia controls. Among them, 1,001 DEGs were found to be significantly upregulated, while 1,102 were downregulated . egulated . In subjegulated . Of thesP = 5e − 49) and FOXO1 ; the turquoise module of 1,151 DEGs had the strongest negative correlation with AD&HD  and FOXO1 , while the brown module of 127 DEGs was positively correlated with AD&HD  and FOXO1 ; and for the grey module, 35 noncoexpressed DEGs were clustered. These data suggest that DEGs in blue and turquoise modules had the strongest correlation with AD and HD.The set of DEGs was used for hierarchical clustering analysis and module-trait heatmap plotting. All samples passed the predefined cut-off line (height = 16) for the next step of bioinformatic analysis . Using Wβ signaling pathway, extracellular matrix (ECM)-receptor interaction, and advanced glycation end product- (AGE-) receptors for AGEs (RAGE) signaling pathway in diabetic complications; for turquoise module, DEGs were involved in pathway of neurodegeneration, GABAergic synapse, FOXO signaling pathway, cellular senescence, and AGE -RAGE signaling pathway in diabetic complications  ications .P = 1.2e − 33; turquoise: correlation coefficient = 0.73, P = 3.8e − 192)  . We extre − 192) . As showe − 192) . Separate − 192) ; and thee − 192) .GSEA was adopted to validate the biological processes in the AD&HD and FOXO1-high groups. There were five biological processes significantly enriched in AD&HD, including B cell-mediated immunity, regulation of protein maturation, regulation of protein processing, immunoglobulin-mediated immune response, and T cell activation involved in immune response . Five biα, IL-1β, IL-6, and IL-8 [The GSEA results showed that the background genes in both AD&HD/control and FOXO1-high/low expression cohorts were enriched in biological processes of protein maturation and processing regulation. The newly synthesized proteins are processed in the endoplasmic reticulum (ER) to form mature proteins with physiological functions. In the presence of cellular crowding, gene mutations, oxidative stress, etc., protein processing in the ER is compromised, resulting in the formation of misfolded proteins. Subsequently, the accumulation of misfolded proteins leads to a state of ER stress to degrade these misfolded proteins. FOXO1 protein participates in such processes by modulating autophagy of misfolded proteins or apoptosis of impaired cells –33, consand IL-8 . These pand IL-8 . Indeed,and IL-8 , along wand IL-8 . The mutand IL-8 –40. Moreand IL-8 . In addiand IL-8 , 43. It and IL-8 . TherefoFOXO factors lie in the center of a complex regulatory network of multiple upstream pathways and downstream target genes, receiving upstream signals simultaneously or sequentially to regulate transcriptional activity of downstream target genes in normal or pathological cells. The PI3K/Akt/FOXO pathway is one of the major FOXO pathways that regulates the activation and localization of FOXO1. Knockdown of the upstream insulin receptor substrate (IRS) of this pathway leads to hyperactivation of FOXO1 . Subsequβ induces excessive mitochondrial production of ROS; conversely, sustained activation of the JNK pathway induces increased expression of β-secretase and γ-secretase under conditions of oxidative stress, which in turn promotes Aβ production [ROS are mainly produced by and act on mitochondria to regulate cell growth and differentiation at low concentrations . JNKs beoduction . In HD poduction , 59, uproduction , 61.β and mHTT, leading to a sustained release of neuroinflammatory factors for inflammatory damage and cell death [Following the process of apoptosis, the removal of apoptotic and senescent cells by phagocytosis contributes to the emergence of phagosomes, which are vesicles formed by the fusion of the cell membrane of the phagocyte around the granule. Fusion with lysosomes results in maturation of phagosome, which not only contains hydrolases and ROS to digest debris but also forms proinflammatory factors via activation of MAPK signaling and PI3K/Akt pathways . Microglll death . Neuroinll death . Similarll death , consistAutophagy is another essential mechanism for eliminating organelles in response to stress or starvation. It is involved in the phosphorylation of the downstream target FOXO1 in dynamic equilibrium through synergistic, inhibitory, or cross-talking action of the AMPK, PI3K/Akt, and JNK pathways . Recent Oxidative stimulation acts on cellular components through different signaling pathways or exerts varying effects via the same pathways, determining diverse or even completely opposite cell fates. The precise mechanism is not fully understood, and the results of related experiments are variable and controversial. Salih and Brunet reported that apoptosis was the preferred cellular mechanism of FOXO1-activated neurons exposed to oxidative stress . Neverthin vivo HD model has also confirmed that SIRT1 activation provides a positive complement to mitochondrial failure, improving motor coordination and learning in HD [Cellular senescence participates in physiological processes including wound healing, tissue repair, and embryonic development, which is also a protective mechanism against tumor propagation triggered by oxidative stress-induced DNA damage or oncogenic signals. Moreover, it has also been observed that increased cellular senescence may contribute to senescence-associated diseases including ND . Senesceng in HD .The glycosylate modification of proteins by sugars and aldehydes in an oxygenated environment can produce AGEs . AGEs biCumulating evidence suggested that GABAergic synaptic dysfunction aggravated cognitive impairment in patients with dementia –90. Althβ signaling pathway plays an important role in maintaining cellular homeostasis through apoptosis, autophagy, and cellular senescence in a variety of cells [β/Smad signaling pathway, thus hampering neuronal growth and participating in the pathogenesis of AD [β/Smad pathway with the PI3K/Akt/FOXO1 pathway [β signaling pathway has implications in the pathology of neurodegeneration by interacting with the FOXO signaling pathway, which is consistent with the results of our functional enrichment analysis in the brown module.The multifunctional TGF-of cells –95. As ais of AD , 97. Alt pathway . Likewisβ deposition [ECM is a highly dynamic, continuously remodeling tissue with a specific structure . Expressposition . In addiposition .β involving the synthesis of ECM proteins [β1 [The onset and progression of AD or HD are the outcomes of the interaction of multiple mechanistic pathways. The cells are always in homeostasis by means of coordination, cross-regulation, and even mutual restraint among various pathways . For insproteins . Furtherproteins . Additioeins [β1 , 105.In the scatter plot between MM and GS, the strongest correlation between DEGs and FOXO1 expression was found in blue and turquoise modules. According to the cross-talking pathways of FOXO1 identified in the global regulatory network of DEGs, FOXO1 plays pleiotropic roles in the physiopathology of AD and HD via cellular senescence, FOXO signaling pathway, and AGE-RAGE signaling pathway in diabetic complications. The result of AUC analysis showed a good diagnostic performance in differentiating AD and HD patients from nondementia controls, which indicated that FOXO1 was possibly a predictive factor for the incidence of AD and HD. Collectively, the findings emerging from this study provide novel possible directions for experiments focusing on the timing and conditions whereby cells enter the senescence or apoptosis program and whether cerebrovascular endothelial integrity influences the progression of AD and HD through the interaction of FOXO1 and AGE-RAGE signaling pathways.In summary, our findings support that the high expression of FOXO1 is responsible for the pathogenesis of AD and HD, possibly mediated by FOXO signaling pathway, cellular senescence, and the AGE-RAGE signaling pathway in diabetic complications.


Huangqi Sijunzi decoction (HQSJZD) is a commonly used conventional Chinese herbal medicine prescription for invigorating Qi, tonifying Yang, and removing dampness. Modern pharmacology and clinical applications of HQSJZD have shown that it has a certain curative effect on Alzheimer's disease (AD).  The active components and targets of HQSJZD were searched in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The genes corresponding to the targets were retrieved using UniProt and GeneCard database. The herb-compound-target network and protein-protein interaction (PPI) network were constructed by Cytoscape. The core targets of HQSJZD were analysed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The main active compounds of HQSJZD were docked with acetylcholinesterase (AChE). In vitro experiments were conducted to detect the inhibitory and neuroprotective effects of AChE.  Compound-target network mainly contained 132 compounds and 255 corresponding targets. The main compounds contained quercetin, kaempferol, formononetin, isorhamnetin, hederagenin, and calycosin. Key targets contained AChE, PTGS2, PPARG, IL-1B, GSK3B, etc. There were 1708 GO items in GO enrichment analysis and 310 signalling pathways in KEGG, mainly including the cAMP signalling pathway, the vascular endothelial growth factor (VEGF) signalling pathway, serotonergic synapses, the calcium signalling pathway, type II diabetes mellitus, arginine and proline metabolism, and the longevity regulating pathway. Molecular docking showed that hederagenin and formononetin were the top 2 compounds of HQSJZD, which had a high affinity with AChE. And formononetin has a good neuroprotective effect, which can improve the oxidative damage of nerve cells.  HQSJZD was found to have the potential to treat AD by targeting multiple AD-related targets. Formononetin and hederagenin in HQSJZD may regulate multiple signalling pathways through AChE, which might play a therapeutic role in AD. Amylnt of AD . At presnt of AD . It is eHedysarum Multijugum Maxim , Panax Ginseng C. A. Mey , Atractylodes Macrocephala Koidz , Poria Cocos S. Wolf , and Licorice . As the major component of HQSJZD, HQ plays a very effective role in the treatment of many diseases. HQ has an optimal therapeutic effect on cholinergic substances, the brain, immune regulation, and cancer when combined with different TCMs [licorice.” GC being one of the most important Chinese herbal medicines in TCM and can reduce toxicity and improve the efficacy of some Chinese herbal medicines in some prescriptions [An increasing number of studies have proven that traditional Chinese medicine (TCM) plays a very important role in the treatment of complex diseases , 6. Huanent TCMs –9. RS alent TCMs , 11. FL ent TCMs , 13. Accriptions , 15. TheNetwork pharmacology is a cutting-edge methodology for discovering new therapeutic drugs and understanding the therapeutic mechanisms of complex diseases . In receTherefore, TCM network pharmacology establishes a link between the molecular targets of networks and disease patterns. We used network pharmacology to identify the effective natural components and key targets of HQSJZD, and then, molecular docking analysis was performed to verify the predicted molecular interactions between the compounds and their targets. Our study determined the relationship among the TCM, ingredients, and targets of HQSJZD and drugs, which is of great significance for discovering the potential mechanism of HQSJZD in the treatment of AD and for future pharmacological research on and clinical applications of HQSJZD. This study provides a new reference basis for HQSJZD.https://tcmspw.com/index.php), which is a systematic pharmacology database and analysis platform for TCM. According to the requirement of their high utilisation in TCM, eligible compounds were selected from the above active components [https://db.idrblab.org/ttd/), which can be queried and downloaded. Moreover, TTD also provides pharmacokinetic information for each compound, and compounds with good drug-like and ADME  characteristics can be selected for further research. To identify drugs with good absorption and chemical suitability, two ADME-related parameters, oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18, were employed to screen potential bioactive ingredients using TCMSP in this study [Using the Chinese pinyin “Huangqi, Renshen, Baizhu, Fuling, and Gancao” as keywords, data were collected from the Traditional Chinese Medicine System Pharmacology Database , DrugBank (http://www.drugbank.ca), and TTD were searched and screened for AD-related targets. Then, the corresponding gene names were found in the UniProt knowledge base (UniProtKB) (https://www.uniprot.org/) by searching for “Homo sapiens” [With “Alzheimer's disease” as the keyword, GeneCard  network were analyzed by using Cytoscape 3.6.1. “Nodes” were used to represent target protein molecules, “edges” were used to represent mutual relationships, and the intermediary center of the PPI network was calculated according to betweenness centrality, closeness centrality, and degree, so that the interaction between each component and the target could be displayed .https://string-db.org/) for further predictions.Using Venn software, the screened targets corresponding to the active ingredients of HQSJZD were crossed with the disease targets corresponding to AD targets, and common targets were obtained through a Venn diagram. Then, the prediction targets were imported into the STRING database (https://metascape.org/) is an online biological knowledge base and an analytic tool used to extract biological information regarding gene functional classification, functional annotation, and enriched pathways [P < 0.05 and KEGG pathways with P < 0.05 were considered being significant [The Metascape database (pathways . The genpathways –28. GO tnificant .http://www.pdb) or the sites of the original ligands of proteins, crystal structures were obtained [Homo sapiens.” Three-dimensional conformers of the candidate compounds were gained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) in MOL format [X, Y, and Z coordinates were −6.141, −43.015, and 26.199 [Molecular docking was performed to determine the binding affinity of the compounds of HQSJZD with AChE. Through a tool used to edit binding sites and search for binding sites that involve receptors and ligands based on information from the RCSB protein database (PDB) . Experiments were in accordance with guidelines for animal care and were approved by the Institutional Animal Care and Utilization Committee of Xinxiang Medical University.AChE activity was assessed by the improved Ellman's method . Donepez2 at 37°C. SH-SY5Y cells were plated at a density of 4,000 cells per well in 96-well plates and cultured for 16 h. To determine the neuroprotective effect of active constituents on SH-SY5Y cells, cells were pretreated with 0.5, 1.0, 1.5, and 2 μM formononetin in HQSJZD for 3 h and then treated with sodium nitroprusside (500 mM) or potassium chloride (80 mM) for 20 h, respectively.Human neuroblastoma SH-SY5Y cells were maintained in a medium consisting of high glucose DMEM supplemented with 10% fetal bovine serum (FBS) in humidified 5% COμL of CCK-8 reagent  was subsequently added to each well for 3 h incubation at 37°C. Finally, cell viability was determined by reading the optical density (OD) at a wavelength of 450 nm using an enzyme-labelled instrument .Cell viability was assessed by cell counting kit-8 (CCK-8) assay according to the manufacturer's instructions. In short, after treatment of sodium nitroprusside , or potassium chloride , 10 P value <0.05 was considered significant.All data are presented as mean ± SEM. Statistical analysis was carried out using the GraphPad Prism version 7.0 software, and the significance of each group was verified with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post hoc test. A A total of 132 active components were isolated and identified in HQSJZD using the TCMSP database . AccordiA total of 255 compound-related targets were identified from the databases after eliminating duplicates. There were 210 targets in HQ, 115 in RS, 18 in BZ, 24 in FL, and 230 in GC, and the composition of jaranol in HQ was used as an example and is presented in To clarify the relationship among the TCM, active ingredients, and potential targets of HQSJZD, the target network of HQSJZD was constructed, as shown in n from the diameter of the connected component plus 1. The radiality of each node is divided by the diameter of the connected component. Thus, it is a number between 0 and 1. Topological Coefficient attribute stores the topological coefficient of n. Nodes with less than 2 neighbors have a topological coefficient of zero [The preliminary screening of 125 targets and AD-related targets intersected, and 60 predicted key targets were got by Venn software in . We cons of zero . TopologP < 0.05), including 1510 items for BP, 73 items for CC, and 125 items for MF. GO analysis showed that most of the potential targets were found in CC, including membrane rafts, postsynaptic complexes, cytoplasmic complexes, the organelle outer membrane, and ion channel complexes, and had good binding properties with neural receptors, enzymes, and peptides. Specifically, in BP, most of the target proteins had rich functions in toxic reactions, drug reactions, synaptic signals, neurotransmitter transmission, neuronal death regulation, blood circulation, proliferation and differentiation regulation, and exogenous apoptosis signalling processes. MF was found to interact with transcription factors, as well as a specific protein, and showed good antioxidant activity. These results indicate HQSJZD has multiple synergistic effects in biological processes  were affected by HQSJZD. The top 10 enriched pathways included pathways involved in cancer, colorectal cancer, amyotrophic lateral sclerosis (ALS), cAMP signalling, vascular endothelial growth factor (VEGF) signalling, serotonergic synapses, calcium signalling, legionellosis, amoebiasis, and retrograde endocannabinoid signalling  in the PPI map generated from the STRING database , and AChE can increase cholinergic defects or aggravate Aβ fibril formation and toxicity [β oxidation pathway of fatty acids, which can improve the clearance of toxic molecules, such as Aβ, by the brain [β plays a key role in the pathophysiology of AD and can significantly decrease the ability of MAPT/Tau to bind and stabilise microtubules [A total of 255 targets and 16 predicted key targets were obtained by assessing the target proteins of HQSJZD in various bioinformatics databases and the generated composite target network. APP and AChE are principally neuronal membrane-bound proteins. APP is the precursor of Atoxicity . We fountoxicity , 42. PTGtoxicity , 44. PPAhe brain . GSK3-β otubules . The choβ25-35 by participating in the PI3K/Akt signaling pathway, increasing the activity of α-secretase and the release of sAPPα, accelerating the non-amyloidosis process of amyloid precursor protein (APP), and reducing the production of Aβ [β-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. The results of an in vitro Aβ aggregation assay suggested that IRA destabilizes Aβ fibrils [Salvia miltiorrhiza, has been proven to inhibit AChE and butyrate cholinesterase (BChE) in vitro [Most clinical cholinesterase inhibitors have achieved some promising results in the treatment of AD patients . In our on of Aβ . Isorhamon of Aβ . In addi fibrils . Flavono fibrils . Hederagin vitro . It has in vitro . TherefoIn order to clarify the relevant signal channel of HQSJZD on AD, we first performed GO enrichment analysis, only kept the first 20 significant enrichments, and found that HQSJZD treatment of AD involves multiple signaling pathway processes, including antioxidative stress process, neurotransmitter signal transmission process, cell proliferation and differentiation, and other processes that synergistically act. In the KEGG pathway database, someone significantly enriched them in several pathways associated with AD such as calcium signalling pathway, Wnt signalling pathway, and type II diabetes mellitus. The dysregulation in cellular calcium signalling pathway homeostasis is the principal driving force of neurodegeneration in AD . Wnt sigMolecular docking can reveal interactions between components and their targets in a network by their evaluating binding energy, improving the accuracy of the network . Figure To evaluate the neuroprotective effects of formononetin during the pathology progression of AD, two cell damaged models were established. As the NO donor, Sodium nitroprusside (SNP) causes neural damage. NO regulates the release of proinflammatory molecules, interacts with ROS leading to the formation of reactive nitrogen species (RNS), and targets vital organelles such as mitochondria, ultimately causing cellular death, a hallmark of AD . PotassiIn summary, multiple compounds in HQSJZD are linked to the potential treatment of AD. Among these compounds in HQSJZD, formononetin and hederagenin may be potential lead compounds in the treatment of AD. For the first time, we used virtual screening and network pharmacology to clarify the material basis of HQSJZD in the treatment of AD. This study is expected to broaden the choice of treatment methods for AD and further demonstrate the feasibility of applying network pharmacology to the analysis of TCM prescriptions.Our study identified the multi-ingredient and multitarget mechanisms of HQSJZD in the treatment of AD through the network pharmacology method, molecular docking technology, and in vitro experiments and provided a theoretical basis for the development of active compounds of HQSJZD as an innovative alternative therapy for AD.


Colorectal cancer (CRC) is the second most common cause of cancer mortality and a major health challenge worldwide. Despite advances in therapeutic and diagnostic methods, there is still a poor prognosis in CRC patients. Tumor recurrence and metastasis are the main causes of high mortality rate in these patients, which are due to late diagnosis in advanced tumor stages. Epithelial-mesenchymal transition (EMT) is known to be the most important cause of CRC metastasis, during which tumor cells obtain metastasis ability by losing epithelial features and gaining mesenchymal features. Long non-coding RNAs (lncRNAs) are pivotal regulators of EMT process. Regarding the higher stability of lncRNAs compared with coding RNAs in body fluids, they can be used as non-invasive diagnostic markers for EMT process. In the present review, we summarized all of the lncRNAs involved in regulation of EMT process during CRC progression and metastasis. It was observed that lncRNAs mainly induced the EMT process in CRC cells by regulation of EMT-related transcription factors, Poly comb repressive complex (PRC), and also signaling pathways such as WNT, NOTCH, MAPK, and Hippo. Colorectal cancer (CRC) is the second most common cause of cancer-related deaths globally . In spitWNT signaling pathway is involved in various cellular processes such as embryogenesis, cell proliferation, differentiation, and migration –33. FrizNotch signaling pathway is a highly maintained cellular process in different species with important roles in cell cycle, differentiation, apoptosis, angiogenesis, hematopoiesis, neurogenesis and proliferation –52. It nβ binds to the receptor extracellular domain, the intracellular domain phosphorylates Smad2/3 and binds to TRAF6, which recruits TAK1 and TAB1 to stimulate JNK and p38 MAPK. Upregulated JNK and p38 MAPK can promote EMT via regulating downstream transcriptional factors in a Smad-dependent or -independent pathway [MAPK pathway has pivotal roles in cell migration, growth, apoptosis, and differentiation. The central components of this signaling pathway are ERK1/2, JNK, p38 MAPK, and ERK5. Among them JNK and p38 MAPK are activated by chemical, physical, and biological stimuli while ERK1/2 are activated by cell growth factors . Tyrosin pathway . TRAF6 k pathway . ERK act pathway . Researc pathway . TGF-β1- pathway . Overall pathway , 78. SLC pathway . As a tr pathway . MMP-14  pathway . Heat sh pathway . There w pathway . There w pathway . H19 and pathway . Tumor m pathway , 87. It  pathway . SNHG16  pathway .Fig. 2RoTransforming growth factor-Beta (TGF-β) signaling is an important pathway involved in cell differentiation, proliferation, apoptosis, and embryogenesis. The TGF-β family of ligands such as Bone morphogenetic proteins (BMPs), Activin, and TGFβs can interact with type I and type II kinase receptors to activate Smad proteins. Smad family proteins includes; receptor-regulated Smad (R-Smad), inhibitory Smad (I-Smad), and the Co-mediator Smad (Co-Smad). After receptor-ligand interaction, the R-Smad proteins make heteromeric complex with Co-Smad proteins. Among ligands, BMPs include BMP2 and 4 have high affinity for receptor I and TGF-β and Activin have high affinity for receptor II. In nucleus both Co-Smads and R-Smads except Smad2 bind to Smad binding element (SBE) on DNA to regulate the target genes. The molecular investigations show relationship between the TGF-β pathway and RAS/MAPK/ERK signaling during development and oncogenesis. The effects of TGF-β pathway on cells depend on cell-type and its link with other pathways . TGF-β, Hippo signaling has mainly anti-tumor roles during tumor progression . It is cPoly comb repressive complex (PRC) as a histone methyltransferase methylates H3K27me3 that is a hallmark of transcriptionally inactive chromatin. It is involved in cellular differentiation, early embryonic development, X chromosome inactivation, DNA damage response, and imprinting. Aberrant PRC2 expression and mutation have been reported in various human cancers , 115. AsHistone acetylation, which is regulated by histone deacetylases (HDACs), is essential during tumor progression , 125. SnEMT process is regulated by a series of specific transcription factors that are activated in downstream of various signaling pathways Fig. . Slug isHistone deacetylases (HDACs) inhibit transcription by removing acetyl groups from lysine residues in histones. HDACs deacetylate non-histone and histone proteins. Several malignancies have been associated with abnormal expression of classical  HDACs . HDAC1 fNuclear HMGB2 facilitates EMT by interacting with OCT4 that enhances the formation of particular transcriptional complexes . OCT4 ovHypoxia is a prevalent event in the microenvironment of solid tumors. Tumor cells have a high proliferation rate, and their oxygen and nutrient supply is considerably lower than that of normal cells. Aberrant vascular system structure and function also result in insufficient blood circulation, which fails to provide tumor cell oxygen and nutrient requirements following the formation of a hypoxic microenvironment , 167. HyMolecular mechanisms involved in EMT process are key targets for development of novel therapeutic modalities to suppress tumor metastasis. LncRNAs regulate epithelial-mesenchymal plasticity through affecting the miRNAs and protein expression profiles. Therefore, targeting the lncRNA and miRNA interactions can be an efficient way to inhibit the EMT and tumor progression in early stage carcinomas. However, targeting the EMT related lncRNA/miRNA axis is not a reliable therapeutic method for the patients with advanced stage and metastatic tumors. A variety of approaches such as RNA interference, Antisense oligonucleotides, and Aptamers are under assessments for lncRNAs. However, these methods are faced with the lack of efficient delivery methods. Although, further investigations are required for safe and efficient delivery method, some of them are currently used in clinical trial studies. It has been reported that lncRNAs mainly function as the inducers of EMT process in CRC through regulation of EMT-related transcription factors, PRC complex, and also signaling pathways including WNT, NOTCH, MAPK, and Hippo. Indeed, the EMT-related transcription factors are critical direct or indirect targets of lncRNAs in regulation of EMT in CRC in which they can be affected directly or in downstream of the mentioned signaling pathways.


Ailurus fulgens): Ailurus fulgens fulgens and Ailurus fulgens styani. The red panda belongs to the endangered species living in the wild only in Asia and is included in the CITES list. Although the biology and diet of this species has been extensively described, the histological structure of the tongue and lingual glands has not yet been characterized in detail in relation to the lifestyle of this mammal under specific conditions and as a basis for comparative anatomical studies of the biodiversity of endemic species. Study samples were collected from two adult males of Ailurus fulgens f. held in Wrocław Zoological Garden. Both tongues were examined macroscopically; moreover, samples with lingual papillae for light microscopy and scanning electron microscopy (SEM) were collected from the apex, body and root of the tongue. Both tongues of the Ailurus fulgens f. males were approximately 9 cm long. The dorsal lingual surface was covered with mechanical and gustatory lingual papillae. Filiform papillae were observed on the apex and the body of the tongue, while small conical papillae were observed on the root of the tongue. An elongated, 1–1.5 cm long cylinder-shaped lyssa was observed in the ventral part of the apex. Moreover, most numerous and largest round in shape fungiform papillae were observed on the apex and on the border of the body and root of the tongue, located directly rostrally to 12–13 round and oval in shape vallate papillae. The SEM study showed that filiform papillae on the apex had several long secondary processes, while filiform papillae on the body of the tongue were taller and their secondary papillae were shorter than the equivalent structures on the apex of the tongue. The SEM study showed numerous taste pores on the surface of the fungiform papilla, while irregular surface of the vallate papillae, however some of them had smoother surface. Mixed glands (comprised of mucous acini and serous acini) were present within the vallum (within the connective tissue core) of the vallate papilla. Beneath the papillae more serous glands were observed, while the posterior lingual glands in the caudal part of the root of the tongue were mucoserous . A characteristic feature of the tongue of Ailurus fulgens f. was the presence of lyssa, which is comparable to other representatives of Carnivora, but the number of vallate papillae was individually variable. The lack of strongly developed mechanical conical papillae probably may be related to the type of plant food that is particularly dominant in red panda. Further differences between Ailurus fulgens f. and Ailurus fulgens s. cannot be excluded. The results of these studies may be useful especially for veterinarians specializing in working with exotic animals and people dealing with wildlife conservation.In the light of recent molecular studies, there are two phylogenetic species of the red panda ( Ailurus fulgens Cuvier, 1825) belongs to the mammal Ailuridae family in the Carnivora order  and the other species Ailurus fulgens styani (Chinese red panda) (The red panda (ra order . Moleculd panda) , 2020. Td panda) .http://cfpcc.gov.np/downloadfile/National-Parks-and-Wildlife-Conservation-Act-2029-1973_english_1517477214.pdf). In English, the red panda (Ailurus fulgens) is also called “lesser panda” or “red cat-bear”.According to the Red List of Threatened Species of IUCN, the red panda is an endangered species (EN). It is also included in The studies to date in the biology of red panda and its diet, in particular, show that despite being a member of the Caniformia suborder, it feeds on leaves, mainly young leaves and shoots of bamboo . FurtherStudies in microstructure of the lingual surface were carried out in many representatives of the Carnivora order, both in domestic species, such as cat , 2000 anPanthera tigris , in the following members of the Felidae family, among others: the tiger a tigris , lion Pahera leo , jaguar era onca , fishings tigris , Persianaxicolor , puma Puconcolor , Asian gmminckii , leoparda pardus , ocelot pardalis ; it was pardalis . For theenaticus , racoon  and fox , black-besomelas , Africann pictus , wolf Cais lupus , family ibetanus , Americaalayanus , giant panoleuca , polar baritimus , family melampus , Japanes anakuma , ferret ius furo , Americaon vison , least w nivalis , Asian scinereus , family crivorus , common on lotor , family a largha , and alsornianus . Moreoveornianus , Persianornianus  or crab-ornianus .Ailurus fulgens f. was analysed using SEM  held in Wrocław Zoological Garden. The post mortem examination of the animals revealed no pathologies of the oral cavity. Registered permissions for the post-mortem collection of specimens was issued by the District Veterinary Officer in Wroclaw . The tongues were rinsed with physiological saline solution. The length and width of the tongues were recorded and the lyssas were prepared. An electronic caliper (with an accuracy of 0.1 mm) was used to take the measurements and a Canon EOS 300 camera was used to document the analysis.The specimens were gathered in the collection of the Division of Animal Anatomy in 2020–2021. The tongues (S1) were collected from two adult males of the red panda  and lyssa were examined using a Zeiss Stemi 2000-C microscope  and were photographed subsequently.Samples for histological and histochemical studies  were taken from the apex, body and root of the tongue. Sections for histological studies included filiform papillae (from the apex and body of the tongue), small conical papillae (from the root of the tongue), fungiform papillae (from the apex and body), vallate papillae and samples of the tongue in transverse cross section from the apex of the tongue including the lyssa. Additional sections were taken from the root of the tongue for histochemical studies. The specimens were fixed in 4% buffered formaldehyde . Next, they were dehydrated in a series of alcohol dilutions, cleared in xylene and impregnated with paraffin. The specimens were cut using a Slide 2003  sliding microtome.Subsequently, the specimens were subjected to histochemical studies and were stained with hematoxylin&eosin (H&E)  and additionally with Masson-Goldner trichrome and Azan trichrome (to assess connective tissue and epithelium), whereas samples for histochemical studies  of the lSamples for SEM examination were collected from the apex, body and root of the tongue. Filiform papillae, small conical papillae, fungiform papillae, vallate papillae and the caudal surface of the root of the tongue without the papillae were analysed in the SEM study. The collected samples were fixed with 2.0% glutaraldehyde dissolved in 0.1 M phosphate buffer at pH 7.4 in small test tubes. The samples were then prepared for SEM analysis using Ailurus fulgens f. in the current study were described using the terms from the The detailed results of the analysis of the tongue structure in Ailurus fulgens f. was approximately 9 cm long. It was composed of a rounded apex, body and root , with the density of 59/cm2  lingual glands were predominant. Interestingly, mucous acini were also observed in the connective tissue core of the vallate papillae  in mucous acini . The PASAilurus fulgens f. allowed us to conclude that in this endemic species there are features typical for the tongue related to the number and structure of the lingual papillae, and the presence of lyssa is another of these characteristic features.The studies to date have paid much attention to the examination of the microstructure of lingual surface in mammals using various scientific methods. Results of many previous studies contribute to the development of comparative anatomy, and may also be included in clinical trials or veterinary surgery. Proper veterinary management of non-domestic species is a big challenge for veterinarians—among others—working in zoological gardens. Therefore, from this standpoint, a detailed knowledge of similarities and differences both in structure and function of particular organs  is of paramount importance. Likewise, a detailed knowledge of the functional anatomy of digestive tract is linked to appropriate selection of food for captive animals. Although the microstructure of lingual surface was analysed in many different species of mammals , and they had 5–6 folds  confirmed the presence of simple sensory nerves in the tissue forming lyssa, and it probably also plays a role of a receptive lingual organ  and vallate papillae area of red panda male 2 without any measurements or letters. These both tongues were used for detailed stereoscopic, LM and SEM analyses in our study.The figure is showing dorsal and ventral surface of the tongue from adult red panda male 1 (Click here for additional data file.


This is a novel study that explored the function of bta-miR-149-5p in the lipid metabolism of bovine adipocytes. Previously, we explored the role of bta-miR-149-5p gene in proliferation and differentiation of bovine adipocytes; however, the underlying mechanism of bta-miR-149-5p function in the regulation of lipid metabolism and adipogenesis in bovine adipocytes is unexplored. A transcriptomic study with integration of bioinformatics study was performed to fully explore the function of bta-miR-149-5p microRNA in bovine adipogenesis. This study will help the scientific community deal with lipogenesis for the breed improvement program of cattle for the provision of healthy meat to consumers. p < 0.01) downregulated the mRNA levels of adipogenic marker genes such as CCND2, KLF6, ACSL1, Cdk2, SCD, SIK2, and ZEB1 in bovine adipocytes. In conclusion, our results suggest that bta-miR-149-5p regulates lipid metabolism in bovine adipocytes. The results of this study provide a basis for studying the function and molecular mechanism of the bta-miR-149-5p in regulating bovine adipogenesis.Intramuscular fat is a real challenge for the experts of animal science to improve meat quality traits. Research on the mechanism of adipogenesis provides invaluable information for the improvement of meat quality traits. This study investigated the effect of bta-miR-149-5p and its underlying mechanism on lipid metabolism in bovine adipocytes. Bovine adipocytes were differentiated and transfected with bta-miR-149-5p mimics or its negative control (NC). A total of 115 DEGs including 72 upregulated and 43 downregulated genes were identified in bovine adipocytes. The unigenes and GO term biological processes were the most annotated unigene contributor parts at 80.08%, followed by cellular component at 13.4% and molecular function at 6.7%. The KEGG pathways regulated by the DEGs were PI3K-Akt signaling pathway, calcium signaling pathway, pathways in cancer, MAPK signaling pathway, lipid metabolism/metabolic pathway, PPAR signaling pathway, AMPK signaling pathway, TGF-beta signaling pathway, cAMP signaling pathway, cholesterol metabolism, Wnt signaling pathway, and FoxO signaling pathway. In addition to this, the most important reactome enrichment pathways were R−BTA−373813 receptor CXCR2 binding ligands CXCL1 to 7, R−BTA−373791 receptor CXCR1 binding CXCL6 and CXCL8 ligands, R−BTA−210991 basigin interactions, R−BTA−380108 chemokine receptors binding chemokines, R−BTA−445704 calcium binding caldesmon, and R−BTA−5669034 TNFs binding their physiological receptors. Furthermore, the expression trend of the DEGs in these pathways were also exploited. Moreover, the bta-miR-149-5p significantly ( Adipose tissue stores energy in the form of neutral triglycerides and performs a vital function in maintaining energy homeostasis . AdipocymiRNAs are a class of endogenous small non-coding, approximately 22 nt, single-stranded RNAs, and play a role in post-transcriptional regulation by targeting mRNA degradation or inhibiting their translation . A singlThe experimental animals were dealt with as per standard operating procedures (SOPs) formulated by the Chinese Council of Animals Care, and further approved by “Experimental Animal Management Committee (EAMC)” of the Northwest Agricultural and Forestry University, notified vide notification no. EAMC/20-23 dated 20.04.2013. The sampling was performed after human killing of the experimental animals at the National Beef Cattle Improvement Research Center, Yangling, Shaanxi, China.g for 10 min, and the cell pellet was washed 2 times with DMEM-F/12 medium  without serum. The isolated cells pellet was suspended within DMEM-F/12 medium supplemented with 10% FBS, then seeded in 60 mm collagen-coated cell culture plates, and finally incubated at 37 °C and 5% CO2 for 1 h. The medium was changed and then washed 3 times with PBS to remove the debris and free-floating dead cells.Bovine preadipocytes were isolated from healthy newborn calves (5 days old) of Qinchuan cattle by using previously described methods ,35,36,372. The cells were transiently transfected with bta-miR-149-5p mimic (50 nM) and mimic negative control (50 nM) at 70–90% confluence and density of 1.2 × 105 cells with growth medium without antibiotics and FBS using lipofectamine 3000 transfection reagent . Briefly, the RNA oligonucleotides and the transfection reagent were diluted separately in Opti-MEM medium (Gibco) and incubated for 10 min at RT. Both the reagents were mixed, combined, and incubated for another 15 min at RT to form the transfection reagent–RNA complexes. The complexes were then added dropwise to the cells and incubated for 24 h before changing to fresh medium. The bovine adipocyte cells were seeded in 24-well plates and cultured in DMEM-F/12  with 10% FBS  and 1% antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin), and incubated at 37 °C with 5% COAt 48 h post-transfection, the cells were differentiated with first differentiation media including 0.5 mM hydro cortisol, 1μM dexamethasone, IBMX (3. isobut-1-methylxanthine), and 167 nM insulin, as previously described . After 2® Premix Ex Taq II kit . For the bta-miR-149-5p expression analysis, we used a mircute miRNA isolation kit . The first strand cDNA library was constructed using miRcute Plus miRNA First-Strand cDNA kit . The bta-miR-149-5p expression was measured through mircute plus miRNA qPCR detection kit . For gene expression analysis, GAPDH and β-actin were used, while for miRNA expression, U6 was used as housekeeping genes. The 2−ΔΔCt method was used for the identification of relative mRNA and miRNA expression levels [After 48 h of adipocyte treatment with bta-miR-149-5p mimics or mimics NC (negative control), the adipocytes were collected and RNA was extracted through RNAiso Plus kit . The RNA integrity both quantity and quality were evaluated through optical density (OD) of 260 and ratio of the OD of 260/280 with Nano Quant plate TM . The RNA quality was further analyzed through 1% agarose gel. Then, the RNA was subjected to reverse transcription for cDNA synthesis through PrimeScriptTM RT reagent kit  with a gDNA eraser . The qRT-PCR was performed using SYBRn levels . The PCRThe total RNA was extracted with Trizol reagent kit , and the RNA quality was analyzed through an Agilent 2100 Bioanalyzer . The quality was further assessed through RNase free agarose gel electrophoresis. After extraction of total RNA, Oligo (dT) beads were used for the enrichment of mRNA, and fragmentation buffer was used for making short fragments, and then reverse-transcribed into cDNA using random primers. The DNA polymerase I, dNTP, RNase H, and buffer were used for the synthesis of second-strand cDNA, and their fragments were purified using QiaQuick PCR extraction kit ; poly(A) was added for end repair and then ligated into Illumina sequencing adapters. The ligation products were size-selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co. .After sequencing, the raw reads including adapters and low quality bases  were filtered through fastp software version 0.18 . The reads were mapped to ribosomal RNA (rRNA) database using the short reads alignment tool Bowtie-2 software version 2.2.8 . Then, tFPkM (A) refers to the expression of gene A, “c” denotes the number of fragments mapped to gene A, “N” is the total number of fragments mapped with reference genes, and “L” is number of bases in gene A. The fragment per kilobase of transcript per million mapped reads (FPKM) value was calculated for quantification and variations using StringTie software ,44. The The DEGs between the 2 groups was analyzed with DESeq2 software , and edghttp://www.geneontology.org/ (accessed on 20 April 2021). The significantly enriched GO terms in the DEGs correspond to the respective biological functions. Hypergeometric test was used for the calculation of gene numbers for each term in comparison with the reference genome using the following formula:N” denotes the number of all transcripts within GO annotation, “n” is the number of DEGs in N, “M” is the number of all transcripts annotated without respective GO terms, and “m” is the number of DEGs in M. The p-value is from FDR correction (FDR ≤ 0.05) and was used as a threshold. The GO terms within these conditions were considered as significantly enriched GO terms in DEGs.The DEGs were mapped into GO terms through GO enrichment analysis using the GO database available at N” denotes the number of all transcripts within KEGG annotation, “n” is the number of DEGs in N, “M” is the number of all transcripts annotated without respective KEGG pathways, and “m” is the number of DEGs in M. The p-value is from FDR correction (FDR ≤ 0.05) and was used as a threshold. The pathways within these conditions were considered as significantly enriched KEGG pathways in DEGs. The reactome is a free online database of biological pathways [The pathway enrichment analysis identified significantly enriched metabolic or signal transduction pathways in DEGs compared with the reference whole genome using the following formula:pathways ,48. The The same formula was used for reactome enrichment analysis.Protein–protein interaction network was identified using String v10 , which dp < 0.05 and ** p < 0.01 were considered to be statistically significant.The ANOVA was used for the statistical analysis through SAS version 8.1 software . The graphs were designed through Graph-Pad Prism6  software, with mean ± SEM. The variations at * p < 0.01) increased as compared to mimic NC  ranged from 7.5 to 10 A. These p < 0.01) reduction in LDs in adipocytes transfected with bta-miR-149-5p mimics as compared with mimic NC. These results illustrate the fact that bta-miR-149-5p inhibited differentiation of bovine adipocytes.The lipid droplets (LDs) on day 9 of differentiation were assessed using BODIPY staining . The adip-value (<0.05). These 115 DEGs contain 72 upregulated and 43 downregulated genes in bovine differentiated adipocytes  downregulated in adipocytes transfected with bta-miR-149-5p mimics. However, upregulation of bta-miR-149-5p significantly (p < 0.01) enriched relative mRNA expression of KLF12 and LIPG genes in bovine adipocytes. These results are in line with the results of sequencing data.The RNA sequence results were validated through qRT-PCR analysis of key marker genes selected from the list of DEGs . The mRNImprovement of intramuscular fat is a challenge for the experts of animal science for the improvement of meat quality traits, and obesity is a global issue—a root cause of certain fatal human disorders . AdipogeIn the present study, deep RNA-sequencing analysis revealed that bta-miR-149-5p significantly affected expression of adipogenic marker genes in differentiated bovine adipocytes transfected with bta-miR-149-5p mimic and mimic NC. The unigenes and GO term biological processes were the most annotated unigene contributor part with 80.08%, followed by cellular component of 13.4%, and molecular function of 6.7%. Interestingly, in the present study, the DEGs were upregulated in all three GO categories, namely, in biological processes, biological function, and cellular component ontologies. These findings show that bta-miR-149-5p regulates the functional roles of DEGs in bovine adipocytes through regulation of GO terms. Furthermore, the pathway analysis of the DEGs was performed to exhibit the pathways and molecular interactions in bovine adipocytes transfected with bta-miR-149-5p mimics or mimics NC. Strikingly, the pathway enrichment analysis explored the KEGG_A_classes, including disease and metabolism regulated by the DEGs in adipocytes transfected with bta-miR-149-5p, which specifically categorized the most important KEGG_B class pathways with proven roles in metabolism and adipogenesis. The pathway-based studies helped to exploit the gene roles in biological functions ,71. The In addition to this, the specific KEGG pathways regulated by these DEGs were PI3K-Akt signaling pathway, calcium signaling pathway, pathways in cancer, MAPK signaling pathway, lipid metabolism/metabolic pathway, cholesterol metabolism, PPAR signaling pathway, AMPK signaling pathway, TGF-beta signaling pathway, cAMP signaling pathway, Wnt signaling pathway, and FoxO signaling pathway. These data indicate the role of bta-miR-149-5p in adipogenesis through interactive regulation of DEGs and their respective KEGG pathways in bovine adipocytes. All these pathways have proven roles in adipogenesis. Previously, it has been reported that PI3K-Akt signaling pathway plays an important role in adipose tissue function and energy metabolism through regulation of the genes expressed in lipid metabolism ,75,76. Pp < 0.05) upregulated LIPG  and KLF12 expression, which perform vital roles in cancer [Interestingly, the pathway enrichment analysis in the present study also exhibited the role of bta-miR-149-5p in cancer. Previously, the roles of miR-149 in osteosarcoma , cancersn cancer ,92. Howen cancer . Howeverp < 0.01) downregulated the mRNA levels of adipogenic marker genes such as CCND2, KLF6, ACSL1, Cdk2, SCD, SIK2, and ZEB1 in bovine adipocytes. These findings validated the transcriptomic data and also proved the functional roles of bta-miR-149-5p in bovine adipogenesis through regulation of these adipogenic marker genes’ expression. The cyclin D2 (CCND2) and Cdk2 are well-known proliferation marker genes, enriched in early differentiation and then again overexpressed in the later stage of adipocyte differentiation [Moreover, the bta-miR-149-5p significantly (ntiation ,95. Krupntiation . ACSL1 intiation . The cruntiation  and fattntiation . SIK2 plntiation . Previountiation . These rThese data indicate the role of bta-miR-149-5p in adipogenesis through interactive regulation of DEGs and their respective KEGG pathways in bovine adipocytes. In conclusion, our results suggest that bta-miR-149-5p regulates lipid metabolism in bovine adipocytes. The results of this study provide a basis for studying the function and molecular mechanism of the bta-miR-149-5p in regulating adipogenesis.


Apis cerana–Nosema ceranae interaction is very limited, though A. cerana is the original host of N. ceranae. Apis cerana cerana is a subspecies of A. cerana and a major bee species used in the beekeeping industry in China and other countries. Here, the effective infection of A. c. cerana workers by N. ceranae was verified, followed by transcriptomic investigation of host responses. Furthermore, immune responses between A. c. cerana and Apis mellifera ligustica were deeply compared and discussed. In total, 1127 and 957 N. ceranae-responsive genes were identified in the infected midguts at 7 d post-inoculation (dpi) and 10 dpi, respectively. Additionally, DEGs in workers’ midguts at both 7 dpi and 10 dpi were associated with six cellular immune pathways and three humoral immune pathways. Noticeably, one up-regulated gene was enriched in the NF-κB signaling pathway in the midgut at 10 dpi. Further analysis indicated that different cellular and humoral immune responses were employed by A. c. cerana and A. m. ligustica workers to combat N. ceranae. Our findings provide a foundation for clarifying the mechanisms regulating the immune response of A. c. cerana workers to N. ceranae invasion and developing new approaches to control bee microsporidiosis.Currently, knowledge regarding Apis cerana cerana workers at 7 d post-inoculation (dpi) and 10 dpi with Nosema ceranae and corresponding un-inoculated midguts. PCR identification and microscopic observation of paraffin sections confirmed the effective infection of A. c. cerana worker by N. ceranae. In total, 1127 and 957 N. ceranae-responsive genes were identified in the infected midguts at 7 dpi and 10 dpi, respectively. RT-qPCR results validated the reliability of our transcriptome data. GO categorization indicated the differentially expressed genes (DEGs) were respectively engaged in 34 and 33 functional terms associated with biological processes, cellular components, and molecular functions. Additionally, KEGG pathway enrichment analysis showed that DEGs at 7 dpi and 10 dpi could be enriched in 231 and 226 pathways, respectively. Moreover, DEGs in workers’ midguts at both 7 dpi and 10 dpi were involved in six cellular immune pathways such as autophagy and phagosome and three humoral immune pathways such as the Toll/Imd signaling pathway and Jak-STAT signaling pathway. In addition, one up-regulated gene (XM_017055397.1) was enriched in the NF-κB signaling pathway in the workers’ midgut at 10 dpi. Further investigation suggested the majority of these DEGs were engaged in only one immune pathway, while a small number of DEGs were simultaneously involved in two immune pathways. These results together demonstrated that the overall gene expression profile in host midgut was altered by N. ceranae infection and some of the host immune pathways were induced to activation during fungal infection, whereas some others were suppressed via host–pathogen interaction. Our findings offer a basis for clarification of the mechanism underlying the immune response of A. c. cerana workers to N. ceranae infection, but also provide novel insights into eastern honeybee-microsporodian interaction.Here, a comparative transcriptome investigation was conducted based on high-quality deep sequencing data from the midguts of  Honeybees are a pivotal pollinator for a great number of wild plants and crops throughout the world . In addiNosema ceranae is a spore-forming unicellular and obligate intracellular fungal parasite that exclusively infects the midgut epithelium cells and not all epithelial cells of the adult honeybee, leading to bee microsporidiosis  was simultaneously enriched in autophagy and lysosome  = −11.16) and lysosomal aspartic protease  = −1.17) were simultaneously enriched in autophagy and lysosome  enriched in this cellular immune pathway were down-regulated  = −10.81) and 10 dpi (log2(fold change) = −12.19). These results suggested that the aforementioned two cellular immune pathways were suppressed by N. ceranae at different stages of fungal infection.In this current work, Venn analysis displayed that the numbers of specific up-regulated genes were 297 and 245, which were much less than those (706 and 582) of specific down-regulated genes in AcCK1 vs. AcT1 and AcCK2 vs. AcT2; additionally, only 124 DEGs were shared by the above-mentioned two comparison groups, including 30 up-regulated and 94 down-regulated genes. It is speculated that some DEGs in d 10 dpi . In addiA. m. ligustica workers at 7 dpi and 10 dpi with N. ceranae, respectively, suggesting that that apoptosis pathway was activated to combat N. ceranae infection [N. ceranae was shown to suppress apoptosis in host midgut epithelial cells [N. ceranae-tolerant A. mellifera workers could escape parasitic manipulation of apoptosis [A. c. cerana worker midguts responding to N. ceranae invasion.One up-regulated gene was previously discovered to be enriched in apoptosis in the midguts of nfection . Intrigunfection . This stnfection ,80. In wal cells ,19,28. Hpoptosis . That fiDrosophila and mosquito. The NF-κB pathway, a conserved signal transduction pathway present in nearly all mammalian cell types, is involved in various biological processes including innate and adaptive immunity, survival, development, proliferation, and cell apoptosis [κB pathway in N. ceranae-challenged A. c. cerana workers’ midgut at 10 dpi, and one up-regulated gene and one down-regulated gene were enriched in the Toll-like receptor signaling pathway  that are uniquely active against different pathogens [A. m. ligustica workers at both 7 dpi and 10 dpi with N. ceranae, one up-regulated gene was enriched in the NF-κB pathway, implying that this humoral immune pathway was activated by N. ceranae infection [κB pathway may play a pivotal role in immune responses of both eastern honeybee workers and western honeybee workers to N. ceranae invasion. In addition, five genes enriched in the Jak-STAT pathway were up-regulated in workers’ midgut at 7 dpi, while three DEGs in workers’ midgut at 10 dpi were engaged in this humoral immune pathway, including two up-regulated genes (Drosophila [Anopheles gambiae responding to bacterial infection [A. c. cerana workers’ midguts was activated due to N. ceranae infection. Intriguingly, there was no DEG enriched in the Jak-STAT pathway in A. m. ligustica workers’ midguts in response to N. ceranae infection [A. c. cerana workers and A. m. ligustica workers to battle the same fungal parasite, N. ceranae.After fungal infection, insect humoral immunity is induced to synthesize AMPs and activate the prophenoloxodase system, which can work together to inactivate or kill the invaders . Currentpoptosis ,83. Here pathway B, wherea pathway B. Drosopathogens ,85,86,87athogens ,89. In tnfection . These red genes B. The Jaosophila ,91. Jak-osophila . This imnfection . It has nfection . TogetheA. c. cerana worker response to N. ceranae invasion. That effort can be expected to provide knowledge for the development of effective and environmentally friendly strategies to control bee nosemosis.Additional work is needed to decipher the function of key immune genes with differential expression in A. c. ceranae workers’ midguts to N. ceranae invasion were comprehensively investigated at a transcriptome-wide level. These results demonstrated that the overall gene expression profile in host midgut was altered by N. ceranae invasion and that some host immune pathways such as the autophagy and Jak-STAT pathway were induced to activation during fungal infection, whereas some were suppressed via host–pathogen interaction. Our findings not only offer a basis for clarification of the mechanism underlying the immune response of A. c. cerana to N. ceranae infection, but also provide novel insights into eastern honeybee–microsporidian interaction.In the present study, cellular and humoral immune responses of