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Background Parkinson’s disease (PD) is a common neurodegenerative disease in the elderly population. However, there are no reliable diagnostic biomarkers for PD, and the pathogenesis of PD still needs further study. The aim of the current study was to identify potential biomarkers and explore the pathogenesis of PD. Methods We conducted an integrative analysis of messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA) expression profiles of PD using data from the Gene Expression Omnibus (GEO). The GSE110720, GSE110719 and GSE133347 data sets were selected and analysed. Gene ontology (GO) enrichment and gene set variation analysis (GSVA) were performed for annotation, visualization, and integrated discovery. Protein–protein interaction (PPI) and competing endogenous RNA (ceRNA) networks were constructed, and hub genes were identified. Meanwhile, the immune infiltration analysis of hub genes was analysed. Moreover, receiver operating characteristic (ROC) curves were generated to verify the diagnostic value of the differentially expressed genes (DEGs). Finally, the genes with high area under the curve (AUC) values were verified by human samples. Results We identified 464 DEGs closely related to PD, including 154 mRNAs, 134 miRNAs, and 176 lncRNAs. The GO analyses indicated that changes in PD were mainly enriched in receptor ligand activity and cytokine receptor binding. The KEGG enrichment analysis showed that these DEGs were significantly involved in cytokine-cytokine receptor interactions, signalling pathways regulating the pluripotency of stem cells and Th17 cell differentiation. GSVA suggested that growth factor binding, IL2-stat5 signalling, and IL6-jak-stat3 signalling were crucial in the development of PD. A total of five hub genes (NPBWR2, CXCL10, CXCL5, S1PR5, and GALR1) were selected via the PPI network. A ceRNA network of the CXCL5, CXCL10 and S1PR5 genes was constructed, and target genes of the three genes were screened. The immune infiltration analysis showed that there were significant differences in a variety of immune cells between the hub genes. The expression of DEGs was validated in clinical human samples by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression levels of hsa-miR6895–5p, hsa-miR6791–5p, hsa-miR518f-5p, hsa-miR455–3p and TEKT4P2 were decreased, while the levels of TPTE2P6 were increased in human samples. These findings are consistent with the bioinformatics analysis results. Conclusion We found that the immune inflammatory response and immune cell regulation were involved in the pathogenesis of PD. Five hub genes involved in the immune infiltration biological processes of PD based on bioinformatics. We verified the DEGs with significant differences by qRT-PCR. These findings might provide new insight into the pathogenesis of PD and the development of diagnostic and therapeutic strategies for PD.

Type 2 diabetes mellitus (T2DM), a chronic low-grade inflammatory disease with high morbidity and mortality, is a serious threat to public health. Previously we demonstrated that a traditional Chinese medicine formulation, Jiedu Tongluo Tiaogan Formula (JDTL), exerted a favorable hypoglycemic effect due to unknown molecular mechanisms involving interactions among JDTL compounds and various cellular components. This study aimed to explore JDTL mechanisms for alleviating hyperglycemia using an integrated strategy incorporating system pharmacology, bioinformatics analysis, and experimental verification. This strategy entailed initial elucidation of JDTL chemical composition using fingerprint analysis via high performance liquid chromatography (HPLC). Next, functions of putative shared target genes and associated pathways were deduced using GO and KEGG pathway enrichment and molecular docking analyses. Ultimately, targets associated with JTDL anti-T2DM effects were found to be functionally associated with biological functions related to lipopolysaccharide and cytokine receptor binding. These results implicated PI3K-Akt signaling pathway involvement in JDTL anti-T2DM effects, as this pathway had been previously shown to play significant roles in glucose and lipid metabolism-related diseases. Furthermore, addition of JDTL to INS-1 and HepG2 cell cultures stimulated cellular mRNA-level and protein-level expression leading to enhanced production of IRS1, Akt, and PI3K. In summary, here JDTL bioactive ingredients, potential targets, and molecular mechanisms underlying JDTL anti-T2DM effects were identified using a multi-component, multi-target, and multi-channel analytical approach, thus providing an important scientific foundation to facilitate development of new drugs mechanistic strategies for preventing and treating T2DM.

Using network pharmacology to explore the mechanism of the ‘invigorating qi and promoting blood circulation’ drug pair Ginseng-Danshen (Salvia miltiorrhiza) on treatment of ischemic heart disease (IHD). The chemical constituents of ginseng and Danshen drug pair were identified by searching the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the potential targets of the pair were identified. The pharmacodynamics of the pair was analyzed using network pharmacology. The targets of IHD were identified by database screening. Using protein-protein interaction network, the interaction targets of Ginseng-Danshen on IHD were constructed. A “constituent-target-disease” interaction network was constructed using Cytoscape software, Gene Ontology (GO) term enrichment analysis and biological pathway enrichment analysis were carried out, and the mechanism of improving myocardial ischemia by the Ginseng-Danshen drug pair was investigated. Seventeen active constituents and 53 targets were identified from ginseng, 53 active constituents and 61 targets were identified from Danshen, and 32 protein targets were shared by ginseng and Danshen. Twenty GO terms were analyzed, including cytokine receptor binding, cytokine activity, heme binding, and antioxidant activity. Sixty Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways were analyzed, including phosphatidylinositol 3-kinase-serine-threonine kinase (PI3K-AKT) signaling pathway, p53 signaling pathway, interleukin 17 signaling pathway, tumor necrosis factor signaling pathway, and the advanced glycation end product (AGE)-the receptor for AGE (RAGE) signaling pathway in diabetic complications. The specific mechanism of Ginseng-Danshen drug pair in treating IHD may be associated with improving the changes of metabolites inbody, inhibiting the production of peroxides, removing the endogenous oxygen free radicals, regulating the expression of inflammatory factors, reducing myocardial cell apoptosis and promoting vascular regeneration.

Objective: To investigate the potential mechanism of Drynariae Rhizoma-Epimedii Folium in the treatment of osteoarthritis (OA) based on network pharmacology. Methods: The potential active constituents and targets of Drynariae Rhizoma-Epimedii Folium were screened through the traditional Chinese medicine (TCM) systems pharmacology database and analysis platform (TCMSP). Genecards database is used to find relevant targets of OA. The targets of “Drynariae Rhizoma-Epimedii Folium” were mapped to the targets of OA, and used Cytoscape software to build a “drug-ingredient-target-di- sease” regulatory network and protein protein interaction (PPI) network. R software was used to analyze the Gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment of traditional Chinese medicine-disease targets. Results: Thirty-four effective ingredients and 130 traditional Chinese medicine-disease targets were screened out for the treatment of OA. The GO functions of traditional Chinese medicine-disease targets mainly included cytokine activity, cytokine receptor binding, nuclear receptor activity, transcription factor activity, proximal promoter DNA-binding transcription activator activity, DNA-binding transcription activator activity, phosphatase binding and so on. KEGG pathways involved in traditional Chinese medicine-disease targets mainly included TLR4 signaling pathway, TNF signaling pathway, IL-17 signaling pathway, MAPK signaling pathway, PI3K/AKT signaling pathway, apoptotic signaling pathway and so on. Conclusion: Network pharmacology may predict the multiple targets and multiple signaling pathways in Drynariae Rhizoma-Epimedii Folium treatment for OA, providing new ideas for future research.

Background. Chuankezhi injection (CKZI) was an effective traditional Chinese medicine (TCM) injection in adjuvant bronchial asthma therapy. In this report, we used a network pharmacology method to reveal the mechanisms of CKZI for the treatment of asthma. Methods. The candidate compounds in CKZI were determined by searching the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and China National Knowledge Infrastructure website (CNKI). The targets of candidate compounds were searched in the TCMSP, DrugBank 5.0, and SwissTargetPrediction. The disease targets were screened from the Online Mendelian Inheritance in Man (OMIM) and GeneCards. The overlapping gene symbols between candidate compounds and disease were filtered via a Venn diagram and were considered as potential targets. A protein-protein interaction (PPI) network and disease-related candidate compound-target-pathway (DC-T-P) network were visualized by Cytoscape 3.6.1. Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed by metascape to determine the pathways related to asthma. Results. A total of 70 overlapping gene symbols were recognized as potential targets. Cytokines (IL6, TNF, and IL1B) and chemokines (CXCL8 and CCL2) could be recognized as hub genes. Asthma-related candidate compounds were mainly flavonoids, such as quercetin, luteolin, and kaempferol. The cytokine-mediated signaling pathway, cytokine receptor binding, and membrane craft were the most significant biological process (BP), molecular function (MF), and cellular component (CC) of GO function results, respectively. The relevant pathways of CKZI against asthma mainly include IL-17, NF-kappa B, HIF-1, calcium, and PI3K-Akt signaling pathways. Conclusion. Our research provided a theoretical basis for further investigating the mechanisms of CKZI in the treatment of asthma.

Background Age-related macular degeneration (AMD) is the most common, progressive, and polygenic cause of irreversible visual impairment in the world. The molecular pathogenesis of the primary events of AMD is poorly understood. We have investigated a transcriptome-wide analysis of differential gene expression, single-nucleotide polymorphisms (SNPs), indels, and simple sequence repeats (SSRs) in datasets of the human peripheral retina and RPE-choroid-sclera control and AMD. Methods and results Adaptors and unbiased components were removed and checked to ensure the quality of the data sets. Molecular function, biological process, cellular component, and pathway analyses were performed on differentially expressed genes. Analysis of the gene expression datasets identified 5011 upregulated genes, 11,800 downregulated genes, 42,016 SNPs, 1141 indels, and 6668 SRRs between healthy controls and AMD donor material. Enrichment categories for gene ontology included chemokine activity, cytokine activity, cytokine receptor binding, immune system process, and signal transduction respectively. A functional pathways analysis identified that chemokine receptors bind chemokines, complement cascade genes, and create cytokine signaling in immune system pathway genes (p value

We aimed to explore the potential biomarkers and susceptible population for early diagnosis and treatment of tuberculosis (TB). Ten hub differentially expressed TB-related genes (DETRGs) from GSE83456 dataset were screened with the "limma" package and the GeneCards database. Unsupervised clustering was utilized to identify susceptible population among TB patients based on 10 hub DETRGs. TRANSFAC, MirTarbase, miRanda and TargetScan was used to predict microRNAs and transcription factors (TFs) and construct TF-miRNA-mRNA regulatory network. The results showed that a total of 266 DEGs were identified. Functional analysis mainly enriched in interferon pathway, cytokine and receptor interaction and host defense response to virus, while the four-module genes screened were closely related to interferon-γ signal transduction pathway as well. Based on 10 DETRGs, TB patients were divided into two clusters with significant differences in neutrophil function and 16 hub miRNAs and 10 hub TFs were predicted. Finally, NFATc1- (miR145) - STAT1 regulatory pathway was identified as the critical regulatory pathway, which mediates cytokine receptor binding, interleukin-1 receptor binding and TNF signaling pathway. Hence, we concluded that immunoheterogeneity exists among TB patients and NFATC1-(miR145)-STAT1 regulatory pathway might be associated with tuberculosis infection, which may be valuable targets for prevention and treatment of tuberculosis.

Objective. To clarify the therapeutic mechanisms of compound Xuanju capsule-treated rheumatoid arthritis (RA) based on network pharmacology tactics. Method. The TCMSP, TCMID and STITCH databases were used to screen the active ingredients and targets in the compound Xuanju capsule; the OMIM, TTD, PharmGKB and GeneCards databases were applied to screen the RA-related disease targets. Then, the obtained targets were imported into Cytoscape 3.7.1 software to construct the active ingredient-target network and the RA-related disease-target network. The active ingredient-target PPI network, the RA-related disease-target PPI network and the common target PPI network were built by using the STRING platform and Cytoscape 3.7.1 software. The GO and KEGG analyses of the common targets were analyzed by using the Metascape and Bioinformatics online tools. Results. A total of 51 active ingredients and 513 corresponding ingredient targets were harvested from the compound Xuanju capsule; 641 RA-related disease targets were obtained. After two PPI networks were constructed and merged, 116 RA-related targets of compound Xuanju capsules were identified and analyzed. 116 RA-related targets of compound Xuanju capsules are mainly involved in the biological processes and molecular functions, such as the cytokine-mediated signaling pathways, the response to lipopolysaccharide and the blood vascular development, the cytokine activity, the cytokine receptor binding and the receptor regulator activity. Furthermore, 116 RA-related targets of compound Xuanju capsules are concentrated in signaling pathways such as the IL-17, TNF, Th17 cell differentiation, Toll receptor and RA signaling pathway. Conclusion. The compound Xuanju capsule had the action characteristics of multiple components, multiple targets, and multiple pathways in the treatment of RA, which might primarily reduce the release of proinflammatory factors (such as IL-6 and TNF-α) and increase the production of anti-inflammatory factors (such as IL-10) by regulating inflammation-related signaling pathways (such as IL-17), thereby alleviating the inflammatory damage and improving the bone tissue repair.

BackgroundThe rapid development of coronavirus disease 2019 (COVID-19) pandemic has become a great threat to global health. Its mortality is associated with inflammation-related airway mucus hypersecretion and dysfunction of expectoration, and the subsequent mucus blockage of the bronchioles at critical stage is attributed to hypoxemia, complications, and even death. Traditional Chinese medicine (TCM) has rich experience in expectorant, including treatment of COVID-19 patients with airway mucus dysfunction, yet little is known about the mechanisms. This study is aiming to explore the potential biological basis of TCM herbal expectorant for treating COVID-19.ObjectiveTo get core herbs with high used frequency applications in the actions of expectoration by using association rule algorithm and to investigate the multitarget mechanisms of core herbs in expectorant formulae for COVID-19 therapies.MethodsForty prescriptions for expectorant were retrieved from TCM Formulae. The ingredient compounds and targets of core herbs were collected from the TCMSP database, Gene-Cards, and NCBI. The protein interaction network (PPI) was constructed by SRING, and the network analysis was done by Cytoscape software. Bioconductor was applied for functional enrichment analysis of targets.ResultsThe core herbs of expectorant could regulate core pathways (MAP kinase activity, cytokine receptor binding, G-protein-coupled receptor binding, etc.) via interactions of ingredients (glycyrol, citromitin, etc.) on mucin family to eliminate phlegm.ConclusionTCM herbal expectorant could regulate MAPK and cytokine-related pathways, thereby modulating Mucin-family to affect mucus generation and clearance and eventually retarding the deterioration of COVID-19 disease.

C2C12 cells were cultured on medium containing fluoride (0, 1, and 2.5 mmol/L) for 48 h to investigate the effect of excessive fluoride on T helper 17 (Th17)-related cytokine expression profile in skeletal muscle cells, and the culture supernatant was collected and subjected for the detection of 18 cytokines via Th17 array. Results showed that compared with the control group, no differential expression proteins (DEPs) were found in the 1 mmol/L fluoride group; however, eight DEPs were upregulated in the 2.5 mmol/L fluoride group, including macrophage inflammatory protein-3α (MIP-3α), interleukin-21 (IL-21), IL-13, IL-17F, IL-28A, transforming growth factor type beta 1 (TGF-β1), IL-23, and IL-17A. In addition, five DEPs (MIP-3α, IL-13, IL-21, TGF-β1, and IL-17F) were upregulated in the 2.5 mmol/L fluoride group compared with the 1 mmol/L fluoride group. Gene ontology analysis revealed that the positive regulation of cytokine production, cytokine activity, receptor ligand activity, and cytokine receptor binding accounted for high percent of DEPs present. Kyoto Encyclopedia of Genes and Genomes analysis showed that these DEPs primarily involved 12 pathways enriched in the cytokine-cytokine receptor interaction and IL-17 signaling pathway after 2.5 mmol/L fluoride treatment. The results indicated that fluoride might induce cytotoxicity by disturbing Th17-related cytokine expression.

Background Studies have shown that extracts from lycium barbarum could play a protective role against colorectal cancer (CRC) cells. We used the network pharmacology approach to establish the effects of lycium barbarum on CRC and to predict core targets and their biological func­tions, pathways, and mechanisms of action. Methods We obtained the active compounds and their targets in lycium barbarum through Traditional Chinese Medicine System Pharmacology Database (TCMSP), retracted the CRC targets from Malacards, TTD, GeneCards, and DisGeNET, and chosen the overlapped targets as the candidate targets. After protein-protein interaction (PPI) network analysis, 20 with the highest node degree were selected as the core targets, and their enrichment and pathway were analyzed. Furthermore, iGEMDOCK was employed to validate the compound-target association. Results Eventually, 103 overlapped targets were chosen as the candidate targets. Targets with the top 20 highest node degree were selected as the core targets. Gene Ontology (GO) enrichment analysis indicated that the core targets were significantly enriched in regulation of cell proliferation, extracellular space, cytokine receptor binding, and so on. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis proved that the core targets were significantly enriched in bladder cancer, pathways in cancer, and the like. The docking results showed that beta-sitosterol, glycitein, and quercetin had a good binding activity to CRC putative targets. Conclusions Our work successfully predicted the active ingredients and potential targets of lycium barbarum for CRC and helped to illustrate the pathways and mechanisms of action on a comprehensive level.

Background Non-alcoholic fatty liver (NAFLD) is a chronic disease worldwide, which poses a huge threat to human health. Xiaochaihu decoction is a well-known traditional Chinese medicine prescription. It has been proven effective in treating NAFLD but its mechanism is still unclear. Objective Multiple mechanisms of Xiaochaihu decoction are explored by identifying and connecting potential targets and active ingredients in the treatment of NAFLD. Methods Active ingredients and related targets of seven herbs were collected from TCMSP database. The related targets of NAFLD were obtained from Genes cards database, TDD and OMIM database. The intersected targets of disease targets and drug targets were input into STRING database to construct protein-protein interaction network. DAVID database was used for GO enrichment analysis and KEGG enrichment analysis. Results After screening and removal of duplicates, a total of 145 active ingredients and 105 potential targets were obtained. PPI network manifested that AKT1, IL6, JUN MAPK8 and STAT3 were the key target proteins. The results of GO enrichment analysis mainly involved cytokine receptor binding, cytokine activity, and heme binding. The results of KEGG analysis suggested that the mechanism mainly involved in AGE-RAGE signaling pathway in diabetic complications, Hepatitis C, fluid shear stress and atherosclerosis. The signaling pathways were further integrated as network manner, including AGE-RAGE signaling pathway in diabetic complications, Fluid shear stress and atherosclerosis, Insulin resistance, HIF-1 signaling pathway, Th17 cell differentiation and IL-17 signaling pathway. The network contained immunity regulation, metabolism regulation and oxidative stress regulation. Conclusion Xiaochaihu decoction plays a key role in the treatment of NAFLD with multiple targets and pathways. Immunity regulation, metabolism regulation and oxidative stress regulation consist of the crucial regulation cores in mechanism. Graphical abstract Design and workflow of this study

Studies have shown that extracts from Lycium barbarum exerted protective effects against colorectal cancer (CRC) cells. We used the network pharmacology method to determine the effects of L. barbarum on CRC and to predict core targets, biological functions, pathways, and mechanisms of action. We obtained the active compounds and their targets in L. barbarum via use of the Traditional Chinese Medicine System Pharmacology Database (TCMSP), gathered the CRC targets from Malacards, TTD, GeneCards, and DisGeNET, and chosen the overlapped targets as the candidate targets. After protein–protein interaction (PPI) network analysis, 20 with the highest node degree were selected as the core targets, and their enrichment and pathways were analyzed. Furthermore, we employed iGEMDOCK to validate the compound-target relation. Eventually, 103 overlapped targets were chosen as the candidate targets. Targets with the top 20 highest node degree were selected as the core targets. Gene Ontology (GO) enrichment analysis indicated that the core targets were enriched in cell proliferation regulation, extracellular space, cytokine receptor binding, and so on. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis proved that the core targets were significantly enriched in bladder cancer, pathways in cancer. The docking results demonstrated that beta-sitosterol, glycitein, and quercetin had good binding activity to CRC putative targets. Our work successfully predicted the functioning ingredients and potential targets of L. barbarum in CRC and illustrated the potential pathways and mechanisms comprehensively. Nevertheless, these results still call for in vitro and in vivo experiments to validate.

Fungal keratitis is a corneal disease with a high blindness rate caused by pathogenic fungal infections. The pathogenesis of fungal keratitis and the immune response after fungal infection are still unclear. Notably, the pathological features of fungal keratitis in tree shrews are similar to those in humans. In the present study, mRNA profiling of tree shrew corneas with fungal keratitis was performed. GO and KEGG enrichment analyses were performed on the differentially expressed mRNAs, and the GO biological process ontology was used to analyze functional trends in the differentially expressed mRNAs. In total, 151 downregulated and 71 upregulated mRNAs were shared among the 7-day, 14-day and 30-day infection groups. These differentially expressed mRNAs were significantly enriched in the GO category immune response (GO: 0002376) and the KEGG pathways cytokine receptor binding (KEGG ID: tup04060) and cell adhesion (KEGG ID: tup04514). The downregulated mRNAs were significantly enriched in the corneal epithelial cell adhesion function. Fifty-eight initially upregulated mRNAs gradually decreased in expression, and these mRNAs were significantly enriched in the functions lipopolysaccharide (LPS) and antibacterial polypeptide recognition, cell differentiation, and cell rearrangement. Zeta chain of T-cell receptor associated protein kinase 70 (ZAP70), lymphocyte cytosolic protein 2 (LCP2), C-C motif chemokine and its receptor showed high degrees of connectivity in the protein-protein interaction (PPI) network. We speculate that the decrease in symptoms of tree shrew fungal keratitis may be related to the upregulation of genes involved in immune regulation and macrophage colony stimulation. This study showed that the C-C motif chemokine and its receptor may play a key role in regulating tree shrew fungal keratitis, providing a theoretical basis for studying the pathogenesis of human fungal keratitis.

Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.

Cytokines activate downstream signaling networks via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered variants of IL-6 with different affinities to the gp130 receptor chain to investigate how cytokine receptor binding kinetics influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes, from minimal to full agonist, and induced biased signaling, with changes in receptor binding kinetics affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This, in turn, leads to a graded gene expression response and substantial differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.

Background The participation of long noncoding RNAs (lncRNAs) in myocardial infarction has recently been noted. However, their underlying roles in the border zone of myocardial infarction remain unclear. This study uses microarrays to determine the profiles of lncRNAs and mRNAs in the border zone. Methods Bioinformatics methods were employed to uncover their underlying roles. Highly dysregulated lncRNAs was further validated via PCR. Results Four hundred seven lncRNAs and 752 mRNAs were upregulated, while 132 lncRNAs and 547 mRNAs were downregulated in the border zone of myocardial infarction. A circos graph was constructed to visualize the chromosomal distribution and classification of the dysregulated lncRNAs and mRNAs. The upregulated mRNAs in the border zone were most highly enriched in cytokine activity, binding, cytokine receptor binding and related processes, as ascertained through Go analysis. Pathway analysis of the upregulated mRNAs showed the most significant changes were in the TNF signaling pathway, cytokine–cytokine receptor interaction and chemokine signaling pathway and similar pathways and interactions. An lncRNA–mRNA co-expression network was established to probe into the underlying functions of the 10 most highly dysregulated lncRNAs based on their co-expressed mRNAs. In the co-expression network, we found 16 genes directly involved in myocardial infarction, including Alox5ap, Itgb2 and B4galt1. The lncRNAs AY212271, EF424788 and MRAK088538, among others, might be associated with myocardial infarction. BC166504 is probably a key lncRNA in the border zone of myocardial infarction. Conclusions The results may have revealed some aberrantly expressed lncRNAs and mRNAs that contribute to the underlying pathophysiological mechanisms of myocardial infarction.

In this study, allergic rhinitis (AR) disease targets and Xiang Ju tablet-associated targets were determined through the use of databases for the identification of putative therapeutic targets and then combined. After the production of a putative therapeutic target interaction network for Xiang Ju tablets against AR, topological analysis was used to determine the core targets of Xiang Ju tablets in AR treatment. For all putative therapeutic targets, analyses of biological function and pathway enrichment were performed to optimize the biological processes and key signaling pathways of Xiang Ju tablets in AR treatment. The top 5 therapeutic targets of Xiang Ju tablets in AR treatment were identified and included CXCL8, IL1B, IL6, IL10, and TNF. The biological processes, molecular functions, and cell composition related to the use of Xiang Ju tablets in AR treatment were predominantly associated with cytokine production, regulation of protein secretion, and regulation of peptide secretion; cytokine activity, cytokine receptor binding, and receptor ligand activity; and platelet alpha granule lumen, collagen-containing extracellular matrix, and platelet alpha granule. In addition, the top 64 key signaling pathways were identified.

Chronic rhinosinusitis with nasal polyps (CRSwNP) refers to chronic inflammation of the sinonasal mucosa. It can either be eosinophilic (ECRSwNP) or non-eosinophilic (non-ECRSwNP). However, immune cell infiltration in the microenvironment and pathogenesis of ECRSwNP and non-ECRSwNP are still unclear. The aim of the present study was to assess the immune cell infiltration and molecular mechanisms of ECRSwNP and non-ECRSwNP. In the present study, 22 immune cell types in ECRSwNP and non-ECRSwNP were investigated by CIBERSORT based on transcriptome data. The core gene related pathophysiology of CRSwNP was analyzed using Weighted Gene Correlation Network Analysis according to the phenotype of the infiltrated eosinophils and nasal polyps (NP). A total of four types of immune cells (mast cells, activated dendritic cells, M2 macrophages and activated natural killer cells) were demonstrated to have a direct and indirect correlation with eosinophilic infiltration in ECRSwNP. M1 macrophages and activated CD4+ memory T cells were correlated with major immune cell types in non-ECRSwNP. NP could affect the expression of ‘olfactory receptor activity’ and ‘channel activity’ genes to impair the olfactory signaling pathway and neuroactive ligand receptor pathway. ‘Cell adhesion molecule binding’, ‘cytokine receptor binding’ and ‘glucocorticoid receptor binding’ were significantly enriched in ECRSwNP, whereas epithelial cell injury, autophagy and the mTOR pathway (hsa04140 and hsa04150) may serve an important role in the pathogenesis of non-ECRSwNP. There were significantly different immune cell infiltration and related core genes expression characteristics between ECRSwNP and non-ECRSwNP. The results of the present study provide an improved basis for elucidation of the mechanism and treatment of CRSwNP.

Background Network pharmacology uses bioinformatics to broaden our understanding of drug actions and thereby advance drug discovery. Here we apply network pharmacology to generate testable hypotheses about the multi-target mechanism of celastrol against systemic lupus erythematosus (SLE). Methods We reconstructed drug–target pathways and networks to predict the likely protein targets of celastrol and the main interactions between those targets and the drug. Then we validated our predictions of candidate targets by performing docking studies with celastrol. Results The results suggest that celastrol acts against SLE by regulating the function of several signaling proteins, such as interleukin 10, tumor necrosis factor, and matrix metalloprotein 9, which regulate signaling pathways involving mitogen-activated protein kinase and tumor necrosis factor as well as apoptosis pathways. Celastrol is predicted to affect networks involved mainly in cytokine activity, cytokine receptor binding, receptor ligand activity, receptor regulator activity, and cofactor binding. Molecular docking analysis showed that hydrogen bonding and π-π stacking were the main forms of interaction. Conclusions This network pharmacology strategy may be useful for discovery of multi-target drugs against complex diseases, specifically, it provides protein targets associated with SLE that may be further tested for therapeutic potential by celastrol.

The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function., The N-terminal domain (NTD) of interleukin-3 receptor α-subunit (IL3Rα) is involved in IL-3 recognition but the underlying mechanism is unknown. Here, the authors present crystal structures of the IL3Rα complex and provide biochemical evidence that the NTD regulates IL-3 binding and signalling complex assembly.

We predicted potential genes and identified pathways associated with hepatitis C. The gene expression profiles of GSE40184 from blood samples and GSE38597 from liver biopsy samples were downloaded from the GEO database. Differentially expressed genes (DEGs) were recognized using the Limma Package. The Pearson correlation test was used to construct the co-expression network of DEGs. Gene set enrichment analysis was used to define significant functions and pathways for DEGs. A total of 165 DEGs in blood samples and 523 DEGs in liver biopsy samples were identified. Eight DEGs were common between these samples. Gene Ontology enrichment analysis showed that 165 DEGs in blood samples were significantly enriched regarding the response to protein binding, receptor binding, G-protein coupled receptor binding, cytokine receptor binding, and cytokine activity. The most significant term of the Kyoto Encyclopedia of Genes and Genomes pathway was the cytokine-cytokine receptor interaction. Protein-protein interaction network analysis indicated that three subnetworks with more nodes and edges were involved in these interactions. We used robust biomarkers that were differentially expressed in hepatitis C and determined their relevance in the biological function, signal pathways, protein-protein interaction network, and co-expression network of hepatitis C.

Bacillus subtilis is a common group of probiotics that have been widely used in the feed industry as they can increase host resistance to pathogens and balance the immune response. However, the regulatory mechanism of Bacillus subtilis on the host immune system remains unclear in teleosts. In this study, we isolated and enriched dendritic cells from white blood cells (WBCs), and then stimulated them with Bacillus subtilis. Morphological features, specific biological functions, and authorized functional molecular markers were used in the identification of dendritic cells. Subsequently, we collected stimulated cells at 0, 4, and 18 h, and then constructed and sequenced the transcriptomic libraries. A transcriptome analysis showed that 2557 genes were up-regulated and 1708 were down-regulated at 4 h compared with the control group (|Fold Change| &ge, 4), and 1131 genes were up-regulated and 1769 were down-regulated between the cells collected at 18 h and 4 h (|Fold Change| &ge, 4). Gene Ontology (GO) annotations suggested many differentially expressed genes (DEGs) (p &lt, 0.05 and |Fold Change| &ge, 4) were involved in immune-related biological functions including immune system progress, cytokine receptor binding, and cytokine binding. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the cytokine&ndash, cytokine receptor interaction pathways were significantly enriched at both time points (p &lt, 0.05), which may play a key role in the response to stimulation. Furthermore, mRNA expression level examination of several pro-inflammatory cytokines and anti-inflammatory cytokines genes by quantitative real-time polymerase chain reaction (qRT-PCR) indicated that their expressions can be significantly increased in Bacillus subtili, which suggest that Bacillus subtilis can balance immune response and tolerance. This study provides dendritic cell (DC)-specific transcriptome data in grass carp by Bacillus subtilis stimulation, allowing us to illustrate the molecular mechanism of the DC-mediated immune response triggered by probiotics in grass carp.

Objective Toll-like receptors (TLRs) are important molecules for detecting both pathogen invasion and tissue damage. The expression of TLR4 is upregulated in ischemic stroke, at least in the subacute stage. However, the TLR downstream pathways in the context of stroke have not been well studied in previous research. The purpose of this study is to elucidate the TLR4 downstream pathways following ischemic stroke. Design and methods In this study, 12 ischemic stroke patients and 12 controls were selected from among 89 ischemic stroke patients and 166 controls. The chosen subjects had the highest levels of TLR4 mRNA in the peripheral blood. The differences in the TLR downstream signaling pathways, which were studied by using an RT2 Profiler TM PCR array system (Qiagen), were analyzed. The differentially expressed genes were analyzed by using GeneSpring GX and visualized based on the TLR pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Results The genes upregulated in stroke patients were found to be involved in the MyD88-independent pathway and in UBE2V1–TRAF6 ubiquitin-mediated proteolysis. The genes were more expressed in extracellular space, receptor binding, and cytokine receptor binding by use of gene ontology (GO) terms than in control patients. Conclusions We found that the MyD88-independent pathway and the ubiquitin-mediated proteolysis pathway, especially TRAF6, may be the most vital molecules among TLR downstream pathways in incidences of ischemic stroke.

Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.

We have developed a cloning vector for the expression of type I cytokine receptor, NO, extracellular domain (ECD)-mouse IgG1 Fc fusion proteins. The vector has a versatile polylinker that allows in-frame cloning of the receptor ECD with the mouse IgG1 sequence to encode a receptor ECD-IgG1 fusion construct. The receptor-IgG1 fusion proteins are transiently expressed in useful amounts following transfection of the expression vector into COS7 cells and G418 selection. The mouse IgG1 portion of the fusion protein provides a universal handle for purification on an affinity matrix and detection by anti-mouse IgG antibodies in ELISA or Western blot formats. The expressed receptor ECD-IgG1 fusion proteins bind their cognate ligands. In order to demonstrate that the fusion proteins have similar ligand binding affinities as the native receptors, the affinity constants (Kd) for EPOR, TNFR, IL-4R, and IL-6R-IgG1 fusion proteins were measured by surface plasmon resonance and shown to be in good agreement with published values. The TNFR-IgG1 fusion protein was employed in a demonstration of a novel ELISA format for detecting cytokine receptor binding to cytokine.

To detect inhibitors of the type-I interleukin-1 receptor in a safe and cost-effective nonisotopic environment, the use of particle concentration fluorescence (PCF) as a detection method for a high volume receptor binding assay (RBA) has been explored. A cell-free PCF-RBA has been developed using a soluble form of the type-I IL-1 receptor (sIL-1R), a nonneutralizing anti-sIL-1R monoclonal antibody and two fluorescein-labelled tracers: IL -1α ∗ and IL-1ra fused to the Escherichia coli maltose binding protein (MBP-IL-1ra∗). Both ligands showed saturable specific binding (KA values in the 109 M−1 range), although signal intensity was about 3-fold higher with MBP-IL-1ra∗. The data presented here suggest that the combined use of recombinant DNA and PCF technologies can replace the traditional cell-based, radioactive RBA when screening for inhibitors of cytokine receptors.

The amine-carboxyboranes and related derivatives have been shown to be potent anti-inflammatory and anti-osteoporosis agents. Their action in part appears to be mediated by the modulation of cytokines, e.g. TNFα or IL-1. Previous studies have demonstrated that LPS induced macrophages release of TNFα maximally at 60 to 90 min. and IL-1 from 5 to 8 hr. The amine-carboxyboranes reduced significantly the release of these cytokines but also blocked TNFα high affinity binding to UMR-106 receptor at 90 min. at 10 μM, and IL-1 high affinity binding at 5 hr. at 12.5 μM. In addition, the agents suppressed IL-8 binding to CHO K1 high affinity receptor at 24 hr. at 50 μM and IL-2 binding to HuT-8 receptors at 25 μM at 90 min. and 5 hr. Correlation of metabolic events associated with osteoporosis showed that at 90 min., when TNFα receptor binding was reduced by the agents, calcium uptake into UMR-106 cells was reduced at 10 μM as well as the acid and alkaline phosphatases, and the prostaglandin cyclo-oxygenase activities and adhesion of leukocytes and macrophages to UMR-106 cell monolayers. At 5hr. when the agents reduced IL-1 binding to UMR-106 receptors, calcitonin and 1,25-dihydrovitamin D3 binding was reduced by the agents as was acid and alkaline phosphatase, and 5′-lipoxygenase activities and white blood cell adhesion. At this time calcium uptake and proline incorporation was increased significantly by the agents. At later times e.g. 18-48 hr. calcium uptake was still increased, and NAG activity was inhibited in the presence of the agents. These effects may be related more to the inhibition of other cytokine receptor binding, e.g. IL-8. Thus, many of the observed metabolic effects of amine-carboxyboranes as antiosteoporosis agents can be correlated with their inhibition of cytokine high affinity binding to target cell receptors.

Jak1 is a tyrosine kinase that noncovalently forms tight complexes with a variety of cytokine receptors and is critically involved in signal transduction via cytokines. Jaks are predicted to have a 4.1, ezrin, radixin, moesin (FERM) domain at their N terminus. FERM domains are composed of three structurally unrelated subdomains (F1, F2, and F3) which are in close contact to one another and form the clover-shaped FERM domain. We generated a model structure of the Jak1 FERM domain, based on solved FERM structures and the alignments with other FERM domains. To destabilize different subdomains and to uncover their exact function, we mutated specific hydrophobic residues conserved in FERM domains and involved in hydrophobic core interactions. In this study, we show that the structural integrity of the F2 subdomain of the FERM domain of Jak1 is necessary to bind the IFN-γRα. By mutagenesis of hydrophobic residues in the hydrophobic core between the three FERM subdomains, we find that the structural context of the FERM domain is necessary for the inhibition of Jak1 phosphorylation. Thus, FERM domain mutations can have repercussions on Jak1 function. Interestingly, a mutation in the kinase domain (Jak1-K907E), known to abolish the catalytic activity, also leads to an impaired binding to the IFN-γRα when this mutant is expressed at endogenous levels in U4C cells. Our data show that the structural integrity of both the FERM domain and of the kinase domain is essential for both receptor binding and catalytic function/autoinhibition.

The presence of a Src homology 2 (SH2) domain sequence similarity in the sequence of Janus kinases (Jaks) has been discussed since the first descriptions of these enzymes. We performed an in depth study to determine the function of the Jak1 SH2 domain. We investigated the functionality of the Jak1 SH2 domain by stably reconstituting Jak1-defective human fibrosarcoma cells U4C with endogenous amounts of Jak1 in which the crucial arginine residue Arg466 within the SH2 domain has been replaced by lysine. This mutant still binds to the receptor subunits gp130 and OSMR. Moreover, the SH2 R466K mutation does not affect the subcellular distribution of Jak1 as assessed by cell fractionation and confocal microscopy of cells expressing endogenous levels of non-tagged or a yellow fluorescent protein (YFP)-tagged Jak1-R466K, respectively. Likewise, the signaling capacity of Jak1 was not affected by this point mutation. However, we found that the SH2 domain is structurally important for cytokine receptor binding and surface expression of the OSMR.

The interaction between receptors and kinases of the Janus kinase (Jak) family is critical for signaling by growth factors, cytokines, and IFNs. Therefore, the characterization of the domains involved in these interactions is pivotal not only in understanding kinase activation but also in the development of drugs that mimic or inhibit signaling. In this report, we have characterized the domains of Jak1 required to associate with distinct cytokine receptor subunits: IFN-αRβL, IFN-γRα, IL-10Rα, IL-2Rβ, and IL-4Rα. We demonstrate that two regions of Jak1 are necessary for the interaction with cytokine receptors. First, a common N-terminal region that includes Jak homology (JH) domain 7 and the first 19 aa of JH6, and, second, a C-terminal region (JH6–3) that was different for distinct receptors. The contribution of the two different regions of Jak1 to cytokine receptor binding was also variable. Deletion of JH7–6 impaired the association of IL-2Rβ and IL-4Rα chains with Jak1 but did not have a major impact on the binding of Jak1 to IFN-αRβL or IL-10Rα. Interestingly, regardless of the effect on receptor binding, removal of JH7–6 completely abrogated kinase activation, indicating that this domain is required for ligand-driven kinase activation and, thus, for proper signaling through cytokine receptors.

Inflammation, regardless of whether it is provoked by infection or by tissue damage, starts with the activation of macrophages which initiate a cascade of inflammatory responses by producing the cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (ref. 1). Three naturally occurring ligands for the IL-1 receptor (IL1R) exist: the agonists IL-1alpha and IL-1beta and the IL-1-receptor antagonist IL1RA (ref. 2). IL-1 is the only cytokine for which a naturally occurring antagonist is known. Here we describe the crystal structure at 2.7 A resolution of the soluble extracellular part of type-I IL1R complexed with IL1RA. The receptor consists of three immunoglobulin-like domains. Domains 1 and 2 are tightly linked, but domain three is completely separate and connected by a flexible linker. Residues of all three domains contact the antagonist and include the five critical IL1RA residues which were identified by site-directed mutagenesis. A region that is important for biological function in IL-1beta, the 'receptor trigger site' is not in direct contact with the receptor in the IL1RA complex. Modelling studies suggest that this IL-1beta trigger site might induce a movement of domain 3.