TNF Pathways Research Articles

Screening of plasma exosomal lncRNAs to identify potential biomarkers for obstructive sleep apnea

BackgroundObstructive sleep apnea (OSA) is highly prevalent, but frequently undiagnosed. The existing biomarkers of OSA are relatively insensitive and inaccurate. Long non-coding RNAs (lncRNAs) have no protein-coding ability but have a role in regulating gene expression. They are stably expressed in exosomes, easily and rapidly measurable. Changes in expression of exosomal lncRNAs can be useful for disease diagnoses. However, there are few reports on the association of exosomal lncRNAs with OSA. We aimed to investigate the exosomal lncRNA profiles to establish the differences between non-OSA, OSA with or without hypertension (HTN) and serve as a potential diagnostic biomarker.MethodsThis diagnostic test included 63 participants: [normal control (NC) =25], (OSA =23), and (HTN-OSA =15). Expression profiling of lncRNAs in isolated exosomes was performed through high-throughput sequencing in 9 participants. Subsequently, OSA/HTN-OSA related lncRNAs were selected for validation by droplet digital polymerase chain reaction (ddPCR), receiver operating characteristic (ROC) curves were used to determine the diagnostic value. The reliabilities of the screened gene were further validated in another independent cohort: (NC =10), (OSA mild =10), (OSA moderate =11), and (OSA severe =10), the correlation between clinical features and its expression was analyzed. The MiRanda software was used to predict the binding sites of interaction between microRNA (miRNA) and target genes regulated by screened lncRNA.ResultsWe identified the differentially expressed lncRNAs and mRNAs in plasma exosomes of the NC, OSA, HTN-OSA groups. Most pathways enriched in differentially expressed lncRNAs and mRNAs had previously been linked to OSA. Among them, ENST00000592016 enables discrimination between NC and OSA individuals [area under curve (AUC) =0.846, 95% confidence interval (CI): 0.72–0.97]. The severity of OSA was associated with changes in the ENST00000592016 expression. Furthermore, ENST00000592016 affected the PI3K-Akt, MAPK, and TNF pathways by regulating miRNA expressions.ConclusionsThis is the first report about differential expression of lncRNA in OSA and HTN-OSA exosomes. ENST00000592016 enables discrimination between NC and OSA individuals. This work enabled characterization of OSA and provided the preliminary work for the study of biomarker of OSA.

A Study on the Mechanism of Herbal Drug FDY003 for Colorectal Cancer Treatment by Employing Network Pharmacology

Colorectal cancer (CRC) originates from the uncontrolled growth of epithelial cells in the colon or rectum. Annually, 1.9 million new CRC cases are being reported, causing 0.9 million deaths worldwide. The suppressive effects of the herbal prescription FDY003, a mixture of Cordyceps militaris, Lonicera japonica Thunberg, and Artemisia capillaris Thunberg, against CRC have previously been reported. Nonetheless, the multiple compound-multiple target mechanisms of FDY003 in CRC cells have not been fully elucidated. In this study, we used network pharmacology (NP) to analyze the polypharmacological mechanisms of action of FDY003 in CRC treatment. FDY003 promoted the suppression of viability of CRC cells and strengthened their sensitivity to anticancer drugs. The NP study enabled the investigation of 17 pharmaceutical compounds and 90 CRC-related genes that were targets of the compounds. The gene ontology terms enriched with the CRC-related target genes of FDY003 were those involved in the control of a variety of phenotypes of CRC cells, for instance, the decision of apoptosis and survival, growth, stress response, and chemical response of cells. In addition, the targeted genes of FDY003 were further enriched in various Kyoto Encyclopedia of Genes and Genomes pathways that coordinate crucial pathological processes of CRC; these are ErbB, focal adhesion, HIF-1, IL-17, MAPK, PD-L1/PD-1, PI3K-Akt, Ras, TNF, and VEGF pathways. The overall analysis results obtained from the NP methodology support the multiple-compound-multiple-target-multiple-pathway pharmacological features of FDY003 as a potential agent for CRC treatment.

Network pharmacology-based pharmacological mechanism prediction on Eucommia ulmoides against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common chronic autoimmune disease characterized by synovial inflammation and progressive joint destruction. Eucommia ulmoides (EU) is a kidney-tonifying Chinese medicine that has been applied to treat RA for decides. The present study aims to explore pharmacological mechanisms of EU against RA using network pharmacology approach. Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to screen active ingredients of EU, and their relative targets were fished from UniProt database. RA-related targets were screened from GeneCards database and DisGeNET database. The overlapping genes between EU and RA were identified by Venn diagram, and further analyzed for protein-protein interaction (PPI), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG). Fifty active ingredients were identified in EU, and corresponded to 207 targets. Meanwhile, 499 targets were closely associated with RA development. A total of 50 overlapping genes between EU and RA were identified, which were regarded as therapeutically relevant. GO enrichment analysis indicated that EU exerted antiRA effects depending on regulating multiple biological processes including inflammatory response, oxidative stress, cell apoptosis and matrix catabolism. Several key pathways such as TNF pathway, IL-17 pathway, T cell receptor pathway, NOD-like receptor pathway and Toll-like receptor pathway, were involved in the above biological processes. Network pharmacology revealed that EU exerts therapeutic effects on RA through multi-ingredients, multi-targets and multi-pathways, which provides basis for its clinical application and promising directions for subsequent research.

Qinzhi Zhudan formula improves memory and alleviates neuroinflammation in vascular dementia rats partly by inhibiting the TNFR1-mediated TNF pathway

The Qinzhi Zhudan formula (QZZD) exhibits a prominent therapeutic effect in the treatment of vascular dementia (VaD). This study combined a network pharmacology approach and experimental validation to identify the underlying biological mechanism of QZZD against VaD. Male Wistar rats received bilateral common carotid artery occlusion (BCCAO) surgery, and after 4 weeks of intragastric administration of QZZD, the therapeutic effect was assessed using the Morris water maze test and cerebral blood flow (CBF) assessment. Hematoxylin and eosin staining, Nissl staining, and electron microscopy were used to measure the histopathological changes in the neurons of rats. The effect of QZZD treatment on hippocampal neurotransmitters was assessed by high-performance liquid chromatography with electrochemical detection and liquid chromatography mass spectrometry. Immunofluorescence was used to observe VaD-induced microglia activation. The inflammatory cytokine levels were assessed by enzyme linked immunosorbent assay. Western blot was used to examine the TNFR1-mediated TNF pathway, which was screened out by network pharmacology analysis. QZZD treatment alleviated pathological changes and neuronal damage in VaD rats and attenuated their cognitive impairment. In addition, QZZD increased CBF and the expression of acetylcholine and 5-hydroxytryptamine in the hippocampal region. Notably, QZZD inhibited microglial activation and the expression of IL-6 and TNF-α. Network pharmacology and western blot indicated that QZZD inhibited the levels of TNFR1, NF-κBp65, p-ERK, TNF-α, and IL-6, which are related to the TNFR1-mediated TNF signaling pathway. QZZD clearly improved learning and memory function, reduced brain pathological damage, elevated CBF and hippocampal neurotransmitter levels, and alleviated neuroinflammation of VaD rats partly by inhibiting the TNFR1-mediated TNF pathway, indicating its potential value in the clinical therapy of VaD.

Changes in the expression of inflammatory and epigenetic-modulatory genes after an intensive meditation retreat

BackgroundMeditation retreats are characterized by intensive or concentrated periods of meditation practice, commonly undertaken in a residential setting. Although research indicates that meditation training can positively influence physical and mental health outcomes, the biological consequences of meditation retreat interventions are relatively understudied. In this study, we examined the influence of a month-long, silent meditation retreat on the expression of genes involved in epigenetic modulation and immune processes. MethodWe assessed gene expression changes in experienced meditators attending a month-long Insight meditation retreat (n = 28), as compared to a community control group (n = 34) of experienced practitioners living their everyday lives. Blood samples were collected on day two of the retreat (Time 1) and again 3 weeks later (Time 2). Control participants were also assessed across a 3-week interval, during which they maintained their regular daily routines. ResultsAs compared to controls, retreat participants showed differential changes in the expression of several genes involved in chromatin modulation and inflammation. The most substantive finding was downregulation of the TNF pathway in retreat participants, which was not observed in controls. ConclusionsThese findings indicate that meditation retreat participation may influence some of the inflammatory mechanisms involved in the development of chronic diseases, and that this style of psychosocial intervention may have therapeutic potential, particularly in experienced practitioners.

Synergistic anti-proliferative activity of JQ1 and GSK2801 in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) constitutes 10–20% of breast cancers and is challenging to treat due to a lack of effective targeted therapies. Previous studies in TNBC cell lines showed in vitro growth inhibition when JQ1 or GSK2801 were administered alone, and enhanced activity when co-administered. Given their respective mechanisms of actions, we hypothesized the combinatorial effect could be due to the target genes affected. Hence the target genes were characterized for their expression in the TNBC cell lines to prove the combinatorial effect of JQ1 and GSK2801.MethodsRNASeq data sets of TNBC cell lines (MDA-MB-231, HCC-1806 and SUM-159) were analyzed to identify the differentially expressed genes in single and combined treatments. The topmost downregulated genes were characterized for their downregulated expression in the TNBC cell lines treated with JQ1 and GSK2801 under different dose concentrations and combinations. The optimal lethal doses were determined by cytotoxicity assays. The inhibitory activity of the drugs was further characterized by molecular modelling studies.ResultsGlobal expression profiling of TNBC cell lines using RNASeq revealed different expression patterns when JQ1 and GSK2801 were co-administered. Functional enrichment analyses identified several metabolic pathways (i.e., systemic lupus erythematosus, PI3K-Akt, TNF, JAK-STAT, IL-17, MAPK, Rap1 and signaling pathways) enriched with upregulated and downregulated genes when combined JQ1 and GSK2801 treatment was administered. RNASeq identified downregulation of PTPRC, MUC19, RNA5-8S5, KCNB1, RMRP, KISS1 and TAGLN (validated by RT-qPCR) and upregulation of GPR146, SCARA5, HIST2H4A, CDRT4, AQP3, MSH5-SAPCD1, SENP3-EIF4A1, CTAGE4 and RNASEK-C17orf49 when cells received both drugs. In addition to differential gene regulation, molecular modelling predicted binding of JQ1 and GSK2801 with PTPRC, MUC19, KCNB1, TAGLN and KISS1 proteins, adding another mechanism by which JQ1 and GSK2801 could elicit changes in metabolism and proliferation.ConclusionJQ1-GSK2801 synergistically inhibits proliferation and results in selective gene regulation. Besides suggesting that combinatorial use could be useful therapeutics for the treatment of TNBC, the findings provide a glimpse into potential mechanisms of action for this combination therapy approach.

KCa3.1 differentially regulates trachea and bronchi epithelial gene expression in a chronic-asthma mouse model.

Ion channels are potentially exploitable as pharmacological targets to treat asthma. This study evaluated the role of KCa3.1 channels, encoded by Kcnn4, in regulating the gene expression of mouse airway epithelium and the development of asthma traits. We used the ovalbumin (OVA) challenge as an asthma model in wild-type and Kcnn4-/- mice, performed histological analysis, and measured serum IgE to evaluate asthma traits. We analyzed gene expression of isolated epithelial cells of trachea or bronchi using mRNA sequencing and gene ontology and performed Ussing chamber experiments in mouse trachea to evaluate anion secretion. Gene expression of epithelial cells from mouse airways differed between trachea and bronchi, indicating regional differences in the inflammatory and transepithelial transport properties of proximal and distal airways. We found that Kcnn4 silencing reduced mast cell numbers, mucus, and collagen in the airways, and reduced the amount of epithelial anion secretion in the OVA-challenged animals. In addition, gene expression was differentially modified in the trachea and bronchi, with Kcnn4 genetic silencing significantly altering the expression of genes involved in the TNF pathway, supporting the potential of KCa3.1 as a therapeutic target for asthma.

Exploration on mechanism of Polygalae Radix and Acori Tatarinowii Rhizoma in treating Alzheimer's disease based on network pharmacology and experimental verification

This study aimed to explore the action targets and mechanisms of Polygala tenuifolia and Acorus tatarinowii in treating Alzheimer's disease(AD) based on network pharmacology, molecular docking, and animal tests. The AD-related targets were collec-ted from GeneCard and the main active ingredients and targets of P. tenuifolia and A. tatarinowii from the TCMSP. Cytoscape was applied to construct the "Chinese herb-active ingredient-target-disease" network, followed by the construction of protein-protein interaction(PPI) network using STRING. GO biological function and KEGG pathway enrichment analysis was performed by DAVID and Metascape. The main active components of P. tenuifolia and A. tatarinowii and their potential core targets were docking using AutoDock Vina. The effects of P. tenuifolia and A. tatarinowii on the cognitive function were verified in mice with scopolamine(SCOP)-induced cognitive impairment. A total of seven active ingredients including kaempferol, onjixanthone Ⅰ, and marmesin and 56 potential targets of P. tenuifolia and A. tatarinowii were screened out, with the core targets covering AKT1, PTGS2, TNF, and NF-κB inflammation pathway mainly involved. The results of molecular docking also showed that the main active components of P. tenuifolia and A. tatarinowii stably bond to the core targets predicted by network pharmacology. The new object recognition experiment suggested that P. tenuifolia and A. tatarinowii improved the learning and memory abilities of mice after SCOP induction. As revealed by pathological section observation and relevant kit assay, P. tenuifolia and A. tatarinowii reduced the damage of central cholinergic neurons and enhanced the antioxidant ability of SCOP-induced mice. Western blot confirmed that P. tenuifolia and A. tatarinowii down-regulated the protein expression levels of TLR4, NF-κB, and related inflammatory factors(TNF-α, IL-1β, and IL-6). All these have suggested that P. tenuifolia and A. tatarinowii inhibits AD via multiple components, multiple targets, and multiple pathways, which has provided an experimental basis for the clinical application of P. tenuifolia and A. tatarinowii for the treatment of AD.

Single-Cell RNA and ATAC Sequencing Reveal Hemodialysis-Related Immune Dysregulation of Circulating Immune Cell Subpopulations.

BackgroundAn increased risk of infection, malignancy, and cardiovascular diseases in maintenance hemodialysis patients is associated with hemodialysis-related immunity disturbances. Although defects in T-lymphocyte-dependent immune responses and preactivation of antigen-presenting cells have been documented in hemodialysis patients, the effects of long-term hemodialysis on the transcriptional program and chromosomal accessibility of circulating immune cell subpopulations remain poorly defined.MethodsWe integrated single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to characterize the transcriptome profiles of peripheral mononuclear cells (PBMCs) from healthy controls and maintenance hemodialysis patients. Validation of differentially expressed genes in CD4+ T cells and monocytes were performed by magnetic bead separation and quantitative real-time PCR.ResultsWe identified 16 and 15 PBMC subgroups in scRNA-seq and scATAC-seq datasets, respectively. Hemodialysis significantly suppressed the expression levels of T cell receptor (TCR) genes in CD4+ T cell subsets (e.g., TRAV4, CD45, CD3G, CD3D, CD3E) and major histocompatibility complex II (MHC-II) pathway-related genes in monocytes (HLA-DRB1, HLA-DQA2, HLA-DQA1, HLA-DPB1). Downstream pathways of TCR signaling, including PI3K-Akt-mTOR, MAPK, TNF, and NF-κB pathways, were also inhibited in CD4+ T cell subpopulations during the hemodialysis procedure. Hemodialysis altered cellular communication patterns between PBMC subgroups, particularly TGF-TGFBR, HVEM-BTLA, and IL16-CD4 signalings between CD4+ T cells and monocytes. Additionally, we found that hemodialysis inhibited the expression of AP-1 family transcription factors (JUN, JUND, FOS, FOSB) by interfering with the chromatin accessibility profile.ConclusionsOur study provides a valuable framework for future investigations of hemodialysis-related immune dysregulation and identifies potential therapeutic targets for reconstituting the circulating immune system in maintenance hemodialysis patients.

Exploration of the System-Level Mechanisms of the Herbal Drug FDY003 for Pancreatic Cancer Treatment: A Network Pharmacological Investigation.

Pancreatic cancer (PC) is the most lethal cancer with the lowest survival rate globally. Although the prescription of herbal drugs against PC is gaining increasing attention, their polypharmacological therapeutic mechanisms are yet to be fully understood. Based on network pharmacology, we explored the anti-PC properties and system-level mechanisms of the herbal drug FDY003. FDY003 decreased the viability of human PC cells and strengthened their chemosensitivity. Network pharmacological analysis of FDY003 indicated the presence of 16 active phytochemical components and 123 PC-related pharmacological targets. Functional enrichment analysis revealed that the PC-related targets of FDY003 participate in the regulation of cell growth and proliferation, cell cycle process, cell survival, and cell death. In addition, FDY003 was shown to target diverse key pathways associated with PC pathophysiology, namely, the PIK3-Akt, MAPK, FoxO, focal adhesion, TNF, p53, HIF-1, and Ras pathways. Our network pharmacological findings advance the mechanistic understanding of the anti-PC properties of FDY003 from a system perspective.

Adiponectin Ameliorates Hyperglycemia-Induced Retinal Endothelial Dysfunction, Highlighting Pathways, Regulators, and Networks.

BackgroundThe pathophysiology of diabetic retinopathy (DR) is multifaced. A low level of circulating adiponectin (APN) in type 2 diabetes is associated with microvasculature complications, and its role in the evolution of DR is complex.AimThis study is designed to explore the potential impact of APN in the pathogenesis of DR, linking the changes in cellular and biological processes with the pathways, networks, and regulators involved in its actions.MethodsHuman microvascular retinal endothelial cells (HMRECs) were exposed to 30mM glucose (HG) and treated with globular adiponectin (30μg/mL) for 24 hours. The cells were evaluated for reactive oxidative stress (ROS) and apoptosis. RT-PCR profile arrays were utilized to evaluate the profile of genes involved in endothelial functions, angiogenesis, extracellular matrix, and adhesion molecules for hyperglycemic HMRECs treated with adiponectin. In addition, the barrier function, leukocyte migration, and angiogenesis were evaluated. The differential expressed genes (DEGs) were outlined, and bioinformatic analysis was applied.ResultsAdiponectin suppresses ROS production and apoptosis in HMRECs under HG conditions. Adiponectin improved migration and barrier functions in hyperglycemic cells. The bioinformatic analysis highlighted that the signaling pathways of integrin, HMGB1, and p38 AMPK, are mainly involved in the actions of APN on HMRECs. APN significantly affects molecular functions, including the adhesion of cells, chemotaxis, migration of WBCs, and angiogenesis. STAT3, NFKB, IKBKB, and mir-8 are the top upstream regulators, which affect the expressions of the genes of the data set, while TNF and TGFB1 are the top regulators.ConclusionAdiponectin significantly counteracts hyperglycemia at various cellular and molecular levels, reducing its impact on the pathophysiological progression towards DR in vitro using HMRECs. Adiponectin ameliorates inflammatory response, oxidative stress, and endothelial barrier dysfunction using a causal network of NFBk complex, TNF, and HMGB1 and integrin pathways.

Abstract 516: Splice Factor Polypyrimidine Tract-binding Protein 1 (PTBP1) Primes Endothelial Inflammation In Atherogenic Disturbed Flow Conditions

NFκB mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through upregulation of adhesion molecules Icam1 and Vcam. The endothelium is primed for cytokine activation of NFκB by exposure to low and disturbed blood flow (LDF). While priming leads to an exaggerated expression of Icam1 and Vcam following cytokine stimulation, the molecular underpinnings are not understood. In a model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including Polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NFκB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam induction by TNFα stimulation as a readout, we performed a CRISPR-Cas9 screen of these factors and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no obvious effect on cell proliferation or viability, but reversed LDF splicing patterns and inhibited NFκB nuclear translocation and transcriptional activation of nearly all downstream targets, including Icam1 and Vcam. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo , endothelial specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice and limited atherosclerosis. Together, our data show that Ptbp1, which is induced in a subset of the endothelium by innate immune cell recruitment at regions of LDF, is required for priming of the endothelium for subsequent NFκB activation, myeloid cell recruitment and atherosclerosis.

Network Pharmacological Analysis and Experimental Study of the Antipharyngitis Mechanism of the Chaiqin Qingning Capsule.

Objective The study aimed to explore the active composition and mechanism of the Chaiqin Qingning capsule (CQQN) against pharyngitis based on the network pharmacology and through using a pharyngitis rat model. Methods The active ingredients and targets of CQQN were queried using the TCMSP database. Disease-related target genes were queried in the DrugBank, GeneCards, OMIM, and DisGeNEt databases using “pharyngitis” as the search term. The STRING database was used to establish a protein-protein interaction (PPI) network. GO function enrichment and KEGG pathway enrichment analyses were performed to identify active components and key targets. Cytoscape software (version 3.7.2) was used to construct an active component/target gene/enrichment pathway network. AutoDock software was used to select the best binding target for molecular docking. The effect of CQQN was verified in the pharyngitis rats. Results Network pharmacology showed 30 active compounds in CQQN with 240 targets, including 54 for the treatment of pharyngitis. Potential active ingredients included quercetin, kaempferol, stigmasterol, saikosaponin D, and isorhamnetin. The key targets involved were AKT1, TNF, IL-6, and IL-1β. Signaling pathways included virus infection, TNF, IL-17, and cancer pathways. The molecular docking results showed that the critical components in CQQN had good potential for binding to key target genes. Animal experiments showed that CQQN could significantly reduce the expression of TNF-α, IL-1β, IL-6, and IL-17 in the serum of rats with pharyngitis (P < 0.05). Hematoxylin and eosin staining showed that the inflammatory state of pharyngeal tissue in rats was significantly reduced compared to that in the model group. Conclusion CQQN can improve pharyngitis by regulating the TNF and IL-17 signaling pathways. The study makes a positive exploration and provides a new idea for a more comprehensive and in-depth excavation of CQQN with an intervention effect on pharyngitis and other upper respiratory diseases in the future.

Analysis of the miRNA-mRNA Regulatory Network Reveals the Biomarker Genes in the Progression of Myocardial Ischemic Reperfusion.

Objective Cardiac injury induced by myocardial ischemic reperfusion (MI/R) is still an intractable question in clinical, and it has been confirmed as a major reason for the development of cardiovascular disease. Bioinformatics analysis has been widely used for revealing the pathogenic mechanism of diseases. This study attempted to identify the biomarkers and reveal the regulation mechanism of MI/R injury via bioinformatics analysis. Methods The GSE67308 and GSE74951 were obtained from the GEO database. The datasets were analyzed with GEO2R tool, and the genes with |logFC| > 2 and p value <0.05 were identified as the differentially expressed genes (DEGs). The enrichment analysis of the DEGs was performed with the DAVID database and R language. Moreover, the protein-protein interaction (PPI) network of DEGs was performed with the STRING database and then visualized with Cytoscape. Result The results showed that 195 downregulated mRNAs and 240 downregulated mRNAs were found in GSE67308, and 11 miRNAs were found in GSE7495. 152 common genes were screened in DEGs of GSE67308 and the targets of 11 miRNAs in GSE7495. Moreover, the enrichment analysis showed that the common genes were related with inflammatory response, immune response, PI3K/AKT, NF-κB, and TNF pathways. Besides, mmu-miR-92a-3p and mmu-miR-27b-3p were identified as the hubs miRNAs, and TNF, IL1B, and IFG1 were screened as the key nodes. Conclusion This study established a miRNA-mRNA network for cardiac injury induced by MI/R and provided the evidence concerning the molecular mechanism of MI/R injury, which provided some reference for MI/R treatment.

Utilising Network Pharmacology to Explore Underlying Mechanism of Astragalus membranaceus in Improving Sepsis-Induced Inflammatory Response by Regulating the Balance of IκBα and NF-κB in Rats.

Objective The purpose of the present study was to explore the mechanism of Astragalus membranaceus in the treatment of sepsis. Methods We searched the active components and targets of Astragalus membranaceus using the TCMSP and BATMAN databases. Then, the GeneCards, MalaCards, and OMIM databases were used to screen out relevant targets of sepsis. The common targets of the former two gene sets were uploaded to the STRING database to create an interaction network. DAVID was used to perform KEGG enrichment analysis of the core targets. Based on the results of KEGG and previous studies, key pathways for the development of sepsis were identified and experimentally validated. Result We obtained 3,370 sepsis-related targets in databases and 59 active components in Astragalus membranaceus through data mining, corresponding to 1,130 targets. The intersection of the two types of targets led to a total of 318 common targets and 84 core targets were obtained after screening again. The KEGG and previous studies showed that these 84 core targets were involved in sepsis by regulating TNF, MAPK, and PI3K pathways. TNF, MAPK8, NF-κB, and IκBα are crucial in sepsis. Experimental validation demonstrated that some markers in sepsis model rats were improved after the intervention with Astragalus granules and their chemical components. Among them, IL-1β, IL-6, and TNF-α in rat serum were reduced. The mRNA and protein expression of TNF-α, IL-6, MMP9, MAPK8, and NF-κB were reduced in rat blood. However, the mRNA and protein expression of IκBα and PI3K were increased in rat blood. Conclusion The AST could affect the TNF, PI3K, and MAPK pathway cascade responses centred on IκBα and NF-κB, attenuate the expression of IL-6 and MMP9, and interfere with the inflammatory response during sepsis.

Single-Cell RNA Sequencing of Peripheral Blood Mononuclear Cells From Pediatric Coeliac Disease Patients Suggests Potential Pre-Seroconversion Markers.

Celiac Disease (CeD) is a complex immune disorder involving villous atrophy in the small intestine that is triggered by gluten intake. Current CeD diagnosis is based on late-stage pathophysiological parameters such as detection of specific antibodies in blood and histochemical detection of villus atrophy and lymphocyte infiltration in intestinal biopsies. To date, no early onset biomarkers are available that would help prevent widespread villous atrophy and severe symptoms and co-morbidities. To search for novel CeD biomarkers, we used single-cell RNA sequencing (scRNAseq) to investigate PBMC samples from 11 children before and after seroconversion for CeD and 10 control individuals matched for age, sex and HLA-genotype. We generated scRNAseq profiles of 9559 cells and identified the expected major cellular lineages. Cell proportions remained stable across the different timepoints and health conditions, but we observed differences in gene expression profiles in specific cell types when comparing patient samples before and after disease development and comparing patients with controls. Based on the time when transcripts were differentially expressed, we could classify the deregulated genes as biomarkers for active CeD or as potential pre-diagnostic markers. Pathway analysis showed that active CeD biomarkers display a transcriptional profile associated with antigen activation in CD4+ T cells, whereas NK cells express a subset of biomarker genes even before CeD diagnosis. Intersection of biomarker genes with CeD-associated genetic risk loci pinpointed genetic factors that might play a role in CeD onset. Investigation of potential cellular interaction pathways of PBMC cell subpopulations highlighted the importance of TNF pathways in CeD. Altogether, our results pinpoint genes and pathways that are altered prior to and during CeD onset, thereby identifying novel potential biomarkers for CeD diagnosis in blood.

Atractyloside-A ameliorates spleen deficiency diarrhea by interfering with TLR4/MyD88/NF-κB signaling activation and regulating intestinal flora homeostasis

PurposeSpleen deficiency diarrhea (SDD) is one of the most common types of diarrhea and is linked to intestinal barrier dysfunction and intestinal flora disorders. Atractyloside-A (AA) is one of the main components of Atractylodes Lancea(Thunb.) DC., which acts on the gastrointestinal tract and has therapeutic effects on diarrhea. Folium sennae is a medicinal plant inducing diarrhea; thus, it is one of the effective methods to obtain a diarrhea model. However, the mechanism of action of AA in the treatment of SDD induced by Folium sennae is unclear. MethodsThe intestinal thrapeutic effect of AA on SDD in mice was evaluated by colon pathology. RNA sequencing (RNA-seq) was used to analyze the colonic transcriptome profiles. In addition, 16S rDNA sequencing and fecal microbiota transplantation (FMT) were carried out to verify the role of AA in the regulation of the intestinal flora. ResultsThe findings revealed that AA alleviated SDD by ameliorating the pathological symptoms while suppressing intestinal inflammatory responses through the TLR4/MyD88/NF-kB signaling and reversing the impairment of mucin synthesis. Furthermore, AA improved the integrity of the intestinal barrier. RNA-seq identified 436 common DEGs out of 1033 DEGs between SDD and AA, and 1933 DEGs between SDD and Ctrl, which are highly enriched in the NF-κB and TNF pathways. Moreover, AA altered the composition of the intestinal flora and FMT reduced SDD. ConclusionAA exerted a therapeutic effect on SDD through the regulation of the intestinal flora and the inflammation by interfering with the TLR4/MyD88/NF-κB signaling pathway.

Vasorelaxant Activities and its Underlying Mechanisms of Magnolia Volatile Oil on Rat Thoracic Aorta Based on Network Pharmacology.

Objective: Magnolia volatile oil (MVO) is a mixture mainly containing eudesmol and its isomers. This study was to investigate the vasorelaxant effects and the underlying mechanism of MVO in rat thoracic aortas. Method: The present study combined gas chromatography–mass spectrometry (GC-MS) and network pharmacology analysis with in vitro experiments to clarify the mechanisms of MVO against vessel contraction. A compound–target network, compound–target–disease network, protein–protein interaction network, compound–target–pathway network, gene ontology, and pathway enrichment for hypertension were applied to identify the potential active compounds, drug targets, and pathways. Additionally, the thoracic aortic rings with or without endothelium were prepared to explore the underlying mechanisms. The roles of the PI3K-Akt-NO pathways, neuroreceptors, K+ channels, and Ca2+ channels on the vasorelaxant effects of MVO were evaluated through the rat thoracic aortic rings. Results: A total of 29 compounds were found in MVO, which were identified by GC-MS, of which 21 compounds with a content of more than 0.1% were selected for further analysis. The network pharmacology research predicted that beta-caryophyllene, palmitic acid, and (+)-β-selinene might act as the effective ingredients of MVO for the treatment of hypertension. Several hot targets, mainly involving TNF, CHRM1, ACE, IL10, PTGS2, REN, and F2, and pivotal pathways, such as the neuroactive ligand–receptor interaction, the calcium signaling pathway, and the PI3K-Akt signaling, were responsible for the vasorelaxant effect of MVO. As expected, MVO exerted a vasorelaxant effect on the aortic rings pre-contracted by KCl and phenylephrine in an endothelium-dependent and non-endothelium-dependent manner. Importantly, a pre-incubation with indomethacin (Indo), N-nitro-L-arginine methyl ester, methylene blue, wortmannin, and atropine sulfate as well as 4-aminopyridione diminished MVO-induced vasorelaxation, suggesting that the activation of the PI3K-Akt-NO pathway and KV channel were involved in the vasorelaxant effect of MVO, which was consistent with the results of the Kyoto Encyclopedia of Genes and the Genomes. Additionally, MVO could significantly inhibit Ca2+ influx resulting in the contraction of aortic rings, revealing that the inhibition of the calcium signaling pathway exactly participated in the vasorelaxant activity of MVO as predicted by network pharmacology. Conclusion: MVO might be a potent treatment of diseases with vascular dysfunction like hypertension. The underlying mechanisms were related to the PI3K-Akt-NO pathway, KV pathway, as well as Ca2+ channel, which were predicted by the network pharmacology and verified by the experiments in vitro. This study based on network pharmacology provided experimental support for the clinical application of MVO in the treatment of hypertension and afforded a novel research method to explore the activity and mechanism of traditional Chinese medicine.

Protective Effects of the Wenfei Buqi Tongluo Formula on the Inflammation in Idiopathic Pulmonary Fibrosis through Inhibiting the TLR4/MyD88/NF-κB Pathway.

Background Idiopathic pulmonary fibrosis (IPF) is a progressive disease with high mortality and poor prognosis. The prognostic signatures related to conventional therapy response remain limited. The Wenfei Buqi Tongluo (WBT) formula, a traditional Chinese medicine (TCM) formula, has been widely utilized to treat respiratory diseases in China, which is particularly effective in promoting inflammatory absorption. In this study, we aim to explore the mechanism of the WBT formula in the inhibition of inflammatory response during IPF, based on network pharmacology and in vivo experiments. Methods Network pharmacology was applied to predict the changes of biological processes and potential pathways for the WBT formula against IPF. Histopathological changes, inflammatory factors (IL-6, IL-1β, and TNF-α), and the proteins of the TLR4/MyD88/NF-κB pathway in bleomycin- (BLM-) induced mice model were examined by hematoxylin-eosin (H&E), Masson or immunohistochemistry staining, Western blot, and enzyme-linked immunosorbent assay analysis. Results A total of 163 possible components and 167 potential targets between the WBT formula and IPF were obtained. The enrichments of network pharmacology showed that inflammation response, TNF, and NF-κB pathways were involved in the treatment of WBT against IPF. The in vivo experiments indicated that the WBT formula could ameliorate inflammatory exudation and collagen deposition at a histopathology level in the BLM-induced mice model. The levels of IL-6, IL-1β, and TNF-α were reduced after the WBT formula treatment. Moreover, the expressions of phosphorylated-NF-κB p65, TLR4, and MyD88 were significantly downregulated by the WBT formula, compared with the BLM-induced group. Conclusion These results indicated that the WBT formula can suppress BLM-induced IPF in a mouse model by inhibiting the inflammation via the TLR4/MyD88/NF-κB pathway. This study provides a new insight into the molecular mechanisms of the WBT formula in the application at the clinic.

DOP86 Mirikizumab-induced upregulation of colonic transcripts correlates with improvements in stool frequency in a phase 2 study of patients with moderately to severely active Ulcerative Colitis

Abstract Background We have previously shown that treatment with mirikizumab (miri), a p19-directed IL-23 antibody, significantly downregulates inflammatory genes associated with disease activity and upregulates genes expressing epithelial transporter proteins in colonic tissue in patients with ulcerative colitis (UC). Here we explored the correlation between the expression of colonic mucosa genes and stool frequency (SF), a symptom reflective of disease activity, during the 12-week induction period of a Phase 2 study of patients with moderately to severely active UC (NCT02589665). Methods Patients were randomised 1:1:1:1 to receive intravenous placebo (PBO), miri 50mg or 200mg with possibility of exposure-based dose increases, or fixed miri 600mg every 4 weeks for 12 weeks. SF was reported daily by patients and transformed on a 4-level ordinal scale [0–3] representing increased SF above their normal or healthy baseline (BL). Patient colonic biopsies (PBO N=58, miri 50mg N=52, 200mg N=51, 600mg N=54) were collected at BL and Week (W)12, and gene expression measured using an Affymetrix HTA2.0 microarray workflow. BL and W12 gene expression or SF values were pooled and associations identified based on non-parametric Kendall’s tau. Pathway analysis (Hallmark and Reactome) of correlated genes was performed using over-representation analysis. p values of enrichment were determined by hypergeometric distribution test and adjusted for multi-testing with Benjamini-Hochberg procedure. Differential gene expression after miri treatment was determined by paired t-test comparing expression levels at BL and at W12 using data from the 200mg treatment group. Results A total of 267 genes were correlated with SF (|tau| &amp;gt;0.3 and qval &amp;lt;0.001). Of these, 212 were positively correlated (high expression associated with high SF) and 55 were negatively correlated (high expression associated with low SF). The 212 transcripts that were positively correlated with SF were uniformly and consistently downregulated with miri treatment, while the 55 transcripts that negatively correlated with SF, were consistently upregulated with miri treatment (Table 1). Biological pathways significantly associated with the miri-responsive transcripts that correlated with SF included inflammatory response, extracellular matrix dysregulation, neutrophil degranulation and cytokine signaling pathways, especially TNF and IL6 pathways (Table 2). Conclusion This is the first study to identify colon-based transcripts that correlate with a clinical disease activity measure, stool frequency, and it demonstrates that treatment with miri may upregulate genes associated with normalization of SF and down regulate genes associated with inflammation in colonic tissue samples of patients with UC.