Toll-like Receptor Signaling Pathway Research Articles

SNASP Mutation Aggravates to the TLR4-Mediated Inflammation in SLE by TAK1 Pathway.

Genetic factors play an important role in the pathogenesis of systemic lupus erythematosus (SLE), and abnormal Toll-like receptor (TLR) signaling pathways are closely related to the onset of SLE. In previous studies, we found that the mutant somatic nuclear autoantigenic sperm protein (sNASP) gene in the mouse lupus susceptibility locus Sle2 can promote the development of lupus model mice, but the mechanism is still unclear. Here, we stimulated mouse peritoneal macrophages with different concentrations of lipopolysaccharide. The results showed that sNASP gene mutations can promote the response of the TLR4-TAK1 signaling pathway but have no significant effect on the TLR4-TBK1 signaling pathway. sNASP mutations enhanced TLR4-mediated nuclear factor-κ-gene binding and mitogen-activated protein kinase activation and IL-6, tumor necrosis factor secretion in murine peritoneal macrophages. Collectively, our study revealed the impact of sNASP gene mutation on the sensitivity of TLR4 receptors in mouse peritoneal macrophages and shed light on potential mechanisms underlying inflammation in autoimmune diseases.

Effects of dietary dihydromyricetin on growth performance, antioxidant capacity, immune response and intestinal microbiota of shrimp (Litopenaeus vannamei)

A 56-day culture trial was conducted to evaluate the effects of dietary dihydromyricetin (DMY) on growth performance, antioxidant capacity, immune response and intestinal microbiota of shrimp (Litopenaeus vannamei). 840 healthy shrimp (1.60 ± 0.21 g) in total were fed with four different levels of DMY diets at 0 (Control), 100 (D1), 200 (D2), and 300 (D3) mg/kg, respectively. Samples were collected after the culture trial, and then, a 7-day challenge experiment against Vibrio parahaemolyticus was conducted. The results demonstrated that DMY significantly enhanced the activity of protease, amylase and lipase as well as the expression of lipid and protein transport-related genes (P < 0.05). The results of plasma lipid parameters indicated that DMY reduced lipid deposition, manifested by significantly (P < 0.05) decreased plasma total cholesterol (T-CHO), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C). The expression of genes involved in fatty acid β-oxidation and triglyceride catabolism was significantly up-regulated (P < 0.05), and genes involved in triglyceride synthesis were significantly down-regulated in DMY groups when compared to control group (P < 0.05). Moreover, dietary DMY also significantly (P < 0.05) increased the total antioxidant capacity (T-AOC), antioxidant enzymes activity and glutathione (GSH) content of shrimp, and a significant increase of total hemocytes count (THC), phagocytic rate (PR), antibacterial activity (AA) and bacteriolytic activity (BA) was observed in DMY groups (P < 0.05). The addition of DMY to the diet significantly augmented immune response by up-regulating the expression of genes related to toll-like receptors (Toll) signaling pathway, immune deficiency (IMD) signaling pathway and intestinal mucin. Furthermore, dietary DMY could modulate the composition and abundance of intestinal microbiota. In conclusion, DMY showed promising potential as a functional feed additive for shrimp to improve the growth performance and physiological health.

The sequencing of the key genes and end products in the TLR4 signaling pathway from the kidney of Rana dybowskii exposed to Aeromonas hydrophila.

Infectious diseases caused by Aeromonas hydrophila (AH) have reduced the populations of Rana dybowskii). However, little is known about the immune response of R. dybowskii against AH infections. The toll-like receptor (TLR) signaling pathway has been identified as a critical component in innate immunity, responsible for identifying pathogen-associated molecular patterns in pathogens. Our study used the next-generation sequencing technique and single-molecule long-read sequencing to determine the structures of transcript isoforms and functions of genes in the kidneys of R. dybowskii, as well as identify and validate the related genes in the TLR4 signaling pathway. In total, 628,774 reads of inserts were identified, including 300,053 full-length non-chimeric reads and 233,592 non-full-length reads. Among the transcriptome sequences, 124 genes were identified as homologs of known genes in the TLR4 pathway especially inflammatory cytokines and receptors. Our findings shed light on the structures and functions of R. dybowskii genes exposed to AH and confirm the presence of both MyD88-dependent and independent pathways in R. dybowskii. Our work reveals how various functional proteins in amphibians at the initial stage of immune response are activated and complete their corresponding functions in a short time.

Network pharmacology-based findings of the immunomodulatory activity of phytocompounds from Withania somnifera and Aloe barbadensis

Immunomodulation constitutes a crucial part of individual organisms&amp;rsquo; defense systems. Moreover, the utilization of plant-based natural products as herbal medicine for immunomodulation has garnered significant interest. Herein, we examined the immunomodulatory potentials of active phytocompounds extracted from Withania somnifera and Aloe barbadensis by employing ADMET screening, network pharmacology, and molecular docking techniques. This study follows the paradigm in drug discovery, which has shifted from a &amp;ldquo;one-target, one-drug&amp;rdquo; mode to a &amp;ldquo;network-target, multiple-component-therapeutics&amp;rdquo; mode. Phyto compounds sourced from W. somnifera and A. barbadensis were mined from online databases, including Dr. Duke&amp;rsquo;s Phytochemical Ethnobotanical Database. After screening these active compounds, their potential targets were predicted through in silico ADMET property prediction models. Network pharmacology was utilized to establish a &amp;ldquo;compound-protein/gene-disease&amp;rdquo; network and reveal the regulatory mechanism of small molecules in a high-throughput manner through STRING, Cytohubba plugin in Cytoscape, and the g: Profiler software. A molecular docking simulation was performed to examine the binding affinity between the selected hub targets and bioactives. The findings showed that phytocompounds derived from the W. somnifera and A. barbadensis exhibit immunomodulatory effects by inhibiting specific protein targets, notably AKT1, HCK, JAK2, PDPK1, KIT, and IL2. Molecular docking analysis further revealed the potential of withanolide G, somniferine, and somniferanolide as promising immunomodulatory compounds against HCK, JAK2, and PDPK1 proteins, which are involved in multiple myeloma pathways, encompassing the PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, and Toll-like receptor signaling pathway. In conclusion, these compounds are recommended for further in vivo and in vitro investigations to ascertain their potential as treatments for multiple myeloma.

Network pharmacology and molecular docking to scientifically validate the potential mechanism of Lonicerae japonicae flos in the clinical treatment of COVID-19

Using network pharmacology and molecular docking, we predicted the potential mechanisms of Lonicerae japonicae flos (LJF) therapy for COVID-19. A total of 493 component-related targets and 6,233 COVID-19-related genes were identified, and 267 core genes with overlapping of the two types of genes were identified. The target AKT1, CASP3, IL1B, IL6, PTGS2, TNF and JUN were the hub genes in PPI network according to MCODE score. Component-Target analysis showed the close relationship between targets and components. The results of functional enrichment analyses revealed that LJF exerted pharmacological effects on COVID-19 by regulating IL-17 signalling pathway, TNF signalling pathway, AGE-RAGE signalling pathway in diabetic complications, and Toll-like receptor signalling pathway. Finally, molecular docking confirmed a strong binding affinity between the 7 main active components with the hub genes. The findings suggested that beta-sitosterol, kaempferol and luteolin might be the promising leading components due to their good molecular docking scores.

Identification and validation of ferroptosis-related biomarkers in steroid-induced osteonecrosis of the femoral head

ObjectivesTreatment of steroid-induced osteonecrosis of the femoral head (SIONFH) is challenging. Due to the limited understanding of its molecular mechanisms, investigating the potential mechanisms of ferroptosis will shed light on SIONFH and provide directions for treating this disease. MethodsThe GSE123568 dataset was utilized to apply various bioinformatics methodologies to identify ferroptosis-related hub genes (FRHGs). Subsequently, the importance of these genes and the reliability of the results were confirmed using protein data-independent acquisition (DIA) and cell experiments. Finally, we assessed the correlation between FRHG expression and immune cell infiltration. ResultsThirty-one hub genes were identified and validated by constructing a protein–protein interaction network and subsequent screening using experimentally determined interactions. These 31 hub genes were enriched in immunity, the AMPK signaling pathway, and the Toll-like receptor signaling pathway. Next, we identified a diagnostic marker comprising two ferroptosis-related genes, NCF2 and SLC2A1. The differential expression of these two genes in healthy and necrotic regions was confirmed by protein DIA analysis. Cell experiments verified the link between FRHGs and ferroptosis and preliminarily explored the potential mechanism of the antioxidant vitexin in promoting osteogenic differentiation in cells. The diagnostic efficiency of these two markers was confirmed by receiver operating characteristic curve (ROC) curves, yielding an area under the curve of 1.0. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated enrichment of FRHGs in the superoxide anion and HIF-1 signaling pathways. A significant correlation was observed between FRHGs and various immune cell populations. ConclusionNCF2 and SLC2A1 are promising ferroptosis-related diagnostic biomarkers of SIONFH. Concurrently, we embarked on a preliminary investigation to elucidate the potential mechanism underlying the promotion of osteogenic differentiation by the antioxidant vitexin. Moreover, these biomarkers are associated with distinct immune cell populations.

Exploration of Key Immune-Related Transcriptomes Associated with Doxorubicin-Induced Cardiotoxicity in Patients with Breast Cancer

Based on a few studies, heart failure patients with breast cancer were assessed to find potential biomarkers for doxorubicin-induced cardiotoxicity. However, key immune-related transcriptional markers linked to doxorubicin-induced cardiotoxicity in breast cancer patients have not been thoroughly investigated. We used GSE40447, GSE76314, and TCGA BRCA cohorts to perform this study. Then, we performed various bioinformatics approaches to identify the key immune-related transcriptional markers and their association with doxorubicin-induced cardiotoxicity in patients with breast cancer. We found 255 upregulated genes and 286 downregulated genes in patients with doxorubicin-induced heart failure in breast cancer. We discovered that in patients with breast cancer comorbidity doxorubicin-induced cardiotoxicity, the 58 immunological genes are elevated (such as CPA3, VSIG4, GATA2, RFX2, IL3RA, and LRP1), and the 60 genes are significantly suppressed (such as MS4A1, FCRL1, CD200, FCRLA, FCRL2, and CD79A). Furthermore, we revealed that the immune-related differentially expressed genes (DEGs) are substantially associated with the enrichment of KEGG pathways, including B-cell receptor signaling pathway, primary immunodeficiency, chemokine signaling pathway, hematopoietic cell lineage, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, MAPK signaling pathway, focal adhesion, dilated cardiomyopathy, cell adhesion molecule, etc. Moreover, we discovered that the doxorubicin-induced immune-related genes are crucially involved in the protein–protein interaction and gene clusters. The immune-related genes, including IFIT5, XCL1, SPIB, BTLA, MS4A1, CD19, TCL1A, CD83, CD200, FCRLA, CD79A, BIRC3, and IGF2R are significantly associated with a poor survival prognosis of breast cancer patients and showed diagnostic efficacy in patients with breast cancer and heart failure. Molecular docking revealed that the survival-associated genes interact with the doxorubicin with appreciable binding affinity. Finally, we validated the expression level of immune-related genes in breast cancer patients-derived cardiomyocytes with doxorubicin-induced cardiotoxicity and found that the level of RAD9A, HSPA1B, GATA2, IGF2R, CD200, ERCC8, and BCL11A genes are consistently dysregulated. Our findings offered a basis for understanding the mechanism and pathogenesis of the cardiotoxicity caused by doxorubicin in breast cancer patients and predicted the interaction of immune-related potential biomarkers with doxorubicin.

Common carp intelectin 3 (cITLN3) plays a role in the innate immune response

Intelectin is a lectin with the capacity to recognize and bind to carbohydrates. In this study, we successfully cloned cITLN3 from common carp, which consists of a signal peptide domain, a FReD domain, and an intelectin domain. The expression levels of cITLN3 were detected in various organs of common carp, including the liver, head kidney, spleen, foregut, midgut, and hindgut, with the highest expression observed in the liver. Following infection with Staphylococcus aureus (S. aureus) or Aeromonas hydrophila (A. hydrophila), the expression level of cITLN3 was significantly upregulated in all organs of common carp. Subsequently, we expressed and purified the recombinant cITLN3 protein using an E. coli expression system. The cITLN3 could aggregate both gram-positive and gram-negative bacteria in the presence of Ca2+, with a stronger affinity for gram-positive bacteria. Moreover, our study demonstrated that cITLN3 displayed a higher binding affinity towards PGN compared to LPS. Furthermore, we observed that cITLN3 had the potential to inhibit bacterial proliferation in common carp and safeguard intestinal integrity during bacterial stimulation. And the results also indicated that cITLN3 might played a role in the Toll-like receptors (TLRs) signaling pathway activation.

Molecular characteristics and therapeutic implications of Toll-like receptor signaling pathway in melanoma

Melanoma is a malignant tumor of melanocytes and is often considered immunogenic cancer. Toll-like receptor-related genes are expressed differently in most types of cancer, depending on the immune microenvironment inside cancer, and the key function of Toll-like receptors (TLRs) for melanoma has not been fully elucidated. Based on multi-omics data from TCGA and GEO databases, we first performed pan-cancer analysis on TLR, including CNV, SNV, and mRNA changes in TLR-related genes in multiple human cancers, as well as patient prognosis characterization. Then, we divided melanoma patients into three subgroups (clusters 1, 2, and 3) according to the expression of the TLR pathway, and explored the correlation between TLR pathway and melanoma prognosis, immune infiltration, metabolic reprogramming, and oncogene expression characteristics. Finally, through univariate Cox regression analysis and LASSO algorithm, we selected six TLR-related genes to construct a survival prognostic model, divided melanoma patients into the training set, internal validation set 1, internal validation set 2, and external validation set for multiple validations, and discussed the correlation between model genes and clinical features of melanoma patients. In conclusion, we constructed a prognostic survival model based on TLR-related genes that precisely and independently demonstrated the potential to assess the prognosis and immune traits of melanoma patients, which is critical for patients’ survival.

Differential developmental and proinflammatory responses of zebrafish embryo to repetitive exposure of biodigested polyamide and polystyrene microplastics

Microplastics (MPs) have attracted global concern and are at the forefront of current research on environmental pollution, whereas, little is known about the degradation of ingested MPs in the gastrointestinal environment and repetitive exposure-associated risk of egested MPs to organisms. The present study revealed that polyamide (PA) and polystyrene (PS) MPs exhibited remarkably differential biodegradations in the gastric and intestinal fluids of a model fish (Siniperca chuatsi). Significant disintegration of the skeleton structure, size reduction (from 27.62 to 9.17 µm), benzene ring scission, and subsequent biogenic corona coating and surface oxidation occurred during in vitro digestion, thus increasing the hydrophilicity and agglomeration of PS. Conversely, PA MPs exhibited high resistance to enzymolysis with slight surface erosions and protein adsorption. Relative to the pristine form, the bioaccumulation of digested PS elevated and the musculoskeletal deformity and mortality of juvenile zebrafish were obviously enhanced, but these changes were unobservable for PA. Lipopolysaccharide-triggered inflammation and apoptosis via Toll-like receptor signaling pathways and reduction of extracellular matrix secretions driven by oxidative stress contributed to the aggravated inhibitory effects of digested PS on larval development. These findings emphasize the necessity of concerning the biota digestion in MP risk assessments in natural waters.

Molecular characterization of MyD88 as a potential biomarker for pesticide-induced stress in Bombyx mori

The widespread use of pesticides hampers the immune system of non-target organisms, however, there is a lack of common biomarkers to detect such effects. Myeloid differentiation primary response factor 88 (MyD88) is a crucial junction protein in the Toll-like receptor signaling pathway, which plays an important role in the inflammatory response. In this study, we investigated MyD88 as a potential biomarker for pesticide-induced stress. Phylogenetic analysis revealed that MyD88 was a conserved protein in the evolution of vertebrates and invertebrates. MyD88s usually have death domain (DD) and Toll/interleukin-1 receptor (TIR) domain. Bombyx mori (B. mori) is an important economic insect that is sensitive to toxic substances. We found microbial pesticides enhanced the expression level of MyD88 in B. mori. Transcriptome analysis demonstrated that MyD88 expression level was increased in the fatbody after dinotefuran exposure, a third-generation neonicotinoid pesticide. Moreover, the expression of MyD88 was upregulated in fatbody and midgut by imidacloprid, a first-generation neonicotinoid pesticide. Additionally, insect growth regulator (IGR) pesticides, such as methoprene and fenoxycarb, could induce MyD88 expression in the fatbody of B. mori. These results indicated that MyD88 is a potential biomarker for pesticide-induced stress in B. mori. This study provides novel insights into screening common biomarkers for multiple pesticide stresses and important implications for the development of more sustainable pest management strategies.

Angong niuhuang wan attenuates LPS-induced acute lung injury by inhibiting PIK3CG/p65/MMP9 signaling in mice based on proteomics

Acute lung injury (ALI) is a serious pulmonary complication that often arises from pneumonia, respiratory tract infections caused by bacteria or viruses, and other factors. It is characterized by acute onset and high mortality. Angong Niuhuang Wan (AGNHW) is a renowned emergency medicine in traditional Chinese medicine, known as the "cool open (febrile disease) three treasures" and regarded as the first of the "three treasures". Previously studies have confirmed that AGNHW has anti-inflammatory effects, improves cerebral circulation, reduces brain edema, and protects vascular endothelium. However, the active components and pharmacological mechanisms of AGNHW in treating ALI remain unclear. In this study, we confirmed that AGNHW can inhibit cytokine storm activity and reduce inflammation induced by LPS in ALI mice. We then analyzed differential proteins using proteomic technology and identified 741 differential proteins. By combining network pharmacological analysis, we deeply discussed the key active components and mechanism of AGNHW in treating ALI. By constructing the interaction network between disease and drug, we identified 21 key active components (such as Quercetin, Kaempferol, and Crocetin) and 25 potential core targets (such as PIK3CG, p65, and MMP9). These candidate targets play an important role in anti-inflammation and immune regulation. Through enrichment analysis of core targets, we found several pathways related to ALI, such as the NF-κB signaling pathway, TNF signaling pathway, and Toll-like receptor signaling pathway. This indicates that AGNHW plays a therapeutic role in ALI through multi-components, multi-targets, and multi-pathways.

Ajpacifastin-like is involved in the immune response of Apostichopus japonicus challenged by Vibrio splendidus.

Pacifastin proteins are previously found to regulate the phenoloxidase system in invertebrates and arthropods. In this study, the immune response that was regulated by Ajpacifastin-like in the sea cucumber Apostichopus japonicus was determined. RNA interference was used to knock down the expression of the Ajpacifastin-like gene in A. japonicus, followed by challenge with Vibrio splendidus, and the colony count showed that the survival of V. splendidus in the si-Ajpacifastin group increased 4.64-fold compared to that of the control group. The purified recombinant Ajpacifastin-like showed an inhibitory effect on the extracellular protease activity of the supernatant collected from the V. splendidus culture. Consequently, a comparative transcriptome analysis of the coelomocytes from the control group and the si-Ajpacifastin group was performed to explore the global regulatory effect of the Ajpacifastin-like. A total of 1486 differentially expressed genes (DEGs) were identified, including 745 upregulated genes and 741 downregulated genes. GO enrichment showed that the DEGs were mainly enriched in translation, cytosolic ribosomal subunit and structural constituent of ribosome. KEGG analysis showed that the DEGs were significantly enriched in the retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, mitogen-activated protein kinase signaling pathway, nuclear factor-kappa B signaling pathway and other immune-related pathways. Furthermore, real-time reverse transcriptase PCR was used to determine the RNA levels of six DEGs, i.e., cathepsinB, CYLD, caspase8, TRAF6, hsp90 and FADD, to verify the RNA-seq results. Overall, our results specified the immune response and pathways of A. japonicus in which Ajpacifastin-like was involved in.

Effects of lactic acid bacteria-fermented formula milk supplementation on colonic microbiota and mucosal transcriptome profile of weaned piglets.

Supplemental probiotic fermented milk as a gut modulator can improve growth performance for weaned piglets by promoting the development of the small intestine in digestion and immune function. The effect on colon health might also play a considerable part in the favourable role of probiotic fermented milk in the growth performance improvement of weaned piglets; however, it has yet to be reported. This study aimed to investigate the effects of supplementation with lactic acid bacteria-fermented formula milk (LFM) on colonic morphology, microbiota composition, and mucosal transcriptome profile in weaned piglets. A total of 24 male weaned piglets were randomly divided into two groups: a control (CON) treatment or the LFM-supplemented treatment. Each group consisted of six replicates (cages) with two piglets per cage, and each piglet in the LFM group was supplemented with 80mL LFM three times a day for 21 d, while the CON group was treated with the same amount of drinking water. Results showed that supplementation of LFM reduced the colonic histological damage scores and significantly increased the number of goblet cells per crypt. Furthermore, LFM consumption decreased the levels of pro-inflammation cytokines in the colonic mucosa. LFM downregulated the expression of inflammatory genes (CXCL9 and CXCL10) involving Toll-like receptor signalling pathway, immune response, and response to bacterium, and up-regulated two active genes (S100A8 and S100A9) involving the IL-17 signalling pathway and Toll-like receptor 4 binding. In addition, LFM could increase the potential probiotic genera containing Lachnospira and Anaerorhabdus furcosa group, which were positively related to short-chain fatty acid (SCFA) production. Correspondingly, LFM-fed piglets had higher total bacterial load and total SCFA concentration in the colonic digesta compared with the CON group. These novel findings support the benefits of LFM in enhancing intestinal homoeostasis and ameliorating weaning stress for weaned piglets, which is associated with the modulation of gut microbiota composition and immune-related genes.

Dietary dehulled adlay ameliorated alcoholic liver disease progression by modulating oxidative stress, inflammation, and gut-liver axis disruption in rats

Alcohol consumption causes alcoholic liver diseases (ALD). The aim of this study investigated whether the mechanisms of the effects of dehulled adlay on ALD by using a chronic ethanol feeding plus single binge model. Rats were pair-fed with a control liquid diets (C), ethanol liquid diet (E), or ethanol + dehulled adlay diet (EA) containing equal energy and dietary fiber for 5 weeks. The results revealed that dietary dehulled adlay ameliorated ethanol-induced increases in oxidative stress and inflammation and modulating nuclear factor erythroid 2-related factor 2-inflammasome and Toll-like receptor-4 signaling pathways. Furthermore, dehulled adlay arrested the decrease in occludin expression in the ileum and mitigated changes in the diversity and composition of the gut microbiota caused by alcohol exposure, which were significantly correlated with ALD biomarkers. Our results suggest that dietary dehulled adlay prevents ALD progression possibly by reducing hepatic oxidative stress and inflammation, and modulating gut dysbiosis.

MiR-146a in myasthenia gravis thymus: from uncontrolled innate immunity to B cell-mediated autoimmunity

The thymus is the main site of autoimmunity development in myasthenia gravis (MG) associated with anti-acetylcholine receptor (AChR) autoantibodies, a prototypic autoimmune disease affecting neuromuscular junction. Most MG patients with early-onset disease presents follicular hyperplastic changes of the thymus, that are critically implicated in the initiation and perpetuation of the autoimmune response against the AChR. Uncontrolled activation of Toll-like receptor (TLR)-mediated innate immune responses, chronic inflammation and ectopic germinal center (GC) formation are key pathological features of hyperplastic thymus in MG, indicating that a close link between innate immunity and B cell-mediated autoimmunity underlies the intra-thymic pathogenesis of MG.&#x0D; MiR-146a, an “immune-miR” acting as key modulator of both innate and adaptive immunity, is a potent inhibitor of TLR signaling pathways, able to prevent and avoid an overstimulation of the inflammatory response by targeting the NF-κB signaling transducers IRAK1 and TRAF6. At the same time, miR-146a modulates the expression of c-REL, ICOS and ICOSL, which are crucial regulators of B cell function and GC response. Dysregulation of miR-146a expression is a common molecular event in several autoimmune disorders. Our recent findings revealed defective expression of miR-146a, associated with over-expression of its TLR- and B cell-related target genes, in follicular hyperplastic MG thymuses, indicating that loss of regulatory functions of this miRNA may significantly contribute to the immunopathological steps leading to MG. Of note, corticosteroids were found to increase the miR-146a expression levels, thus suggesting the miRNA capacity to mediate the effects of these drugs in inducing immunosuppression and autoimmunity control. &#x0D; In this review, we discuss the role of miR-146a as a molecular bridge between innate and adaptive immunity, and summarize the current knowledge on the miRNA contribution to the pathogenesis of MG. Based on our and literature data, we highlight the miR-146a potential as biomarker for therapeutic monitoring, as well as target of future advanced RNA-based therapies to modulate the immune system and counteract the autoimmune response in MG.

Transcriptome Sequencing Reveals That Intact Expression of the Chicken Endogenous Retrovirus chERV3 In Vitro Can Possibly Block the Key Innate Immune Pathway.

Endogenous retroviruses (ERVs) are viral sequences that have integrated into the genomes of vertebrates. Our preliminary transcriptome sequencing analysis revealed that chERV3 is active and is located on chromosome 1:32602284-32615631. We hypothesized that chERV3 may have a role in the host innate immune response to viral infection. In this study, using reverse genetics, we constructed the puc57-chERV3 full-length reverse cloning plasmid in vitro. We measured the p27 content in culture supernatant by enzyme-linked immunosorbent assay (ELISA). Finally, transcriptome analysis was performed to analyze the function of chERV3 in innate immunity. The results showed that chERV3 may generate p27 viral particles. We found that compared to the negative control (NC) group (transfected with pMD18T-EGFP), the chERV3 group exhibited 2538 up-regulated differentially expressed genes (DEGs) and 1828 down-regulated DEGs at 24 hours (h) and 1752 up-regulated DEGs and 1282 down-regulated DEGs at 48 h. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the down-regulated DEGs were enriched mainly in immune-related processes such as the inflammatory response, innate immune response, and Toll-like receptor signaling pathway. GSEA showed that the Toll-like receptor signaling pathway was suppressed by chERV3 at both time points. We hypothesized that chERV3 can influence the activation of the innate immune pathway by blocking the Toll-like receptor signaling pathway to achieve immune evasion.

Integrated network pharmacology analysis and in vitro validation revealed the underlying mechanism of Xiyanping injection in treating coronavirus disease 2019.

Coronavirus disease 2019 (COVID-19) has spread rapidly worldwide, leading to a pandemic. In China, Xiyanping injection (XYP) has been recommended as a drug for COVID-19 treatment in the Guideline on Diagnosis and Treatment of COVID-19 by the National Health Commission of the People Republic of China and National Administration of Traditional Chinese Medicine (Trial eighth Edition). However, the relevant mechanisms at the molecular-level need to be further elucidated. In this study, XYP related active ingredients, potential targets and COVID-19 related genes were searched in public databases. Protein-protein interaction network and module analyzes were used to screen for key targets. gene ontology and Kyoto encyclopedia of genes and genomes were performed to investigate the potentially relevant signaling pathways. Molecular docking was performed using Autodock Tools and Vina. For the validation of potential mechanism, PolyI:C was used to induce human lung epithelial cells for an inflammation model. Subsequently, CCK-8 assays, enzyme-linked immunosorbent assay, reverse transcription quantitative polymerase chain reaction and western blot were employed to determine the effect of XYP on the expression of key genes. Seven effective active ingredients in XYP were searched for 123 targets in the relevant databases. Furthermore, 6446 COVID-19 disease targets were identified. Sodium 9-dehydro-17-hydro-andrographolide-19-yl sulfate was identified as the vital active compounds, and IL-6, TNF, IL-1β, CXCL8, STAT3, MAPK1, MAPK14, and MAPK8 were considered as the key targets. In addition, molecular docking revealed that the active compound and the targets showed good binding affinities. The enrichment analysis predicted that the XYP could regulate the IL-17, Toll-like receptor, PI3K-Akt and JAK-STAT signaling pathways. Consistently, further in vitro experiments demonstrated that XYP could slow down the cytokine storm in the lung tissue of COVID-19 patients by down-regulating IL-6, TNF-α, IL-1β, CXCL8, and p-STAT3. Through effective network pharmacology analysis and molecular docking, this study suggests that XYP contains many effective compounds that may target COVID-19 related signaling pathways. Moreover, the in vitro experiment confirmed that XYP could inhibit the cytokine storm by regulating genes or proteins related to immune and inflammatory responses.

MyD88-dependent Toll-like receptor 2 signaling modulates macrophage activation on lysate-adsorbed Teflon™ AF surfaces in an in vitro biomaterial host response model.

The adsorbed protein layer on an implanted biomaterial surface is known to mediate downstream cell-material interactions that drive the host response. While the adsorption of plasma-derived proteins has been studied extensively, the adsorption of damage-associated molecular patterns (DAMPs) derived from damaged cells and matrix surrounding the implant remains poorly understood. Previously, our group developed a DAMP-adsorption model in which 3T3 fibroblast lysates were used as a complex source of cell-derived DAMPs and we demonstrated that biomaterials with adsorbed lysate potently activated RAW-Blue macrophages via Toll-like receptor 2 (TLR2). In the present study, we characterized the response of mouse bone marrow derived macrophages (BMDM) from wildtype (WT), TLR2-/- and MyD88-/- mice on Teflon™ AF surfaces pre-adsorbed with 10% plasma or lysate-spiked plasma (10% w/w total protein from 3T3 fibroblast lysate) for 24 hours. WT BMDM cultured on adsorbates derived from 10% lysate in plasma had significantly higher gene and protein expression of IL-1β, IL-6, TNF-α, IL-10, RANTES/CCL5 and CXCL1/KC, compared to 10% plasma-adsorbed surfaces. Furthermore, the upregulation of pro-inflammatory cytokine and chemokine expression in the 10% lysate in plasma condition was attenuated in TLR2-/- and MyD88-/- BMDM. Proteomic analysis of the adsorbed protein layers showed that even this relatively small addition of lysate-derived proteins within plasma (10% w/w) caused a significant change to the adsorbed protein profile. The 10% plasma condition had fibrinogen, albumin, apolipoproteins, complement, and fibronectin among the top 25 most abundant proteins. While proteins layers generated from 10% lysate in plasma retained fibrinogen and fibronectin among the top 25 proteins, there was a disproportionate increase in intracellular proteins, including histones, tubulins, actins, and vimentin. Furthermore, we identified 7 DAMPs or DAMP-related proteins enriched in the 10% plasma condition (fibrinogen, apolipoproteins), compared to 39 DAMPs enriched in the 10% lysate in plasma condition, including high mobility group box 1 and histones. Together, these findings indicate that DAMPs and other intracellular proteins readily adsorb to biomaterial surfaces in competition with plasma proteins, and that adsorbed DAMPs induce an inflammatory response in adherent macrophages that is mediated by the MyD88-dependent TLR2 signaling pathway.

Erythropoietin Suppresses the Hepatic Fibrosis Caused by Thioacetamide: Role of the PI3K/Akt and TLR4 Signaling Pathways.

Erythropoietin (EPO) is recognized for its function in erythropoiesis; however, its potential antifibrotic effect against liver fibrosis remains unknown. This study examined whether EPO affects thioacetamide (TAA)-induced liver fibrosis by concentrating on the Toll-like receptor 4 (TLR4) cascade and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway as possible pathways. Male Wistar rats were randomized into four groups, which included: the negative control group, the TAA group (intraperitoneal; TAA 100 mg/kg three times per week for 2 weeks), and EPO-treated groups (150 and 300 IU/kg, i.p.) for 2 weeks after TAA injections. EPO attenuated hepatic fibrosis in a dosage-dependent way, as manifested by the diminution in serum alanine aminotransferase and aspartate aminotransferase activities, as well as the increase in albumin level. EPO inhibited the increase in tissue levels of tumor necrosis factors-α, interleukin-1β, transforming growth factor-β1, and TLR4 and raised tissue levels of PI3K and p-PI3K. EPO antioxidant properties were demonstrated by restoring hepatic glutathione and superoxide dismutase by preventing the accumulation of hepatic malondialdehyde. Further, EPO increased the protein expression of PI3K and Akt and decreased TLR4 protein expression. Immunohistochemically, EPO treatment altered tissue histology and downregulated mitogen-activated protein kinase protein expression. Overall, the research suggested that EPO could prevent TAA-induced hepatic fibrosis through upregulating the PI3K/Akt signaling cascade and downregulation the TLR4 downstream axis.