NOD-like Receptor Signaling Pathway Research Articles

TMIC-70. CORRELATION OF LLT-1 AND NLRC4 INFLAMMASOME AND ITS EFFECT ON GLIOBLASTOMA PROGNOSIS

Abstract PURPOSE LLT-1 is a well-known ligand for the natural killer (NK) cell inhibitory receptor NKRP1A. Here, we examined NLRC4 inflammasome components and LLT-1 expression in glioblastoma (GBM) tissues to elucidate potential associations and interactions between these factors. METHODS Nine GBM tissues were collected for experiments and RNA sequencing, while fifty-two tumor core tissues were collected for RNA sequencing. Expression of LLT-1 and other proteins was assessed by immunofluorescence. Computational analyses utilized RNA-seq data from 296 and 52 patients from the Chinese Glioma Genome Atlas and CHA medical records, respectively, using survival, non-negative matrix factorization clustering, Gene Ontology enrichment, and protein-protein interaction analyses. Receptor-ligand interactions between tumor and immune cells were confirmed by single cell RNA sequencing analysis. RESULTS In GBM tissues, LLT-1 was predominantly colocalized with Glial fibrillary acidic protein (GFAP) -expressing astrocytes, but not with microglial markers like Iba-1. Additionally, LLT-1 and activated NLRC4 inflammasomes were co-expressed mainly in intratumoral astrocytes, suggesting an association between LLT-1, NLRC4, and glioma malignancy. High LLT-1 expression correlates with poor prognosis, particularly in the mesenchymal subtype, and is associated with TNF and NOD-like receptor signaling pathway enrichment, indicating a potential role in tumor inflammation and progression. At the single-cell level, mesenchymal-like malignant cells were highly enriched in the TNF, NLR, and IL-1 signaling pathways compared to other malignant cell types. CONCLUSION We revealed an association between NLRC4 inflammasome activity and LLT-1 expression, suggesting a novel regulatory pathway involving TNF, inflammasomes, and IL-1, potentially offering new anti-glioma approaches by enhancing NK cell-based treatments.

Evaluation of the Biological Effect of a Nicotinamide-Containing Broad-Spectrum Sunscreen on Photodamaged Skin.

UVA-UVB increases skin matrix metalloproteinases and breaks down extracellular proteins and fibrillar type1 collagen, leading to photodamage. Topical application of nicotinamide prevents UV-induced immunosuppression. Several studies have demonstrated the importance of protection against UV. This study aims to determine the biological effect of a high broad-spectrum UVB-UVA sunscreen containing nicotinamide and panthenol (SSNP) on photodamaged skin using linear confocal optical coherence tomography (LC-OCT), immunohistochemistry, and RNA profiling. Two areas of severely photodamaged forearm skin (L01 and L02) and one less sun-damaged (naturally protected) area on the inner part of the forearm (L03) were identified in 14 subjects. These areas were imaged using LC-OCT and L01 and L03 were biopsied at baseline. After 4weeks of treatment with SSNP, L02 was reimaged using LC-OCT, and biopsied. Histology, immunostaining with p21, p53, PCNA, and CPD, and RNA sequencing were performed in all samples. LC-OCT analysis showed that epidermis thickness and the number of keratinocytes is higher in the sun-exposed areas than in the non-exposed areas. Comparing before and after treatment, even though there is a trend towards normalization, the differences were not statistically significant. The expression of p21, PCNA, p53, and CPD increased in severely photodamaged skin compared to less-damaged skin. When comparing before and after treatment, only p21 showed a trend to decrease expression. RNA sequencing analysis identified 1552 significant genes correlating with the progression from non-visibly photodamaged skin to post-treatment and pre-treatment samples; in the analysis comparing pre- and post-treatment samples, 5429 genes were found to be significantly associated. A total of 1115 genes are common in these two analyses. Additionally, nine significant genes from the first analysis and eight from the second are related to collagen. Six of these collagen genes are common in the two analyses. MAPK and cGMP-PKG signalling pathways are upregulated in the progression to photodamage analysis. In the pre- and post-treatment analysis, 32 pathways are downregulated after treatment, the most statistically significant being the ErbB, Hippo, NOD-like receptor, TNF, and NF-kB signalling pathways. This study demonstrates the role of SSNP in collagen generation, highlights the relevance of the cGMP-PKG and MAPK signalling pathways in photodamage, and shows the ability of SSNP to downregulate pathways activated by UV exposure. Additionally, it deepens our understanding of the effect of SSNP on immune-related pathways.

Integrated strategy of mass spectrometry imaging and LC/MS-based GNPS for spatial characterization of alkaloids from Menispermi Rhizoma and study on potential anti-inflammatory mechanism by network pharmacology and molecular docking

Menispermi Rhizoma (MR) is the dried rhizome of Menispermum dauricum DC, which has been used to treat sore throat, enteritis, and rheumatic arthralgia. These therapeutic effects are attributed to its alkaloid ingredient. However, the chemical composition, anti-inflammatory mechanisms and the spatiotemporal distribution of the bioactive ingredients in MR have seldom been investigated. This study aims to clarify the anti-inflammation mechanism, material basis, and their spatial distribution of MR. Here, a LC/MS-based Global Natural Products Social Molecular Networking (GNPS) strategy was used to rapidly exhibit alkaloid molecular clusters that improve annotation accuracy and discover more unknown compounds. Then, a high-sensitive air flow-assisted ionization mass spectrometry imaging (AFAI-MSI) method was developed to visualize the spatial distributions alkaloids in different botanical parts of MR. The anti-inflammation mechanism was investigated based on network pharmacology and verified by molecular docking experiment. Finally, a total of 106 alkaloids including 24 aporphines, 23 monobenzylisoquinolines, 20 morphinanes, 20 proberberines, 12 bisbenzylisoquinolines, and 7 amides were identified in MR as well as 24 alkaloids were visualized in the medullary ray, cortex and epidermis regions. Moreover, the targets with a higher degree in the PPI network were TNF, GAPDH, AKT1, ALB, STAT3. GO and KEGG analysis revealed that MR in anti-inflammatory mechanism mainly involved plasma membrane, ATP binding, cytoplasm, identical protein binding and ATP binding. The signaling pathways mainly included NOD-like receptor signaling pathway, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic complications. The molecular docking results indicated that stepharanine-2-O-glucoside, bianfugedine, bianfugecine, dehydrostephanine had excellent affinity with SRC, MMP9 AKT1, STAT3 and TNF. This study comprehensively characterized the alkaloid ingredients and spatial distribution of MR, and revealed potential mechanism of MR in inflammation, which provides a reference for development and application of MR and other Traditional Chinese medicines (TCMs).

Paired single-cell RNA sequencing and T cell receptor sequencing reveals both pro- and anti-inflammatory roles of T cells in patients with calcific aortic valve disease

Abstract Background Calcified aortic valve disease (CAVD) is a chronic unresolvable inflammatory disorder that affects 25% of adults over 65 years old, leading to aortic stenosis, heart failure, and death [1]. Since the molecular mechanisms of its pathogenesis are not well understood, therapies to treat root causes of the disease remain to be developed, with valve replacement as the major intervention strategy currently. Increasing evidence indicates that substantial T cells infiltrate in the aortic valve during calcification, and clonal expanded CD8+ T cells have been identified in the calcificed aortic valve by bulk T cell receptor (TCR) repertoire sequencing [2]. Purpose To decipher T cell heterogenity and understand the precise mechanisms of T cell subsets involving CAVD progression as a basis for novel therapeutic targets. Methods In Cohort 1, we collected aortic valves from CAVD (n=3) and healthy (n=2) patients for single-cell RNA sequencing (scRNA-seq). In Cohort 2, we performed paired scRNA-seq and TCR sequencing (scTCR-seq) on aortic valves obtained from CAVD (n=3) and healthy (n=3) patients to evaluate both gene expression and clonality. Pathway enrichment analysis and cell-cell interaction analysis elucidated the function of T cells in the calcificied valves. Immunohistochemistry and fluorescence-activated cell sorting were recruited to verify the key findings. Results scRNA-seq demonstrated a conversion from anti-inflammatory regulatory T (Treg) cells towards pro-inflammatory T helper 17 (Th17) cells in the calcificied aortic valve. Paired scRNA-seq and scTCR-seq revealed CAVD-related signaling pathways (osteoclast differentiation, NOD-like receptor signaling pathways, IL-17 signaling pathways) significantly upregulated in highly expanded CD8+ T cells in the calcified valve, with upregulation of pro-inflammatory mediators such as IFNG, CCL4 and CCL5, suggesting pro-inflammation functions in clonally expanded T cells. Moreover, we characterized tissue-resident memory T cells, which resembled similar transcriptome and TCR clonality as exhausted T cells in calcified valves. These cells up-regulated anti-inflammation-related transcriptomes but their cellular fraction decreased in calcified valves, implying anti-inflammatory functions of these tissue-resident memory and exhausted T cells. Conclusion This study elucidates transcriptomic and immune profiling of T cells in the calcified aortic valve, therefore shedding light on the pathophysiological mechanisms underlying aortic valve calcification from the novel perspective of tissue-specific T lymphocytes, providing promising targets for immune checkpoint-based therapeutic interventions.

Huang-Pu-Tong-Qiao Formula Alleviates Hippocampal Neuron Damage by Inhibiting NLRP3 Inflammasome-mediated Pyroptosis in Alzheimer's Disease.

Huang-Pu-Tong-Qiao (HPTQ), a Traditional Chinese Medicine formula, has achieved remarkable efficacy in clinically treating Alzheimer's disease (AD). Pyroptosis refers to the inflammatory necrosis of cells, which contributes to AD pathological progression. However, it is unclear whether the therapeutic effect of HPTQ on AD is related to reducing pyroptosis. In this study, the network pharmacology analysis was used to predict the molecular mechanism of HPTQ in treating AD and validated our hypothesis through mice and cell experiments. APP/PS1 transgenic mice and Aβ25-35-injured HT22 cells were used as AD models in vivo and in vitro. The pharmacological effects and mechanisms of HPTQ on AD were evaluated by Morris water maze, Y-maze, transmission electron microscope, immunofluorescence, Hoechst/PI staining, western blot, and ELISA. Network pharmacology reveals the correlation between the therapeutic effect of HPTQ on AD and the NOD-like receptor signaling pathway. In APP/PS1 mice, HPTQ reduced the escape latency and maintained cell membrane integrity. In HT22 cells, 15% HPTQ-medicated serum and 10 µM MCC950 increased cell viability and decreased PI positive rate compared with the Model group. In addition, HPTQ treatment in AD animal and cell models reduced the protein expressions of NLRP3, ASC, cleaved caspase-1, GSDMD, GSDMD-N, IL-1β, and IL-18. The experimental results of MCC950 specifically inhibiting the NLRP3 expression suggested that HPTQ might reduce neuronal pyroptosis by reducing NLRP3 inflammasome. Network pharmacology and experimental validation suggested that HPTQ alleviated NLRP3 inflammasome-mediated neuronal pyroptosis in AD, which could provide valuable candidate drugs for AD clinical treatment.

Prognostic model for gastric cancer patients with COVID-19 and network pharmacology study on treatment by lentinan

Patients with gastric cancer (GC) are more susceptible to coronavirus disease 2019 (COVID-19), which further worsens their already challenging prognosis. However, there are no effective treatment options for these patients. Lentinan is a potent bioactive component with antiviral and antitumor effects. We hypothesized that lentinan might exert powerful pharmacological effects in patients with GC and COVID-19. In this study, a prognostic model of patients with GC/COVID-19 was constructed and used to apply a network pharmacology approach to reveal biological functions, drug targets, and molecular mechanisms of the action of lentinan against GC/COVID-19. Clinical analysis revealed key prognostic genes in patients with GC/COVID-19. The results of network pharmacology analysis suggested that the therapeutic effect of lentinan on GC/COVID-19 mainly involves the modulation of several neutrophil-related biological processes, as well as the nucleotide-binding and oligomerization domain-like receptor and interleukin-17 signaling pathways. In addition, C-X-C motif chemokine ligand 8, vascular endothelial growth factor A, ribonuclease 3, and F2 were identified as key genes of lentinan against GC/COVID-19. Key prognostic genes were identified in patients with GC/COVID-19 through the construction of a prognostic model. Pharmacological functions and signaling pathways of lentinan against GC/COVID-19 were revealed. These included the regulation of neutrophils and NOD-like receptor signaling pathways. The findings provide the first published evidence of the potential value of lentinan as a complementary therapy for GC/COVID-19.

Identification of Biomarkers and Mechanisms Associated with Apoptosis in Recurrent Pregnancy Loss.

In this study, we employed bioinformatics techniques to identify genes associated with apoptosis in recurrent pregnancy loss (RPL). We retrieved the RPL expression profile datasets GSE165004 and GSE73025 from the Gene Expression Omnibus (GEO) database. We also obtained data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway of Apoptosis (hsa04210) to identify apoptosis-related genes. In addition, we performed Friends analysis to explore the interactions between differential apoptosis genes and other genes in the functional pathway. We identified six differentially expressed genes related to apoptosis, including CTSZ, BCL2, PIK3CD, KRAS, GADD45G, and CASP8, with GADD45G as the most gene. Functional fertility analysis revealed that differentially expressed genes primarily regulated protein stability, cell number homeostasis, myeloid cell homeostasis, hematopoietic progenitor cell differentiation, lytic vacuole and lysosome functions, vacuolar and lysosomal membranes, transmembrane transporter binding, protein domain-specific binding, G-protein beta-subunit binding, phospholipid binding, and were involved in pathways such as Rap1 signaling, regulation of actin cytoskeleton, and NOD-like receptor signaling. KRAS exhibited the highest mutation rate in RPL-related cancer CESC. There was also a positive correlation between differentially expressed genes and B cell memory, CD4 memory resting T cells, follicular helper T cells, naïve B cells, and resting dendritic cells. We identified six differentially expressed genes related to apoptosis in RPL, with GADD45G as the most important. NOD-like receptor signaling pathway and regulation of actin cytoskeleton could be therapeutic targets for RPL.

Nanopore-based full-length transcriptome sequencing of the skin in Pseudopleuronectes yokohamae identifies novel antimicrobial peptide genes

The marbled flounder (Pseudopleuronectes yokohamae) is highly esteemed for its exceptional nutritional value and delicious taste. However, this species has extremely limited transcriptome data, which can offer priceless information for disease protection. In the study, the skin transcriptomic sequencing of P. yokohamae revealed 7.72 GB of clean data using the Nanopore sequencing platform. The results revealed 30,498 transcripts of functional annotations in the P. yokohamae transcriptome. All transcripts were searched in eight functional databases. A total of 10,337 ORFs were obtained, of which 6081 complete ORFs accounted for 58.83% of all predicted CDS. Moreover, 10,195 SSRs were detected. Meanwhile, the non-pecific immunity pathways were investigated for better understanding of the immunological reaction in P. yokohamae, and seven innate immune pathways were identified. The innate-immune related genes were highly expressed in the NOD-like receptor signaling pathway, followed by the C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and Cytosolic DNA-sensing pathway. In this study, four families of antimicrobial peptides (AMPs) in P. yokohamae were analysed for the first time, including piscidins, hepcidins, liver-expressed antimicrobial peptide and defensins. Seven AMPs, including Pypleurocidin-like WF3, Pypleurocidin-like WFX, Pyhepcidin 1, Pyhepcidin-like 1, PyLEAP-2, Pybeta-defensin and Pybeta-defensin-like 1, were further identified. The seven AMPs showed a highly identity in their cDNA and genomic structures and an inducible expression pattern preferable to skin in response to pathogens. The transcriptomic data and investigation of AMPs from P. yokohamae promote a deeper awareness of fish mucosal immunity and provide information in the prevention of fish diseases.

Single-cell transcriptional profiling reveals cell type-specific responses to duck reovirus infection in the Bursa of Fabricius of Cairna moschata

Duck reovirus (DRV) is a universal waterfowl virus that causes significant economic losses in the duck industry. However, the role of the host innate immune response of the Bursa of Fabricius to DRV infection is largely unknown. In the present study, we constructed a single-cell resolution transcriptomic atlas of the Bursa of Fabricius of Cairna moschata after infection with HN10 (a novel DRV). Ten cell-type marker genes were used to annotate the cell type, indicating a high degree of cell heterogeneity in the Bursa of Fabricius. Most of the innate and adaptive immune system-related genes were highly expressed in T cells, B cells, neutrophils, macrophages, and DCs. In the Bursa of Fabricius, the proportions of DCs and macrophages were largely increased by HN10 infection at 14 d, suggesting that DCs and macrophages play important roles in the long-term viral response. Notably, a number of innate and adaptive immune system-related genes were highly expressed at 24 h after HN10 infection, indicating that the Bursa of Fabricius has a very strong immune function even in the early developmental stage. In the immune system, the NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway were significantly activated at the early stage of HN10 infection, while the Toll-like receptor signaling pathway was significantly activated at the late stage. Enrichment analysis suggested that different immune signaling pathways play roles in specific developmental stages. Our data provide an opportunity to reveal the immune response to DRV infection at the single-cell level.

Trophic transfer induced gut inflammation, dysbiosis, and inflammatory pathways in zebrafish via Artemia franciscana: A differential analysis of nanoplastic toxicity

Rising glbal population and plastic consumption have caused a dramatic increase in plastic waste, leading to micro- and nanoplastic ingestion by aquatic organisms and subsequent bioaccumulation in their tissues. This transfer to higher trophic levels raises nanoplastic concentrations and bioavailability, potentially harming organisms' health and development. This poses a risk to human health via seafood. To address these issues, this study assesses the toxicological impacts of nanoplastics (NPs) on brine shrimp (Artemia franciscana) and their trophic transfer to zebrafish. The research unveiled concentration-dependent bioaccumulation of NPs in zebrafish and Artemia franciscana (A. franciscana). Polystyrene nanoplastics (PS-NPs) exhibited higher accumulation in A. franciscana whereas PP-NPs showed greater accumulation in zebrafish gut. Histopathological analysis under PS-NPs exposure revealed significant tissue alterations, indicative of inflammatory responses and impaired mucosal barrier integrity. Gene expression analyses confirmed these findings, showing activation of the P38-MAPK pathway by PS-NPs, which correlated with increased inflammatory cytokines. Additionally, PE-NPs activated the JNK-MAPK pathway, while PP-NPs exposure triggered the NOD-like receptor signaling pathway. Moreover, the composition of gut microbiota shifted to a dysbiotic state, characterized by an increase in pathogenic bacteria in the PS-NPs and PP-NPs groups, elevating the risk of developing Inflammatory Bowel Disease (IBD). PS-NPs were regarded as the most toxic due to their lower stability and higher aggregation tendencies, followed by PP-NPs and PE-NPs. Taken together, the overall study highlighted the complex interactions between NPs, gut microbiota, and host health, emphasizing the importance of thoroughly assessing the ecological and physiological impacts of nanoplastic pollution.

Unveiling the Innate and Adaptive Immunity Interplay: Global Transcriptomic Profiling of the Host Immune Response in Candida albicans Endophthalmitis in a Murine Model.

Intraocular fungal infection poses a significant clinical challenge characterized by chronic inflammation along with vision impairment. Understanding the host defense pathways involved in fungal endophthalmitis will play a pivotal role in identifying adjuvant immunotherapy. Clinical isolates of Candida albicans (15,000 CFU/μL) were intravitreally injected in C57BL/6 mice followed by enucleation at 24 and 72 h postinfection. Histopathological analysis was performed to evaluate the retinal changes and the disease severity. RNA-seq analysis was conducted on homogenized eyeballs to assess the relevant gene profiles and their differentially expressed genes (DEGs). Pathway enrichment analysis was performed to further annotate the functions of the DEGs. Histopathological analysis demonstrated a higher disease severity with increased inflammatory cells at 72 hpi and transcriptome analysis revealed 27,717 DEGs, of which 1493 were significant (adj p value ≤0.05, FC ≥ 1.5). Among these, 924 were upregulated, and 569 were downregulated. Majority of the upregulated genes were associated with the inflammatory/host immune response and signal transduction and enriched in the T-cell signaling pathway, natural killer cell-mediated cytotoxicity, C-type receptor signaling pathway, and NOD-like receptor signaling pathway. Furthermore, inflammation-associated genes such as T-cell surface glycoprotein CD3, cathelicidin antimicrobial peptide, and lymphocyte cell-specific protein tyrosine kinase were enriched, while pathways such as MAPK, cAMP, and metabolic pathways were downregulated. Regulating the T-cell influx could be a potential strategy to modulate excessive inflammation in the retina and could potentially aid in better vision recovery in fungal endophthalmitis.

Unlocking the secrets of NPSLE: the role of dendritic cell-secreted CCL2 in blood-brain barrier disruption.

This study employed RNA-seq technology and meta-analysis to unveil the molecular mechanisms of neuropsychiatric systemic lupus erythematosus (NPSLE) within the central nervous system. Downloaded transcriptomic data on systemic lupus erythematosus (SLE) from the Gene Expression Omnibus (GEO) and analyzed differential genes in peripheral blood samples of NPSLE patients and healthy individuals. Employed WGCNA to identify key genes related to cognitive impairment and validated findings via RNA-seq. Conducted GO, KEGG, and GSEA analyses, and integrated PPI networks to explore gene regulatory mechanisms. Assessed gene impacts on dendritic cells and blood-brain barrier using RT-qPCR, ELISA, and in vitro models. Public databases and RNA-seq data have revealed a significant upregulation of CCL2 (C-C motif chemokine ligand 2) in the peripheral blood of both SLE and NPSLE patients, primarily secreted by mature dendritic cells. Furthermore, the secretion of CCL2 by mature dendritic cells may act through the RSAD2-ISG15 axis and is associated with the activation of the NLRs (Nod Like Receptor Signaling Pathway) signaling pathway in vascular endothelial cells. Subsequent in vitro cell experiments confirmed the high expression of CCL2 in peripheral blood dendritic cells of NPSLE patients, with its secretion being regulated by the RSAD2-ISG15 axis and inducing vascular endothelial cell pyroptosis through the activation of the NLRs signaling pathway. Clinical trial results ultimately confirmed that NPSLE patients exhibiting elevated CCL2 expression also experienced cognitive decline. The secretion of CCL2 by dendritic cells induces pyroptosis in vascular endothelial cells, thereby promoting blood-brain barrier damage and triggering cognitive impairment in patients with systemic lupus erythematosus.

Galectin-9 activates host immune response and improve immunoprotection of Onychostoma macrolepis against Aeromonas hydrophila infection

Galectin-9 (Gal-9) belongs to a family of the glycan-binding proteins (GBPs) and is known to restrict bacterial activity via interacting with pathogen associated molecular pattern (PAMPs). However, the underlying immune mechanism of endogenous Gal-9 on fish against bacterial infection is still unclear. In this study, effect of Gal-9 from Onychostoma macrolepis (OmGal-9) on expression of immune related-genes were measured by HEK293T. The immune response of O. macrolepis with OmGal-9 overexpression to Aeromonas hydrophila (A. hydrophila) infection (1.65×108 CFU/mL) were evaluated by tissue bacterial load, fish survival rate and transcriptome analysis. The results showed that OmGal-9 displayed a punctate distribution in the nucleus and cytoplasm of HEK293T cells. Compared to cells transfected with the empty vector (EV) group, recombinant plasmid pEGFP-Gal9 treatment (Gal9) significantly down-regulated the expression of immune-related genes TNFα, STAT3, MyD88, LCK, and p52 of HEK293T cells stimulated with LPS at 24 h, while up-regulated IκBα and caspase1 (P < 0.05). The activities of catalase (CAT), superoxide dismutase (SOD), the total antioxidant capacity (T-AOC), alkaline phosphatase (AKP), acid phosphatase (ACP), and lysozyme (LZM) of O.macrolepis were significantly increased on 7 days in Gal9 group compared to EV group (P < 0.05). The bacterial load of liver, spleen, and kidney of O.macrolepis infected with A. hydrophila in Gal9 group at 24 h significantly lower than that in EV group (P < 0.05), and the survival rate has increased from 15% to 35%. A comparative transcriptome analysis between the Gal9 and EV group identified 305 differentially expressed genes (DEGs). The analysis showed that OmGal-9 might play important regulatory in glycolysis / gluconeogenesis, fatty acid degradation, and ascorbate and aldarate metabolism. Moreover, the immune-related DEGs were predominantly enriched in eleven pathways, with the most important three of them being linked to innate immunity: NOD-like, C-type lectin and Toll-like receptor signaling pathway. Taking together, OmGal-9 can enhance the resistance of fish to bacterial diseases by improving immune system function and activating immune-related pathways.

Screening and identification of the hub genes in severe acute pancreatitis and sepsis.

Severe acute pancreatitis (SAP) is accompanied with acute onset, rapid progression, and complicated condition. Sepsis is a common complication of SAP with a high mortality rate. This research aimed to identify the shared hub genes and key pathways of SAP and sepsis, and to explore their functions, molecular mechanism, and clinical value. We obtained SAP and sepsis datasets from the Gene Expression Omnibus (GEO) database and employed differential expression analysis and weighted gene co-expression network analysis (WGCNA) to identify the shared differentially expressed genes (DEGs). Functional enrichment analysis and protein-protein interaction (PPI) was used on shared DEGs to reveal underlying mechanisms in SAP-associated sepsis. Machine learning methods including random forest (RF), least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE) were adopted for screening hub genes. Then, receiver operating characteristic (ROC) curve and nomogram were applied to evaluate the diagnostic performance. Finally, immune cell infiltration analysis was conducted to go deeply into the immunological landscape of sepsis. We obtained a total of 123 DEGs through cross analysis between Differential expression analysis and WGCNA important module. The Gene Ontology (GO) analysis uncovered the shared genes exhibited a significant enrichment in regulation of inflammatory response. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the shared genes were primarily involved in immunoregulation by conducting NOD-like receptor (NLR) signaling pathway. Three machine learning results revealed that two overlapping genes (ARG1, HP) were identified as shared hub genes for SAP and sepsis. The immune infiltration results showed that immune cells played crucial part in the pathogenesis of sepsis and the two hub genes were substantially associated with immune cells, which may be a therapy target. ARG1 and HP may affect SAP and sepsis by regulating inflammation and immune responses, shedding light on potential future diagnostic and therapeutic approaches for SAP-associated sepsis.

Evaluation of the clinical efficacy of Pediococcus acidilactici CCFM6432 in alleviating depression

Aim: Accumulating evidence highlights the crucial role of the “gut-brain axis” and emphasizes the potential of dietary interventions to improve brain health through this pathway. This study assesses the effects of the probiotic Pediococcus acidilactici CCFM6432 on mood, sleep, and gastrointestinal function in patients with depressive disorder. Methods: This clinical trial is a randomized, placebo-controlled study (Registration: ChiCTR2300071025). It enrolled 39 adult patients diagnosed with depressive disorder, who were randomly assigned to either the placebo control group (n = 19) or the CCFM6432 intervention group (n = 20). The intervention period spanned four weeks. Assessments were conducted at both the beginning and end of the trial, including comprehensive questionnaire evaluations and the collection of serum and fecal samples. Results: In comparison to the placebo, treatment with CCFM6432 significantly decreased depression and anxiety scores, as well as ameliorated gastrointestinal dysfunction and poor sleep quality commonly associated with mood disorders. Microbiota analysis revealed an increase in species richness without notable changes in overall diversity, yet Pediococcus species was found to be more abundant post-treatment. Functional analysis indicated reduced activity in the NOD-like receptor signaling pathway, suggesting anti-inflammatory effects induced by the probiotic. Metabolomic profiling identified elevated levels of fecal lactic acid, which correlated with lower Hospital Anxiety and Depression Scale (HADS) scores, thereby linking probiotic metabolism to mood enhancement. Conclusion: These findings imply that CCFM6432 may improve brain function by modulating gut microbiota and their mediated immune homeostasis, underscoring its potential as an adjunctive treatment for mental disorders.

Deciphering the mechanisms of the IL-6/CXCL1 and NOD-like receptor signaling pathways in otitis media with effusion in rodents

BackgroundOtitis media with effusion (OME) often leads to pediatric hearing loss and is influenced by innate and adaptive immune responses. Innate immunity serves as the non-specific first line of defense against OME. MethodsWe induced OME in rats using ovalbumin. We administered IL-6 monoclonal antibodies intranasally to inhibit IL-6, and we injected an NF-κB inhibitor intraperitoneally to explore the role of IL-6 in innate immunity and its interaction with the NOD-like receptor signaling pathway. We analyzed RNA-sequencing data with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways to assess signaling pathways involved in OME. We also utilized Western blot, quantitative real-time PCR, and immunohistochemistry on middle ear samples and used microscopy to identify immune cells in ear wash fluids. ResultsOur study suggests a pivotal role for IL-6 in the immune pathways of rats with OME via the regulation of CXCL1-mediated pathways. Increased levels of IL-6 and CXCL1 were observed in the middle ear tissues, and activation of the NLRP3 inflammasome in OME rats led to an immune response via NF-κB, thus promoting IL-6 and CXCL1 production, which was reduced by IL-6 antibody treatment. ConclusionsOur findings confirm that IL-6 and CXCL1 play significant roles in the innate immune response in OME in rodents, predominantly via the NOD-like receptor signaling pathway and NLRP3 inflammasome activation. This research sheds light on OME pathogenesis and its immune-related mechanisms.

Single-cell RNA sequencing analysis of peripheral blood mononuclear cells in PD-1-induced renal toxicity in patients with lung cancer

BackgroundAlthough the patient survival rate for many malignancies has been improved with immune checkpoint inhibitors (ICIs), some patients experience various immune-related adverse events (irAEs). IrAEs impact several organ systems, including the kidney. With anti-programmed cell death protein 1 (PD-1) therapy (pembrolizumab), kidney-related adverse events occur relatively rarely compared with other irAEs. However, the occurrence of AKI usually leads to anti-PD-1 therapy interruption or discontinuation. Therefore, there is an urgent need to clarify the mechanisms of renal irAEs (R-irAEs) to facilitate early management. This study aimed to analyse the characteristics of peripheral blood mononuclear cells (PBMCs) in R-irAEs.MethodsPBMCs were collected from three patients who developed R-irAEs after anti-PD-1 therapy and three patients who did not. The PBMCs were subjected to scRNA-seq to identify cell clusters and differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses were performed to investigate the most active biological processes in immune cells.ResultsFifteen cell clusters were identified across the two groups. FOS, RPS26, and JUN were the top three upregulated genes in CD4+ T cells. The DEGs in CD4+ T cells were enriched in Th17 differentiation, Th1 and Th2 cell differentiation, NF-kappa B, Nod-like receptor, TNF, IL-17, apoptosis, and NK cell-mediated cytotoxicity signaling pathways. RPS26, TRBV25-1, and JUN were the top three upregulated genes in CD8+ T cells. The DEGs in CD8+ T cells were enriched in Th17 cell differentiation, antigen processing and presentation, natural killer cell-mediated cytotoxicity, the intestinal immune network for IgA production, the T-cell receptor signalling pathway, Th1 and Th2 cell differentiation, the phagosome, and cell adhesion molecules.ConclusionsIn conclusion, R-irAEs are associated with immune cell dysfunction. DEGs and their enriched pathways identified in CD4+ T cells and CD8+ T cells play important roles in the development of renal irAEs related to anti-PD-1 therapy. These findings offer fresh perspectives on the pathogenesis of renal damage caused by anti-PD-1 therapy.

514 Roles of core regulatory noncoding RNAs in bovine Staphylococcus aureus subclinical mastitis

Abstract Staphylococcus aureus (S. aureus) is an opportunistic pathogen frequently associated with subclinical mastitis and accounts for a large proportion of the economic losses due to mastitis on Canadian dairy farms. Despite a plethora of investigations on the molecular mechanisms of mastitis, little information is available on the roles of regulatory noncoding RNAs (ncRNA). This study aimed to uncover the regulatory roles of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in the response to subclinical mastitis due to S. aureus of cows. Transcriptome sequencing (RNA-Seq and miRNA-Seq) was conducted on milk somatic cells from 13 cows with S. aureus subclinical mastitis [high somatic cell counts (SCC) of ≥350,000 cells/mL for ≥3 consecutive months and positive for S. aureus only], and 5 healthy cows (low SCC&amp;lt; 100,000 cells/mL for ≥3 consecutive months and negative for mastitis pathogens). RNASeq and smRNASeq nf-core bioinformatics pipelines were used to process the data. Results showed that 97 miRNAs (36 up-regulated and 61 down-regulated) and 1,565 lncRNAs (617 up-regulated and 948 down-regulated) were differentially expressed (DE; FDR&amp;lt; 0.05) between S. aureus subclinical mastitic cows and healthy controls. Competing endogenous networks comprising co-expressed differentially expressed mRNAs (DEGs), miRNAs (DEMs), and lncRNAs (DELs) were constructed. CytoHubba, a Cytoscape plugin, was utilized to identify the top hub regulatory ncRNAs including bta-miR-2387, bta-miR-331, bta-miR-95, bta-miR-744, bta-miR-455, bta-novel-miR-60, bta-miR-3533, bta-miR-500, bta-miR-1249 and bta-novel-miR-66). The results revealed that the main hubs (downregulated bta-miR-2387 and upregulated bta-miR-331) potentially regulate numerous DEGs during S. aureus subclinical mastitis such as NFKB1, IL10RA, IL17RA, TLR10, LOC112442665, LOC112448481 and LOC112442015, etc., and TUNAR, LOC112445429, LOC112444484 and LOC101907369, respectively. Functional analysis of bta-miR-331 target genes revealed roles in several KEGG pathways (e.g., tight junction, bacterial invasion of epithelial cells) and biological processes gene ontology (GO) terms (e.g., cell adhesion, cell locomotion and cell motility). Similarly, roles for bta-miR-2387 in KEGG pathways like JAK-STAT signaling pathway, NOD-like receptor signaling pathway and inflammatory bowel disease, and biological process GO terms such as inflammatory response, neutrophil migration, leucocyte migration and response to cytokine were revealed. MiR-331 has been found to promote cell proliferation, migration, and invasion of breast cancer. Furthermore, bta-miR-2387 has been shown to regulate several immune pathways which aid in improving the movement of spermatids across the epithelium and preleptotene spermatocytes across the blood-testis barrier during spermatogenesis. This supports our findings and suggests that bta-miR-2387 and bta-miR-331 could be pivotal regulators of immune-related genes and pathways during S. aureus subclinical mastitis. Thus, they hold potential as biomarkers for the development of therapeutic and diagnostic tools for managing subclinical mastitis.

Salidroside promotes liver regeneration after partial hepatectomy in mice by modulating NLRP3 inflammasome-mediated pyroptosis pathway

Insufficient residual liver tissue after partial hepatectomy (PH) may lead to serious complications such as hepatic failure and small-for-size syndrome. Salidroside (SAL) is obtained from Rhodiola rosea through modernized separation and extraction and has been validated for treating various liver diseases. It's yet unknown, nevertheless, how SAL affects liver regeneration after PH. This study aimed to determine whether SAL could promote liver regeneration after PH in mice. We demonstrated that SAL could attenuate liver injury after PH and promote hepatocyte proliferation and liver mass recovery. Mechanistically, SAL inhibited the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome, attenuating pyroptosis. RNA-seq analysis indicated that SAL downregulated the transcription of NLRP3 and GSDMD genes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the NOD-like receptor signaling pathway was significantly enriched in down-regulated signaling pathways. Notably, SAL in combination with the NLRP3 inhibitor MCC950 did not further inhibit NLRP3 inflammasome and promote liver mass recovery. In summary, our findings proved that SAL could be a potential agent for improving liver function and promoting liver regeneration after PH.

Extract of Tinospora sinensis alleviates LPS-induced neuroinflammation in mice by regulating TLR4/NF-κB/NLRP3 signaling pathway

Ethnopharmacological relevanceThe dried rattan stem of Tinospora sinensis (Lour.) Merr. is valued for its efficacy of clearing heat and removing toxicity, calming and soothing the nerves. It is widely used in Tibetan medicine for the treatment of rheumatic and aging diseases. Studies have confirmed its anti-inflammatory and ameliorating effects on Alzheimer's disease; however, the anti-neuroinflammation efficacy and mechanism remain unclear. AimThis study aimed to explore the anti-neuroinflammation efficacy, major effective ingredients, and potential mechanism of extract of Tinosporae sinenisis (TIS). MethodsUPLC-Q-TOF/MS was used to identify the compounds of TIS and the plasma components of rats after gastric administration of TIS. C57BL/6 J mice were continuously intraperitoneally injected with lipopolysaccharide (LPS) (250 μg/kg) for 14 d to establish a neuroinflammation model. The effects of TIS (4.5 g/kg, 9 g/kg) on the learning and memory abilities in mice with neuroinflammation was evaluated using spontaneous activity, novel object recognition, and Morris water maze tests. Pathological changes in the hippocampus were observed using hematoxylin and eosin staining. Gene and protein levels of inflammatory factors in the brain were detected using qRT-PCR and ELISA kits. Iba-1 levels in the brain were detected using immunofluorescence to assess the degree of microglial activation. Network pharmacology, based on the components absorbed into plasma of TIS, was used to predict potential targets and pathways. Proteomics was used to study the differentially expressed proteins and related pathways in the brain tissue of mice with neuroinflammation. Finally, correlation analysis was performed on the results of network pharmacology and proteomics, and proteins related the anti-neuroinflammatory effect of TIS were detected by western blot. ResultsA total of 39 compounds were identified in TIS: genipingentiobioside, isocorydin, reticuline, (−)-argemonine, tinosineside A, tinosinenside A, and costunolide were absorbed into the plasma. After continuous intraperitoneal injection of LPS into C57BL/6 J mice, microglia in the brain tissue were activated and the gene and protein levels of IL-1β, TNF-α, IL-6, and iNOS were increased in the brain tissue, suggesting that the neuroinflammation model was successfully established. TIS reduced Iba-1 levels and gene expression and protein levels of inflammatory factors in the brain of mice with neuroinflammation. Furthermore, TIS improved the pathological changes in the hippocampus and learning and memory abilities caused by neuroinflammation. Network pharmacology has predicted that TNF, IL-1β, and IκBKB are closely related to neuroinflammation. Proteomics identified key differentially expressed proteins, including TNF, NF-κB2, NF-κBIA, and TLR4. Toll-like receptor (TLR), NF-κB, and NOD-like receptor (NLR) signaling pathways are involved in neuroinflammation-related pathways. Correlation analysis revealed TLR, TNF and NLR signaling pathways were closely related to the anti-neuroinflammatory effects of TIS. We observed that TIS alleviated neuroinflammation by inhibiting the TLR4/NF-κB/NLRP3 pathway. ConclusionThirty-nine compounds were identified from TIS, among which seven were absorbed into the plasma as prototype components. TIS alleviated LPS-induced neuroinflammation in mice, and its mechanism was related to inhibition of TLR4/NF-κB/NLRP3 signaling pathway.