NOD-like Receptor Signaling Pathway Research Articles

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

BackgroundSevere 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.MethodsWe 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.ResultWe 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.ConclusionARG1 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< 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< 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.

Impact of ovalbumin allergy on oral and gut microbiome dynamics in 6-week-old BALB/c mice.

The gut microbiota is known to have a significant impact on the development of food allergy, and several recent studies have suggested that both oral microbiota, which first come into contact with allergenic foods, may have a profound influence on the development of food allergy. In this study, we have established an ovalbumin-sensitive mice model by utilizing ovalbumin as a sensitizing agent. Subsequently, we performed a comprehensive analysis of the gut and oral microbiota in ovalbumin-sensitive mice and the control mice using full-length 16S rRNA sequencing analysis. Interestingly, both the gut and oral microbiota of ovalbumin-sensitized mice exhibited significant dysbiosis. The relative abundance of s__Lactobacillus_intestinalis in the gut microbiota of ovalbumin-sensitive mice exhibited a significant decrease, whereas the abundance of s__Agrobacterium_radiobacter and s__Acinetobacter_sp__CIP_56_2 displayed a significant increase. Furthermore, the relative abundance of s__unclassified_g__Staphylococcus, s__Streptococcus_hyointestinalis, and s__unclassified_g__Dechloromonas in the oral microbiota of ovalbumin-sensitive mice revealed a significant decrease. In contrast, the abundance of 63 other species, including s__Proteiniclasticum_ruminis, s__Guggenheimella_bovis, and s__Romboutsia_timonensis, demonstrated a significant increase. The random forest classifier achieved the best accuracy in predicting the outcome of food allergy using three gut and three oral biomarkers, with accuracies of 94.12 and 100%, respectively. Based on the predictions of the PICRUSt2 analysis, the only consistent finding observed across multiple samples from both the groups of mice was a significant up-regulation of the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway in the ovalbumin-sensitized mice. Our study demonstrates that ovalbumin-sensitized mice experience substantial alterations in both gut and oral microbial composition and structure, and specific strains identified in this study may serve as potential biomarkers for food allergy screening. Moreover, our findings highlight that the oral environment, under the same experimental conditions, exhibited greater precision in detecting a larger number of species. Additionally, it is worth noting that the NOD-like receptor signaling pathway plays a vital role in the pathogenesis of OVA (ovalbumin)-induced allergy. These findings will generate novel concepts and strategies in the realm of food allergy prevention and treatment.

Differential Gene Expression and Immune Cell Infiltration in Patients with Steroid-induced Necrosis of the Femoral Head.

The study aimed to study the differential gene expression and immune cell infiltration in patients with steroid-induced necrosis of the femoral head (SANFH), identify the key genes and immune cells of SANFH, and explore the relationship between immune cells and SANFH. The high-throughput gene chip dataset GSE123568 was downloaded from the GEO database, and the differential gene expression was analyzed with the R language. The STRING database and Cytoscape software were used to analyze the protein interaction network and screen key genes, and enrichment analysis was carried out on key genes. The infiltration of immune cells in SANFH patients was analyzed and verified by immunohistochemistry. EP300, TRAF6, STAT1, JAK1, CASP8, and JAK2 are key genes in the pathogenesis of SANFH, which mainly involve myeloid cell differentiation, cytokine-mediated signaling pathway, tumor necrosis factor-mediated signaling pathway, and cellular response to tumor necrosis factor through JAK-STAT, NOD-like receptor, toll-like receptor, and other signaling pathways, leading to the occurrence of diseases; immune infiltration and immunohistochemical results have shown the expression of memory B cells and activated dendritic cells as reduced in SANFH patients, while in the same SANFH samples, M1 macrophages have been positively correlated with monocytes, and neutrophils have been negatively correlated with monocytes expression. EP300, TRAF6, STAT1, JAK1, CASP8, and JAK2 have exhibited significant differences in SANFH (spontaneous osteonecrosis of the femoral head). Memory B cells, activated dendritic cells, M1 macrophages, monocytes, and neutrophils have shown abnormal expression in SANFH.

Single-cell transcriptome landscape of the kidney reveals potential innate immune regulation mechanisms in hybrid yellow catfish after Aeromonas hydrophila infection

Aeromonas hydrophila, the pathogen that is the causative agent of motile Aeromonas septicemia (MAS) disease, commonly attacks freshwater fishes, including yellow catfish (Pelteobagrus fulvidraco). Although the kidney is one of the most important organs involved in immunity in fish, its role in disease progression has not been fully elucidated. Understanding the cellular composition and innate immune regulation mechanisms of the kidney of yellow catfish is important for the treatment of MAS. In this study, single-cell RNA sequencing (scRNA-seq) was performed on the kidney of hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) after A. hydrophila infection. Nine types of kidney cells were identified using marker genes, and a transcription module of marker genes in the main immune cells of hybrid yellow catfish kidney tissue was constructed using in-situ hybridization. In addition, the single-cell transcriptome data showed that the differentially expressed genes of macrophages were primarily enriched in the Toll-like receptor and Nod-like receptor signaling pathways. The expression levels of genes involved in these pathways were upregulated in macrophages following A. hydrophila infection. Transmission electron microscopy and TUNEL analysis revealed the cellular characteristics of macrophages before and after A. hydrophila infection. These data provide empirical support for in-depth research on the role of the kidney in the innate immune response of hybrid yellow catfish.

RNA-Seq Analysis of Glycolysis Regulation of Avian Leukosis Virus Subgroup J Replication.

Avian Leukosis virus (ALV) is a widely spread virus that causes major economic losses to the global poultry industry. This study aims to investigate the effect of glycolysis on the replication of the ALV-J virus and identify the key circular RNAs that regulate the replication of the ALV-J virus. We found that glucose uptake, pyruvate content, and lactate content in DF1 cells were increased after ALV-J infection. Moreover, inhibiting the glycolysis of ALV-J-infected DF1 cells reduced the replication of the ALV-J virus. To further study the mechanism of glycolysis in the replication of the ALV-J virus, we performed RNA-seq on ALV-J-infected and ALV-J-infected cells treated with glycolysis inhibition. RNA-seq results show that a total of 10,375 circular RNAs (circRNAs) were identified, of which the main types were exonic circular RNAs, and 28 circRNAs were differentially expressed between ALV-J-infected and ALV-J-infected cells treated with glycolysis inhibition. Then, we performed functional enrichment analysis of differentially expressed circRNA source and target genes. Functional enrichment analysis indicated that some circRNAs might be involved in regulating the replication of the ALV-J virus by influencing some pathways like glycolysis/gluconeogenesis, the NOD-like receptor signaling pathway, MAPK signaling pathway, p53 signaling pathway, Toll-like receptor signaling pathway, Insulin signaling pathway, and Apoptosis. This study revealed the effect of glycolysis on the replication of the ALV-J virus in DF1 cells and its possible regulatory mechanism, which provided a basis for understanding the factors influencing the replication of the ALV-J virus and reducing the rate of infection of the ALV-J virus in poultry.

Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication

The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus–host interactions and potential antiviral strategies.

Identification and expression analysis of NOD-like receptor gene family in response to Aeromonas hydrophila and poly I:C challenge in Chinese soft-shelled turtle (Pelodiscus sinensis)

Nucleotide-binding domain (NOD)-like receptors (NLRs), a type of pattern recognition receptor (PRR), specifically recognize pathogen-associated molecular patterns and damage-associated molecular patterns, playing crucial roles in the immune system. Herein, the composition, expression pattern, and co-expressed pathways of NLRs in Pelodiscus sinensis (PsNLRs) were analyzed. Eight PsNLRs, located on seven chromosomes, were first identified from the genome. Phylogenetic analysis revealed that PsNLRs were most closely related to the NLRs of the western painted turtle and the green sea turtle, and could be clustered into four subfamilies. Besides, the expression profile of PsNLRs exhibited tissue-specificity after infection with Aeromonas hydrophila or poly I:C. Nonetheless, PsCIITA and PsNLRP3.2 displayed infection-specific expression patterns, whereas other PsNLRs showed similar expression patterns during A. hydrophila and poly I:C infections. Weighted gene co-expression network analysis found that six PsNLRs were generated in the turquoise module, in which several immune-related signaling pathways were enriched, including “NOD-like receptor signaling pathway”, “NF-κB signaling pathway”, “PI3K-Akt signaling pathway”, “Chemokine signaling pathway”, “Antigen processing and presentation”, and ”Toll-like receptor signaling pathway”. Additionally, three immune-related genes, NF-κB1, TRAF3, and PIK3CG, were identified as hub genes. These results indicated that PsNLRs may play key roles in regulating immune responses in P. sinensis. Overall, an in-depth study of the NLR gene family in P. sinensis would yield insights into developing strategies to combat bacterial and viral infections.

Establishment and characterization of a gill cell line from Takifugu obscurus and transcriptome analysis of its gene expression profiles upon low temperature

Takifugu obscurus is a farmed fish of great economic importance in China. The rapid development of T. obscurus aquaculture industry has been accompanied by disease and low-temperature stress, resulting in huge economic losses. Cell lines are used extensively in teleost physiology and pathology as the most cost-effective platform for in vitro research. A novel gill cell line of T. obscurus (named TOG) was first successfully established, and passed through 52 generations. The optimal conditions for TOG growth were 20 % FBS concentration and 24 °C, TOG could be grown in both hypotonic (150 mOsmol-kg-1) and hypertonic (600 mOsmol-kg-1) environments. TOG was determined to be derived from T. obscurus by sequencing the mitochondrial COI gene. Karyotype analysis revealed that the chromosome number of TOG was 44 (2n = 44). Transfection experiment showed that TOG was able to express foreign genes. Furthermore, several immune-related genes were significantly up-regulated in TOG after LPS and poly (I:C) stimulation, including tlr3, isg15, il1β and il10. Additionally, transcriptome analysis of TOG under low-temperature stress (24 °C, 18 °C, 12 °C, 10 °C and 8 °C) found that differentially expressed genes (DEGs) were significantly clustered in several immunological and energy metabolic pathways, and cold stress could disrupt the immune barrier and reduce immunity by downregulating the immune-related pathways. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that bule module and turquoise module, which were closely correlated with low temperature and the degree of fish damage, were both predominantly found in PPAR, NOD-like receptor and Toll-like receptor signaling pathway. Hub genes were identified in these two modules, including mre11, clpb, dhx15, ddx18 and utp15. TOG cell line will become an effective experimental platform for genetic and immunological research, and our results would help us gain a deeper insight into the molecular mechanism of cold tolerance in teleost.

Analysis of Key Genes Related to Systemic Lupus Erythematosus and COVID-19.

Systemic Lupus Erythematosus (SLE) is a multifactorial and complex immune disease; however, the relevance of COVID-19 infection in SLE patients remains uncertain. This study aims to explore the key candidate genes and pathways in patients with SLE. It also seeks to employ bioinformatics analysis to unravel the molecular signatures inherent in both SLE and COVID-19 patients. The ultimate aim is to identify potential targets and markers specifically relevant to SLE patients who contract SARS-CoV-2. Datasets (GSE12374, GSE20864, GSE61635, GSE81622, and GSE144390) from the Gene Expression Omnibus (GEO) database were analyzed using Robust Rank Aggregation (RRA) method to identify differential expression genes (DEGs) in SLE patients compared to healthy individuals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, tissue-specific gene analysis, and Protein-protein interaction (PPI) network were performed. Finally, the Venn diagram was employed to identify the intersections of COVID-19 genes, serving as potential targets for SLE patients with COVID-19 infection. A total of 154 DEGs were discovered, with GO enrichment indicating a predominant involvement in the defense response against the virus (P<0.001). KEGG pathway analysis showed enrichment in the NOD-like receptor signaling pathway and coronavirus disease, specifically COVID-19 (P<0.001). Tissue-specific genes related to the hematological and immune systems were emphasized (74%). The PPI network highlighted 22 genes, and 5 key genes, namely, IFIT1, IFIT3, MX1, MX2, and OAS3, which were identified after intersecting with COVID-19 patients' data. IFIT1, IFIT3, MX1, MX2, and OAS3 exhibiting differential expression, as well as the pathways associated with COVID-19, could potentially function as biomarkers and therapeutic targets for individuals with SLE infected with COVID-19.

Mesoporous manganese-doped nano-antioxidant suppresses pyroptosis for effective ischemic stroke therapy

Pyroptosis mediates neuroinflammation following ischemic stroke and contributes to its development, indicating that targeting pyroptosis-related pathways could be a potential therapeutic strategy for stroke. In this study, a mesoporous manganese-doped cerium oxide (MMC) nano-antioxidant encapsulated with caspase-1 inhibitor VX765 is rationally designed and engineered to relieve ischemic stroke by integrating the suppression of pyroptosis activity with reactive oxygen species (ROS) scavenging capability. Through the surface modification of Angiopoietin-2 (Ang2), the nano-antioxidant can specifically bind to the low-density lipoprotein receptor-related protein, cross the blood–brain barrier, and ultimately aggregate in ischemic neuronal tissues. In mice subjected to middle cerebral artery occlusion/reperfusion, the nano-antioxidant alleviates oxidative stress, inhibits cell pyroptosis, reduces inflammatory cytokines, and regulates microglial polarization toward M2, ultimately mitigating neuroinflammation and reperfusion injury in brain tissue. Mechanistic studies reveal that the pyroptosis-associated NOD-like receptor signaling pathway, the inflammation-associated TNF signaling pathway, the neutrophil chemotaxis-associated Toll-like receptor signaling pathway, and the oxidative stress-associated NF-kappa B signaling pathway are involved in the antioxidant-mediated stroke therapy. Endowed with pyroptosis-reversing ability and nano-antioxidant activities, this engineered antioxidant presents a novel therapeutic paradigm for minimizing reperfusion injury and enhancing stroke treatment efficacy.

Sodium-iodate injection can replicate retinal and choroid degeneration in pigmented mice: Using multimodal imaging and label-free quantitative proteomics analysis

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the elderly population. Sodium iodate (NaIO3), a stable oxidizing agent, has been injected to establish a reproducible model of oxidative stress-induced RPE and photoreceptor death. The aim of our study was to evaluate the morphological and molecular changes of retina and retinal pigment epithelium (RPE)-choroid in NaIO3-treated mouse using multimodal fundus imaging and label-free quantitative proteomics analysis. Here, we found that following NaIO3 injection, retinal degeneration was evident. Fundus photographs showed numerous scattered yellow-white speckled deposits. Optical coherence tomography (OCT) images indicated disruption of the retinal layers, damage of the RPE layer and accumulation of hyper-reflective matter in multiple layers of the outer retina. Widespread foci of a high fundus autofluorescence (FAF) signal were noticed. Fundus fluorescein angiography (FFA) revealed diffuse intense transmitted fluorescence mixed with scattered spot-like blocked fluorescence. Indocyanine green angiography (ICGA) presented punctate hyperfluorescence. Due to the atrophy of the RPE and Bruch's membrane and choroidal capillary complex, the larger choroidal vessels become more prominent in ICGA and optical coherence tomography angiography (OCTA). Transmission electron microscope (TEM) illustrated abnormal material accumulation and damaged mitochondria. Bioinformatics analysis of proteomics revealed that the differentially expressed proteins participated in diverse biological processes, encompassing phototransduction, NOD-like receptor signaling pathway, phagosome, necroptosis, and cell adhesion molecules. In conclusion, by multimodal imaging, we described the phenotype of NaIO3-treated mouse model mimicking oxidative stress-induced RPE and photoreceptor death in detail. In addition, proteomics analysis identified differentially expressed proteins and significant enrichment pathways, providing insights for future research, although the exact mechanism of oxidative stress-induced RPE and photoreceptor death remains incompletely understood.

Anti-inflammatory and DNA Repair Effects of Astragaloside IV on PC12 Cells Damaged by Lipopolysaccharide.

This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide (LPS) and to examine the effects of astragaloside IV on key targets using high-throughput sequence technology and bioinformatics analyses. PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25, 0.5, 0.75, 1, and 1.25 mg/mL for 24 h. Cell morphology was evaluated, and cell survival rates were calculated. A neurocyte inflammatory model was established with LPS treatment, which reached a 50% cell survival rate. PC12 cells were treated with 0.01, 0.1, 1, 10, or 100 µmol/L astragaloside IV for 24 h. The concentration of astragaloside IV that did not affect the cell survival rate was selected as the treatment group for subsequent experiments. NOS activity was detected by colorimetry; the expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting. The differentially expressed genes (DEGs) between the groups were screened using a second-generation sequence (fold change>2, P<0.05) with the following KEGG enrichment analysis, RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells. The viability of PC12 cells was not altered by treatment with 0.01, 0.1, or 1 µmol/L astragaloside IV for 24 h (P>0.05). However, after treatment with 0.5, 0.75, 1, or 1.25 mg/mL LPS for 24 h, the viability steadily decreased (P<0.01). The mRNA and protein expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS, and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h (P<0.01); however, these changes were reversed when PC12 cells were pretreated with 0.01, 0.1, or 1 µmol/L astragaloside IV in PC12 cells and then treated with 1 mg/mL LPS for 24 h (P<0.05). Second-generation sequencing revealed that 1026 genes were upregulated, while 1287 genes were downregulated. The DEGs were associated with autophagy, TNF-α, interleukin-17, MAPK, P53, Toll-like receptor, and NOD-like receptor signaling pathways. Furthermore, PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2, CCL11, CCL7, MMP3, and MMP10, which are associated with the IL-17 pathway. RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results. LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage. astragaloside IV plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.

The immune-inflammation factor is associated with diabetic nephropathy: evidence from NHANES 2013–2018 and GEO database

Diabetic nephropathy (DN) is a common secondary kidney disease. Immune and inflammatory responses play an influential role in the development of DN. This study aims to explore the role and mechanisms of immune- and inflammatory-related factors in DN. Participants from the NHANES 2013–2018 were included to evaluate the association between the SII and DN. Considering the skewed distribution of SII, log SII was used for subsequent analysis. Then, the DEGs were extracted from the GSE96804 dataset by the “limma” package of R, which were further screened out genes in the key module based on WGCNA. The intersection genes between DEGs and key module genes were the key genes for the following mechanism exploration. The CyTargetlinker plug-in of Cytoscape software was used to construct the drug-genes network. Molecular docking was used to calculate the binding affinity between potential drugs and the hub genes. Among the 8236 participants from NHANES 2013–2018, Log SII was significantly associated with DN (p < 0.05). DEG and WGCNA revealed 30 DN-related genes, which mainly regulated immune- and inflammation pathways, and the NOD-like receptor signaling pathway was the core pathway highly involved in the DN occurrence. Moreover, NAIP, ZFP36, and DUSP1 were identified as hub genes in DN progression and there was a strong binding interaction between resveratrol and DUSP1.In conclusion, immune inflammation plays an influential role in the occurrence and development of DN. SII is an effective diagnostic marker for DN and resveratrol might have potential value in treating DN.

Insights into the microscopic heterogeneity of whey proteins between yak colostrum and mature milk based on 4D lable-free quantitative phosphoproteomics

This study aimed to reveal the change patterns of the phosphorylation modification status of yak whey phosphoproteins during lactation and their physiological effects. Herein, we comprehensively characterized whey phosphoproteome in yak colostrum and mature milk using an ultra-high throughput phosphoproteomics approach incorporating trapped ion mobility technology. A total of 344 phosphorylation sites from 206 phosphoproteins were identified, with individual site modification predominating. Notably, 117 significantly different phosphorylation sites were distributed on 89 whey phosphoproteins. Gene ontology analysis indicated that these significantly different whey phosphoproteins (SDWPPs) were mainly annotated to carbohydrate metabolic process, membrane, extracellular region and calcium ion binding. Metabolic pathway enrichment analysis demonstrated that SDWPPs were critically involved in protein processing in endoplasmic reticulum, NOD-like receptor signaling pathway and N-glycan biosynthesis. Our results elucidate the phosphorylation profiles of yak whey phosphoproteins at different lactations and their adaptive regulatory role in meeting the nutritional requirements of yak calves during development.