NF-κB Signaling Research Articles

Fluorinated dendrimer-mediated miR-30a delivery regulates the inflammation of macrophages and mitigates the symptoms of rheumatoid arthritis

Abnormal expression of microRNAs (miRNAs) plays a significant role in the pathogenesis of rheumatoid arthritis (RA), and thus miRNA-based therapy has emerged as a promising approach for the RA treatment. Herein, miR-30a was successfully screened and identified to be an essential mediator for the inflammation of RA. MiR-30a could directly target the Snai1 gene and further regulate the Cad11 expression to inhibit the NF-κB and MAPK signaling pathways, contributing to the anti-inflammatory effect. To enhance the therapeutic outcome of miR-30a, fluorinated polyamidoamine dendrimer (FP) was developed as the carrier to achieve the miR-30a delivery in the mice of collagen-induced arthritis. The carrier FP and miR-30a formed stable nanocomplexes and effectively mediated the transfection of miR-30a to execute the anti-inflammatory response in lipopolysaccharide-stimulated macrophages. Further, the intravenous administration of FP/miR-30a showed obvious accumulation in the inflamed joints and inhibited the inflammatory response via the Snai1/Cad11 axis, thereby contributing to the anti-arthritic efficacy. In addition, the FP/miR-30a nanocomplexes displayed favorable biocompatibility, as they did not cause the damage of organs following the systemic administration. Taken together, our study demonstrated that miR-30a is an effective anti-inflammatory oligonucleotide and the fluorinated dendrimer-mediated miR-30a delivery possesses the potential to be a promising approach for the treatment of RA and other autoimmune diseases.

Exploring the Promising Role of Guggulipid in Rheumatoid Arthritis Management: An In-depth Analysis.

Guggulipid, an oleo-gum resin extracted from the bark of Commiphora wightii of the Burseraceae family, holds a significant place in Ayurvedic medicine due to its historical use in treating various disorders, including inflammation, gout, rheumatism, obesity, and lipid metabolism imbalances. This comprehensive review aims to elucidate the molecular targets of guggulipids and explore their cellular responses. Furthermore, it summarizes the findings from in-vitro, in-vivo, and clinical investigations related to arthritis and various inflammatory conditions. A comprehensive survey encompassing in-vitro, in-vivo, and clinical studies has been conducted to explore the therapeutic capacity of guggulipid in the management of rheumatoid arthritis. Various molecular pathways, such as cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), PI3-kinase/AKT, JAK/STAT, nitric oxide synthase (iNOS), and NFκB signaling pathways, have been targeted to assess the antiarthritic and anti-inflammatory effects of this compound. The research findings reveal that guggulipid demonstrates notable antiarthritic and anti-inflammatory effects by targeting key molecular pathways involved in inflammatory responses. These pathways include COX-2, VEGF, PI3-kinase/AKT, JAK/STAT, iNOS, and NFκB signaling pathways. in-vitro, in-vivo, and clinical studies collectively support the therapeutic potential of guggulipid in managing rheumatoid arthritis and related inflammatory conditions. This review provides a deeper understanding of the therapeutic mechanisms and potential of guggulipid in the management of rheumatoid arthritis. The collective evidence strongly supports the promising role of guggulipid as a therapeutic agent, encouraging further research and development in guggulipid-based treatments for these conditions.

Cannabiorcol as a novel inhibitor of the p38/MSK-1/NF-κB signaling pathway, reducing matrix metalloproteinases in osteoarthritis

BackgroundThe bioactivity and potential medicinal applications of cannabiorcol, a lesser-known derivative of Cannabis sativa, require further investigation. Osteoarthritis (OA) is a chronic joint condition marked by gradual degradation of the cartilage and commonly associated with elevated levels of matrix metalloproteinases (MMPs). However, the influence of cannabiorcol on OA and its underlying mechanisms remains unclear. MethodsIn silico analysis investigated the key transcription factors that regulate MMP expression. A chondrocyte cell model [interleukin (IL)-1β and IL-1⍺-treated C20A4 cell line] was established and treated with cannabiorcol. Associated cytotoxicity was assessed using a WST-8 assay. A monoiodoacetate-induced OA rat model was established and treated with cannabiorcol. Protein translocation and transactivation analyses were conducted using immunofluorescence and dual-luciferase reporter assays, respectively. Western blotting and real-time PCR analyzed relevant markers to examine cannabiorcol's effects on OA and its fundamental mechanisms. ResultsCannabiorcol inhibits the expression of IL-1β-induced MMPs compared to other cannabis-related compounds. In silico analysis revealed that the nuclear factor-kappa β (NF-κβ) and mitogen-activated protein kinase (MAPK) pathways are associated with MMP expression as key regulators. In vitro, cannabiorcol inhibits the NF-κB and p38 MAPK pathways independently cannabinoid receptors and transient receptor potential vanilloids. In vivo, cannabiorcol reduces MMP expression and ameliorates monoiodoacetate-induced OA traits in rats. ConclusionCannabiorcol inhibits IL-1β-induced MMP expression in vitro and alleviates OA in an MIA-induced OA rat model by reducing MMP expression and inhibiting the p65/p38 axis.

Network Pharmacology, Molecular Docking and Experimental Verification Revealing the Mechanism of Fule Cream against Childhood Atopic Dermatitis.

The Fule Cream (FLC) is an herbal formula widely used for the treatment of pediatric atopic dermatitis (AD), however, the main active components and functional mechanisms of FLC remain unclear. This study performed an initial exploration of the potential acting mechanisms of FLC in childhood AD treatment through analyses of an AD mouse model using network pharmacology, molecular docking technology, and RNA-seq analysis. The main bioactive ingredients and potential targets of FLC were collected from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and SwissTargetPrediction databases. An herb-compound-target network was built using Cytoscape 3.7.2. The disease targets of pediatric AD were searched in the DisGeNET, Therapeutic Target Database (TTD), OMIM, DrugBank and GeneCards databases. The overlapping targets between the active compounds and the disease were imported into the STRING database for the construction of the protein-protein interaction (PPI) network. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the intersection targets were performed, and molecular docking verification of the core compounds and targets was then performed using AutoDock Vina 1.1.2. The AD mouse model for experimental verification was induced by MC903. The herb-compound-target network included 415 nodes and 1990 edges. Quercetin, luteolin, beta-sitosterol, wogonin, ursolic acid, apigenin, stigmasterol, kaempferol, sitogluside and myricetin were key nodes. The targets with higher degree values were IL-4, IL-10, IL-1α, IL-1β, TNFα, CXCL8, CCL2, CXCL10, CSF2, and IL-6. GO enrichment and KEGG analyses illustrated that important biological functions involved response to extracellular stimulus, regulation of cell adhesion and migration, inflammatory response, cellular response to cytokine stimulus, and cytokine receptor binding. The signaling pathways in the FLC treatment of pediatric AD mainly involve the PI3K-Akt signaling pathway, cytokine‒cytokine receptor interaction, chemokine signaling pathway, TNF signaling pathway, and NF-κB signaling pathway. The binding energy scores of the compounds and targets indicate a good binding activity. Luteolin, quercetin, and kaempferol showed a strong binding activity with TNFα and IL-4. This study illustrates the main bioactive components and potential mechanisms of FLC in the treatment of childhood AD, and provides a basis and reference for subsequent exploration.

Zebrafish FKBP5 facilitates apoptosis and SVCV propagation by suppressing NF-κB signaling pathway

FK506-binding protein 5 (FKBP5), encoded by FKBP5 gene, has been reported as a scaffolding protein in various mammalian pathways related to immunity, inflammation, apoptosis and autophagy. However, the role of FKBP5 in lower vertebrates remains unknown. In this study, we identified zebrafish FKBP5 (DrFKBP5), an ortholog of mammalian FKBP5, which shows high homology with its counterpart in Anabarilius grahami based on amino acid alignment and phylogenetic analysis. DrFKBP5 was found to express ubiquitously across all tested tissues. Its expression were significantly upregulated in eye, intestine, gill, skin, heart, liver and kidney following SVCV treatment. A similar expression pattern was also observed in EPC and ZFIN cells. DrFKBP5 decreased the promoter activitiy of NF-κB and IL-6 rather than IFN I. It also inhibited the expression of inflammatory factor genes such as IL-6, IL-1β and TNF-α. In molecular mechanism, we found that DrFKBP5 interacted with IKKβ (an activator of NF-κB pathway), but not with IKKα or IKKγ, suggesting that DrFKBP5 regulates NF-κB pathway by targeting IKKβ. Then, DrFKBP5 significantly reduced the phosphorylation of IKKβ. Furthermore, it inhibited SVCV-induced nuclear translocation, phosphorylation of p65 and promoted SVCV replication in ZFIN cells. Finally, DrFKBP5 activated the expression of apoptosis-related genes, including BAX, Bcl2, caspase-3 and induced apoptosis under SVCV treatment.

Zang Siwei Qingfei Mixture Alleviates Inflammatory Response to Attenuate Acute Lung Injury by the ACE2/NF-κB Signaling Pathway in Mice.

Acute lung injury (ALI) is a serious lung disease characterized by acute and severe inflammation. Upregulation of ACE2 and inhibition of the NF-κB signaling pathway attenuate LPS-induced ALI. To explore whether Zang Siwei Qingfei Mixture inhibits the development of ALI through the ACE2/NF-κB signaling pathway. Alveolar type II epithelial cells (AEC II) were identified by immunofluorescence staining and flow cytometry. C57BL/6J mice were treated with LPS to establish an ALI model. Cell viability was assessed using CCK8 assays. The levels of ACE, ACE2, p-p38/p38, p- ERK1/2/ERK1/2, p-JNK/JNK, p-IκBα/IκB-α, p-NF-κBp65 were analyzed by Western blotting. ELISA was applied to detect the levels of TNF-a, IL-6, AGT, and Ang1-7. HE staining was used to observe lung injury. The mRNA expression of ACE, ACE2, and Mas was measured by RT-qPCR. AEC II cells were successfully isolated. Treatment with the Zang Siwei Qingfei Mixture resulted in a decrease in ACE, p-p38/p38, p-ERK1/2/ERK1/2, p-JNK/JNK, p-IκBα/IκB-α, p-NF-κBp65 levels, while increasing ACE2 levels. Zang Siwei Qingfei mixture also led to a reduction in TNF-α, IL6, and AGT levels, while increasing Ang1-7 level. Histological analysis showed that Zang Siwei Qingfei Mixture treatment improved the alveolar structure of ALI mice and reduced inflammatory infiltration. The pretreatment with MLN-4760, an ACE2 inhibitor, resulted in opposite effects compared to Zang Siwei Qingfei Mixture treatment. Zang Siwei Qingfei mixture attenuates ALI by regulating the ACE2/NF-κB signaling pathway in mice. This study provides a theoretical foundation for the development of improved ALI treatments.

Effect of Perilla frutescens polyphenols against tight junction alterations and intestinal inflammation: a Caco-2/macrophage co-culture model-based study integrated with network pharmacology

The intestinal barrier plays important roles in the uptake of nutrients and prevention of harmful pathogens. However, intestinal inflammation causes barrier dysfunction, which may contribute to inflammatory bowel disease (IBD). Polyphenols from Perilla frutescens (L.) Britt. exhibit various bioactivities, though their protective effect on the intestinal barrier remains unclear. To address this, the study investigated the effect of Perilla frutescens (L.) Britt. polyphenols (PFP) against tight junction alterations and intestinal inflammation using an intestinal-like Caco-2/macrophage co-culture model. LC-MS/MS analysis identified 15 polyphenols in the PFP extract. Furthermore, PFP were transported across the apical Caco-2 monolayer, with apigenin, genistin, syringic acid and cinnamic acid showing relatively high transport rates (> 70%). The lipopolysaccharide (LPS)-induced tight junction alterations, evaluated by trans-epithelial electrical resistance (TEER) analysis, qPCR, and immunofluorescence assay, were significantly restored by medium and high doses of PFP (P < 0.05). In addition, network pharmacology analysis demonstrated that the core targets of PFP for intestinal inflammation were mostly located in the MAPK and NF-κB signaling pathways. The expression levels of the key proteins in these pathways were further validated by qPCR and western blot analyses. Overall, PFP exhibits great potential as a novel functional food component for treating intestinal inflammation.

FAM83H regulated by glis3 promotes triple-negative breast cancer tumorigenesis and activates the NF-κB signaling pathway.

Triple-negative breast cancer (TNBC) is a highly aggressive and invasive form of breast cancer (BC) with a high mortality rate and a lack of effective targeted drugs. Family with sequence similarity 83 member H (FAM83H) is critically implicated in tumorigenesis. However, the potential role of FAM83H in TNBC remains elusive. Here, we discovered that FAM83H exhibited high expression in tumor tissues of patients with TNBC and was associated with TNM stage. Gain- or loss-of-function experiments were conducted to explore the biological role of FAM83H in TNBC. Subsequently, functional enrichment analysis confirmed that FAM83H overexpression promoted TNBC cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT), accompanied by upregulation of cyclin E, cyclin D, Vimentin, N-cadherin and Slug. As observed, FAM83H knockdown showed anti-cancer effects, such as fostering apoptosis and inhibiting tumorigenicity and metastasis of TNBC cells. Mechanistically, FAM83H activated the NF-κB signaling pathway. Moreover, a dual-luciferase reporter assay demonstrated that GLIS family zinc finger 3 (GLIS3) bound to the promoter of FAM83H and enhanced its transcription. Notably, overexpression of GLIS3 significantly stimulated TNBC cell proliferation and invasion, and all of this was reversed by rescue experiments involving the knockdown of FAM83H. Overall, FAM83H exacerbates tumor progression, and in-depth understanding of FAM83H as a therapeutic target for TNBC will provide clinical translational potential for intervention therapy.

Mechanism of Action of Tongjiang Mixture for Treating Reflux Esophagitis: A Study Using Serum Pharmacochemistry and Network Pharmacology.

Tongjiang Mixture (TJM) is a traditional Chinese formula for treating reflux esophagitis (RE). Nevertheless, its active ingredients and potential pharmacological mechanisms are not yet clearly elucidated. This study will identify the active ingredients of TJM using serum pharmacochemistry and to elucidate the mechanism on RE through network pharmacology. The blood-borne ingredients of TJM are identified by the Ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometer. Subsequently, a "compound-target-disease" network is established and obtained core targets associated with TJM and RE. Then, the potential signaling pathways are forecasted through the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Finally, the rat model of RE is established to verify the results predicted by network pharmacology through animal experiments. Fifteen blood-borne ingredients of TJM are identified, with eight active ingredients-namely Tangeretin, Tricin, Palmati, Berberine, Limonin, Evodiamine, Tetrahydropalmatine and Rutecarpine - making significant contributions to its efficacy. Moreover, TJM is predicted to act on 193 targets related to RE, involving AKT1, HSP90AA1, PIK3CA, and other targets, which enriches mainly in PI3K/AKT /NF-κB signaling. Additionally, TJM can alleviate inflammation of the esophageal mucosa, reduce pathological damage, and increase gastric pH. It can downregulate PI3K, AKT, and NF-κB mRNA transcription levels and reduce the protein expression of PI3K, AKT, and NF-κB. Furthermore, it can inhibit the overproduction of IL-6, TNF-α and IL-17. TJM can alleviate immune-inflammatory responses and ameliorate RE by restraining the PI3K/AKT pathway and its downstream NF-κB.

Sappanone A Ameliorates Concanavalin A-induced Immune-Mediated Liver Injury by Regulating M1 Macrophage Polarization.

Sappanone A (SAP), a high-isoflavone compound derived from the traditional Chinese medicine Sumu, exhibits various pharmacological activities, including anti-inflammatory and anti-oxidant effects. However, its protective effects on the liver have rarely been reported. The aim of this study was to investigate the effects of SAP on immune-mediated liver injury induced by concanavalin A (Con A) in mice and to explore the underlying molecular mechanisms. Mice were administered SAP intraperitoneally (50mg/kg body weight). Three hours later, Con A (18mg/kg) was injected via the tail vein to induce liver damage. Livers and blood were collected 12h after Con A challenge. Liver cell apoptosis, oxidative stress, and M1 macrophage activation in vivo were investigated. Bone marrow-derived macrophages were used to confirm the effects of SAP on M1 polarization in vitro. The results indicated thatSAP decreased transaminase levels, inhibited apoptosis, and improved oxidative stress in mouse livers. Furthermore, SAP significantly reduced the proportion of macrophages, inhibited the expression of CD86, and downregulated the expression of M1 macrophage-related inflammatory cytokines. Moreover, SAP-treated macrophages alleviated liver damage caused by Con A compared to non-SAP-treated macrophages. Mechanistically, SAP inhibited the phosphorylation of key molecules in the MAPK and NF-κB signaling pathways in macrophages, resulting in an inhibitory effect on M1 macrophage activation. Taken together,SAP alleviates immune-mediated liver injury induced by Con A by suppressing M1 macrophage polarization, which is partially associated with NF-κB and MAPK signaling pathways.

Breast Cancer Subtype-Specific Organotropism is Dictated by FOXF2-Regulated Metastatic Dormancy and Recovery.

Breast cancer subtypes display different metastatic organotropism. Identification of the mechanisms underlying subtype-specific organotropism could help uncover potential approaches to prevent and treat metastasis. Herein, we found that FOXF2 promoted the seeding and proliferative recovery from dormancy of luminal breast cancer (LumBC) and basal-like breast cancer (BLBC) cells in the bone by activating the NF-κB and BMP signaling pathways. Conversely, FOXF2 suppressed the seeding and proliferative recovery of BLBC cells in the lung by repressing the TGF-β signaling pathway. FOXF2 directly upregulated RelA/p65 transcription and expression in LumBC and BLBC cells by binding to the RELA proximal promoter region, and RelA/p65 bound to the FOXF2 proximal promoter region to upregulate expression, forming a positive feedback loop. Targeting the NF-κB pathway efficiently prevented the metastasis of FOXF2-overexpressing breast cancer cells to the bone, while inhibiting TGF-β signaling blocked the metastasis of BLBC with low FOXF2 expression to the lung. These findings uncover critical mechanisms of breast cancer subtype-specific organotropism and provide insight into precision assessment and treatment strategies.

Immunomodulation through the Circadian Clock: Impacts on Inflammation and Immunity

The circadian clock, an intrinsic timekeeping system regulating physiological functions over a 24-hour cycle, profoundly influences immune responses and inflammation. Immune cells, including neutrophils, macrophages, and lymphocytes, exhibit circadian rhythms in their numbers, function, and migration. These rhythms are regulated by both central and peripheral clocks, synchronizing immune cell activity with environmental cues. The circadian clock modulates key immune processes such as leukocyte trafficking, cytokine production, and phagocytosis, affecting the body’s ability to respond to pathogens and tissue injury. Furthermore, circadian rhythms tightly control inflammatory pathways, including NF-κB signaling, and regulate the balance between pro- and anti-inflammatory states in macrophages. Disruptions to circadian rhythms—due to factors like shift work or sleep disorders—are associated with heightened inflammation and increased risk for inflammatory diseases, such as autoimmune disorders, metabolic syndromes, and cancer. Understanding how circadian rhythms govern immune functions has significant implications for chronotherapy, which aims to optimize the timing of treatments to improve efficacy and reduce adverse effects. This review explores the mechanisms through which the circadian clock modulates immune responses and inflammation, highlighting the potential for circadian-based therapies in managing inflammatory and immune-related diseases. Keywords: Circadian clock, immunity, inflammation, immune cells, chronotherapy.

Bioinformatics, expression, purification, and inflammation-inducing effect of Mycoplasma genitalium GroEL protein

To preliminarily understand the pathogenic mechanism of Mycoplasma genitalium (Mg) GroEL protein, we used bioinformatics tools to predict the structure and function of Mg GroEL protein and then constructed the recombinant plasmid pET-28a-GroEL. The protein expression was induced by 0.2 mmol/L IPTG, and the expressed protein was purified by Ni-iminodicitic acid (IDA) column affinity. Tohoku Hospital Pediatrics-1 (THP-1) cells were exposed to 2 μg/mL Mg rGroEL. The levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the cell supernatant were measured by ELISA, and that of IL-6 was measured by an automatic chemiluminescence instrument. The activation of the nuclear factor-kappa B (NF-κB) signaling pathway was visualized by immunofluorescence and Western blotting. The results showed that Mg GroEL was a stable hydrophilic protein composed of 543 amino acid residues, with the relative molecular mass of 58.44 kDa, an isoelectric point of 5.68, and a molecular formula of C2568H4300N700O825S8. The secondary structure was mainly composed of α-helices and random coils. Mg GroEL contained 12 B-cell dominant epitopes and 10 T-cell dominant epitopes. It exhibited high homology with the GroEL proteins from Mycoplasma pneumoniae, M. agalactiae, M. arthritidis, M. hyopneumoniae, and M. bovis. Mg rGroEL activated the NF-κB signaling pathway and promoted the secretion of IL-1β, IL-6, and TNF-α in THP-1 cells. These results suggest that Mg GroEL exhibits substantial antigenicity and possesses the capability of triggering inflammation in host cells. This study establishes a theoretical basis for future investigations pertaining to the role and pathogenic mechanisms of Mg GroEL.

Multi-Omic Data Integration Suggests Putative Microbial Drivers of Aetiopathogenesis in Mycosis Fungoides

Background: Mycosis fungoides (MF) represents the most prevalent entity of cutaneous T cell lymphoma (CTCL). The MF aetiopathogenesis is incompletely understood, due to significant transcriptomic heterogeneity and conflicting views on whether oncologic transformation originates in early thymocytes or mature effector memory T cells. Recently, using clinical specimens, our group showed that the skin microbiome aggravates disease course, mainly driven by an outgrowing, pathogenic S. aureus strain carrying the virulence factor spa, which was shown by others to activate the T cell signalling pathway NF-κB. Methods: To explore the role of the skin microbiome in MF aetiopathogenesis, we here performed RNA sequencing, multi-omic data integration of the skin microbiome and skin transcriptome using Multi-Omic Factor Analysis (MOFA), virome profiling, and T cell receptor (TCR) sequencing in 10 MF patients from our previous study group. Results: We observed that inter-patient transcriptional heterogeneity may be largely attributed to differential activation of T cell signalling pathways. Notably, the MOFA model resolved the heterogenous activation pattern of T cell signalling after denoising the transcriptome from microbial influence. The MOFA model suggested that the outgrowing S. aureus strain evoked signalling by non-canonical NF-κB and IL-1B, which in turn may have fuelled the aggravated disease course. Further, the MOFA model indicated aberrant pathways of early thymopoiesis alongside enrichment of antiviral innate immunity. In line with this, viral prevalence, particularly of Epstein–Barr virus (EBV), trended higher in both lesional skin and the blood compared to nonlesional skin. Additionally, TCRs in both MF skin lesions and the blood were significantly more likely to recognize EBV peptides involved in latent infection. Conclusions: First, our findings suggest that S. aureus with its virulence factor spa fuels MF progression through non-canonical NF-κB and IL-1B signalling. Second, our data provide insights into the potential role of viruses in MF aetiology. Last, we propose a model of microbiome-driven MF aetiopathogenesis: Thymocytes undergo initial oncologic transformation, potentially caused by viruses. After maturation and skin infiltration, an outgrowing, pathogenic S. aureus strain evokes activation and maturation into effector memory T cells, resulting in aggressive disease. Further studies are warranted to verify and extend our data, which are based on computational analyses.

Transcriptome analysis revealed that ischemic post-conditioning suppressed the expression of inflammatory genes in lung ischemia-reperfusion injury

IntroductionIschemic post-conditioning (I-post C) is a recognized therapeutic strategy for lung ischemia/reperfusion injury (LIRI). However, the specific mechanisms underlying the lung protection conferred by I-post C remain unclear. This study aimed to investigate the protective mechanisms and potential molecular regulatory networks of I-post C on lung tissue.MethodsTranscriptome analysis was performed on rat lung tissues obtained from Sham, ischemia-reperfusion (IR), and I-post C groups using RNA-seq to identify differentially expressed genes (DEGs). Subsequently, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and gene set enrichment analysis (GSEA) were conducted to elucidate significantly enriched pathways in the IR and I-post C groups. Additionally, protein-protein interaction (PPI) network analysis was carried out to examine associations among the DEGs. Pathological changes in lung tissues were assessed using hematoxylin-eosin (H&amp;amp;E) staining. The expression levels of CXCL1 and CXCL6 in the IR and I-post C groups were evaluated through immunofluorescence and Western blotting.ResultsOur results showed that I-post C significantly attenuated both pulmonary edema and inflammatory cell infiltration. Transcriptome analysis identified 38 DEGs in the I-post C group compared to the IR group, comprising 21 upregulated and 17 downregulated genes. Among these, seven inflammation-related DEGs exhibited co-expression patterns with the Sham and IR groups, with notable downregulation of Cxcl1 and Cxcl6. GO analysis primarily linked these DEGs to neutrophil activation, chemotaxis, cytokine activity, and CCR chemokine receptor binding. KEGG analysis revealed enriched pathways, including the IL-17, TNF, and NF-κB signaling pathways. GSEA indicated downregulation of neutrophil chemotaxis and the IL-17 signaling pathway, correlating with reduced expression of Cxcl1 and Cxcl6. Validation of Cxcl1 and Cxcl6 mRNA expression via immunofluorescence and Western blotting supported the RNA-seq findings. Furthermore, a PPI network was constructed to elucidate interactions among the 29 DEGs.ConclusionsThrough RNA-Seq analysis, we concluded that I-post C may reduce inflammation and suppress the IL-17 signaling pathway, thereby protecting against lung damage caused by LIRI, potentially involving neutrophil extracellular traps.

Transglutaminase 2 regulates endothelial cell calcification via IL-6-mediated autophagy

IntroductionEndothelial cell (EC) calcification is an important marker of atherosclerotic calcification. ECs play a critical role not only in atherogenesis but also in intimal calcification, as they have been postulated to serve as a source of osteoprogenitor cells that initiate this process. While the role of transglutaminase 2 (TG2) in cellular differentiation, survival, apoptosis, autophagy, and cell adhesion is well established, the mechanism underlying the TG2-mediated regulation of EC calcification is yet to be fully elucidated.MethodsThe TG2 gene was overexpressed or silenced by using siRNA and recombinant adenovirus. RT-PCR and WB were used to analyze the relative expression of target genes and proteins. 5-BP method analyzed TG2 activity. mCherry-eGFP-LC3 adenovirus and transmission electron microscopy analyzed EC autophagy level. Calcium concentrations were measured by using a calcium colorimetric assay kit. Alizarin red S staining assay analyzed EC calcification level. Elisa analyzed IL-6 level. Establishing EC calcification model by using a calcification medium (CM).ResultsOur findings demonstrated that CM increased TG2 activity and expression, which activated the NF-κB signaling pathway, and induced IL-6 autocrine signaling in ECs. Furthermore, IL-6 activated the JAK2/STAT3 signaling pathway to suppress cell autophagy and promoted ECs calcification.DiscussionECs are not only critical for atherogenesis but also believed to be a source of osteoprogenitor cells that initiate intimal calcification. Previous research has shown that TG2 plays an important role in the development of VC, but the mechanism by which it exerts this effect is not yet fully understood. Our results demonstrated that TG2 forms complexes with NF-κB components inhibition of autophagy promoted endothelial cell calcification through EndMT. Therefore, our research investigated the molecular mechanism of EC calcification, which can provide new insights into the pathogenesis of atherosclerosis.

Anti-Inflammatory Effects of Apostichopus japonicus Extract in Porphyromonas gingivalis-Stimulated RAW 264.7 Cells

Apostichopus japonicus has been used both as a food and in traditional medicine. However, its anti-inflammatory effects in periodontal diseases have not been studied. We examined the anti-inflammatory properties of Apostichopus japonicus extract in RAW 264.7 cells stimulated by Porphyromonas gingivalis. The cytotoxicity of Apostichopus japonicus extract was evaluated using the MTS assay. Its effect on NO production was then measured using the NO assay. The mRNA expression of inducible nitric oxide synthase (iNOS) and the pro-inflammatory cytokines IL-1β and IL-6 were assessed using quantitative real-time PCR (qRT-PCR). Western blotting was performed to investigate the expression of regulatory proteins involved in the NF-κB and MAPK signaling pathways. Apostichopus japonicus extract significantly inhibited NO production without cytotoxicity in RAW 264.7 cells. Following Porphyromonas gingivalis stimulation, treatment with the extract decreased iNOS mRNA expression and protein levels, which are responsible for NO production. The extract also suppressed the mRNA expression of pro-inflammatory cytokines. Additionally, Apostichopus japonicus extract inhibited NF-κB activation by regulating signaling molecules such as IKK and IκBα, while also preventing the phosphorylation of MAPK, including ERK, p38, and JNK, showing anti-inflammatory potential. Therefore, it may be a promising natural candidate for the development of new preventive and therapeutic strategies for periodontitis.

A Novel Heterozygous NFKB2 Variant in a Multiplex Family with Common Variable Immune Deficiency and Autoantibodies Against Type I IFNs.

We studied a family with three male individuals across two generations affected by common variable immune deficiency (CVID). We identified a novel missense heterozygous variant (c.2602T>A:p.Y868N) of NFKB2 in all patients and not in healthy relatives. Functional studies of the mutant allele in an overexpression system and of the patients' cells confirmed the deleteriousness of the NFKB2 variant and genotype, respectively, on the activation of the non-canonical NF-κB signaling pathway. Impaired processing of p100 into p52 underlies p100 accumulation, which results in gain-of-function (GOF) of IκBδ inhibitory activity and loss-of-function (LOF) of p52 transcriptional activity. The three patients' plasma contained autoantibodies that neutralized IFN-α2 and/or IFN-ω, accounting for the severe or recurrent viral diseases of the patients, including influenza pneumonia in one sibling, and severe COVID-19 and recurrent herpes labialis in another. Our results confirm that NFKB2 alleles that are IκBδ GOF and p52 LOF can underlie CVID and drive the production of autoantibodies neutralizing type I IFNs, thereby predisposing to severe viral diseases.

Single-cell sequencing reveals PTX3 involvement in ovarian cancer metastasis

BackgroundPentraxin 3 (PTX3) has been associated with the development and progression of various malignant tumors. However, its roles and the mechanisms underlying its involvement in ovarian cancer (OC) peritoneal metastasis remain unclear.MethodsSingle-cell RNA sequencing (scRNA-seq) and immunohistochemistry (IHC) were conducted to determine the expression profiles, potential functionalities, and underlying mechanisms of PTX3 within the context of OC. To assess the proliferative response of OC cells, we utilized both EdU (5-ethynyl-2’ -deoxyuridine) and CCK8 assays. The role of PTX3 in facilitating cell migration and invasion was quantified through the use of Transwell assays. The protein expression levels were meticulously analyzed via Western blotting. Furthermore, to explore the interactions between proteins, we conducted immunofluorescence (IF) staining and co-immunoprecipitation (Co-IP) experiments. To determine the factors responsible for the upregulation of PTX3, we performed both coculture and suspension assays, providing a comprehensive approach to understanding the regulatory mechanisms involved.ResultsThis study confirmed, for the first time, that the expression of PTX3 in OC metastatic lesions is greater than that in primary lesions and that tumor cells with high PTX3 expression have greater metastatic ability. PTX3 can activate the EMT and NF-κB signaling pathways in OC cells and can interact with the TLR4 and CD44 receptors in OC cells. Additionally, PTX3’s modulation of the EMT and NF-κB pathways is partially dependent on its interaction with TLR4. Furthermore, this study revealed the intercellular regulatory network related to PTX3 in OC cells via bioinformatic analysis. High levels of PTX3 in OC cells potentially enhance the attraction of dendritic cells (DCs) and CD4 + T cells while diminishing the recruitment of B cells and CD8 + T cells. Finally, this study indicated that PTX3 upregulation was driven by multiple factors, including specific transcription factors (TFs) and modifications within the tumor microenvironment (TME).ConclusionsOur research revealed the contribution of PTX3 to the peritoneal dissemination process in OC patients, identifying a novel potential biomarker and therapeutic target for this disease.

Interleukin-2-mediated NF-κB-dependent mRNA splicing modulates interferon gamma protein production.

Interferon-gamma (IFNγ) is a pleiotropic cytokine produced by natural killer (NK) cells during the early infection response. IFNγ expression is tightly regulated to mount sterilizing immunity while preventing tissue pathology. Several post-transcriptional effectors dampen IFNγ expression through IFNG mRNA degradation. In this study, we identify mRNA splicing as a positive regulator of IFNγ production. While treatment with the combination of IL-12 and IL-2 causes synergistic induction of IFNG mRNA and protein, defying transcription-translation kinetics, we observe that NK cells treated with IL-12 alone transcribe IFNG with introns intact. When NK cells are treated with both IL-2 and IL-12, IFNG transcript is spliced to form mature mRNA with a concomitant increase in IFNγ protein. We find that IL-2-mediated intron splicing occurs independently of nascent transcription but relies upon NF-κB signaling. We propose that while IL-12 transcriptionally induces IFNG mRNA, IL-2 signaling stabilizes IFNG mRNA by splicing detained introns, allowing for rapid IFNγ protein production. This study uncovers a novel role for cytokine-induced splicing in regulating IFNγ through a mechanism potentially applicable to other inflammatory mediators.