Activation Of NF-κB Pathway Research Articles

Gastrodin Alleviates Lumbar Intervertebral Disc Degeneration by Suppressing the NF-κB and MAPK Pathways.

Intervertebral disc degeneration (IDD) is the main pathological factor resulting in low back pain (LBP), the leading cause of disability globally. Inflammatory response and extracellular matrix (ECM) degradation are critical pathological features in the development of IDD. Gastrodin (GAS), a phenol compound isolated from Gastrodia elata Blume, plays an anti-inflammatory role in experimental models of multiple human diseases. Our study aimed to elucidate whether GAS alleviates TNF-α-induced inflammation in nucleus pulposus (NP) cells and IDD in vivo. The cytotoxicity of GAS was assessed by CCK-8 assay. Rat primary NP cells were stimulated with TNF-α to induce inflammatory response. The expression of proinflammatory cytokines, catabolic genes, and anabolic genes was detected by RT-qPCR, western blotting, and immunofluorescence staining. NF-κB and MAPK pathway activation was determined through western blotting and immunofluorescence staining. The IDD rat model was established by using percutaneous needle puncture. The therapeutic effects of GAS were confirmed by histology analysis. We found that TNF-α stimulation enhanced proinflammatory cytokine (COX2, iNOS, IL-6, and IL-1β) expression in NP cells, which was reversed by GAS treatment. GAS offset TNF-α-induced upregulation in catabolic gene (MMP3, MMP9, and MMP13) expression and downregulation in anabolic gene (Collagen II, SOX9, and Aggrecan) expression. The loss of ECM in TNF-α-treated NP cells was mitigated by GAS treatment. Mechanically, GAS abolished TNF-α-induced increase in p-IKKα, p-IKKβ, p-IκBα, p-p65, p-ERK, p-p38, and p-JNK protein levels in NP cells. In puncture-induced IDD rat models, GAS administration improved intervertebral disc (IVD) structure, increased Collagen II expression, and reduced the levels of proinflammatory factors in IVDs. Overall, GAS alleviates the inflammation and ECM degradation in NP cells via inhibiting NF-κB and MAPK pathway activation and alleviates IDD in vivo, which may be a novel treatment strategy for IDD.

Long noncoding RNA FAM111A-DT promotes aggressiveness of papillary thyroid cancer via activating NF-κB signaling.

Long noncoding RNAs (lncRNAs) play crucial regulatory roles in the tumorigenesis and progression of various cancers. However, the functional roles of lncRNAs in papillary thyroid cancer (PTC) remain unclear. In this study, we investigated the functional role of the lncRNA FAM111A-DT in PTC progression and the underlying mechanisms. Different expression levels of lncRNAs in PTC were compared via analysis of the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Bioinformatics analyses and qRT‒PCR were used to investigate the expression of FAM111A-DT in PTC. Cell proliferation was measured via CCK8, EdU, colony formation, and flow cytometry assays. Cell migration and invasion were examined by wound healing and Transwell assays. Apoptosis was detected via flow cytometry. RNA sequencing, qRT‒PCR, Western blot, immunofluorescence and dual-luciferase reporter assays were performed to assess the underlying mechanisms involved. FAM111A-DT was highly expressed in PTC and associated with poor prognosis, thyroid dedifferentiation, various clinical features and the BRAFV600E mutation in PTC patients. Overexpression of FAM111A-DT enhanced the proliferation, migration and invasion of PTC cells while reducing their degree of apoptosis. The NF-κB signaling pathway was activated in FAM111A-DT-overexpressing PTC cells. The NF-κB inhibitor PDTC attenuated the promotive effects of FAM111A-DT on aggressive phenotypes and NF-κB pathway activity in PTC cells. FAM111A-DT is upregulated in PTC, and its expression is associated with poor clinical outcomes. FAM111A-DT plays an oncogenic role by, at least partially, activating the NF-κB signaling pathway.

Hybridization Design and High-Throughput Screening of Peptides with Immunomodulatory and Antioxidant Activities

With the increasing recognition of the role of immunomodulation and oxidative stress in various diseases, designing peptides with both immunomodulatory and antioxidant activities has emerged as a promising therapeutic strategy. In this study, a hybridization design was applied as a powerful method to obtain multifunctional peptides. A total of 40 peptides with potential immunomodulatory and antioxidant activities were designed and screened. First, molecular docking was employed to screen peptides with a high binding affinity to MD2, a key receptor protein in the NFκB immune pathway. For the in vitro high-throughput screening, we constructed a reporter gene-based stable cell line, IPEC-J2-Lucia ARE cells, which was subsequently used to screen peptides with antioxidant activity. Furthermore, the biocompatibility, immunomodulatory, and antioxidant activities of these peptides were assessed. Among the candidates, the hybrid peptide VA exhibited the strongest immune-enhancing activity through the activation of the NF-κB pathway and significant antioxidant activity via the Nrf2-ARE pathway. Additionally, VA demonstrated protective effects against H2O2-induced oxidative damage in HepG2 cells. This study not only demonstrates the potential of peptide hybridization, but also develops a screening platform for multifunctional peptides. It provides a new tool for the treatment of autoimmune diseases and oxidative stress-related diseases.

Monotropein attenuates renal cell carcinoma cell progression and M2 macrophage polarization by weakening NF-κB.

The study aimed to investigate the effect and mechanism of monotropein on renal cell carcinoma (RCC). After monotropein and NF-κB receptor activator (RANKL) treatment, cell proliferation, invasion, and apoptosis were evaluated using CCK-8, Transwell, and flow cytometry. Primary macrophages co-cultured with monotropein-treated RCC cells were analyzed to evaluate macrophage polarization using qRT-PCR, western blot, and ELISA assays by detecting the expression of M2 markers (CD206, CD168) and cytokines (IL-10, TGF-β). Additionally, the therapeutic efficacy of monotropein was examined using an RCC mouse xenograft model. Monotropein could inhibit the proliferation, invasion, and M2 macrophage polarization and accelerate the apoptosis of RCC cells. Mechanistically, monotropein suppressed NF-κB pathway activation in RCC cells and reduced the expression of NF-κB downstream targets, including Bcl-2, c-Myc, and MMP9. RANKL could eliminate the effect of monotropein on RCC progression. In primary macrophages co-cultured with monotropein-treated RCC cells, monotropein downregulated M2 polarization markers and cytokines, further supporting its role in modulating the tumor microenvironment. In mouse models, monotropein reduced RCC tumor growth, induced apoptosis, and blocked NF-κB pathway. Monotropein prevents RCC malignant progression and reduces M2 macrophage polarization by suppressing the NF-κB pathway, suggesting that monotropein may serve as a potential therapeutic agent for RCC by targeting both tumor cells and the tumor microenvironment.

Icariside II Alleviates Chondrocyte Inflammatory Injury by Inhibiting the TNIP2/NF-κB Pathway.

Icariside II exerts protective effects against various diseases; however, its specific effects on osteoarthritis (OA) remain unclear. Therefore, in this study, we aimed to investigate the effects of icariside II in an in vitro model of OA and analyze its action mechanisms. We established an in vitro OA model by treating a human chondrocyte cell line (CHON-001) with interleukin (IL)-1β, followed by treatment with different concentrations of icariside II. Cell viability was measured using the methyl thiazolyl tetrazolium assay, and the level of lactate dehydrogenase (LDH) released from cells was determined using the appropriate kit. Tumor necrosis factor (TNF)-α, IL-6, and IL-8 levels were determined via enzyme-linked immunosorbent assay. Flow cytometry was used to assess apoptosis. Apoptosisrelated protein expression levels and TNFAIP3-interacting protein 2 (TNIP2)/nuclear factor (NF)-κB signaling pathway were analyzed via reverse transcription-quantitative polymerase chain reaction and western blotting. Furthermore, TNIP2-small interfering RNA (siRNA) was used to determine whether the TNIP2/NF-κB pathway influences the effects of icariside II on OA. Results indicated that Icariside II did not exert any significant toxic effects on CHON-001 cells. It inhibited IL-1β-induced apoptosis and increase in LDH levels and enhanced the inflammatory response. Additionally, icariside II reversed the IL-1β-induced decrease in TNIP2 levels and increase in NF-κB phosphorylation. TNIP2-siRNA revealed that the TNIP2/NF-κB signaling pathway influenced the alleviating effects of icariside II on OA. In conclusion, our results revealed that icariside II attenuated IL-1β-induced inflammatory injury in chondrocytes by increasing TNIP2 expression and inhibiting NF-κB pathway activation, highlighting its therapeutic potential for OA.

2-Trifluoromethyl-2H-chromene ethers: The dual triumph of anti-inflammation and analgesia with minimal ulcer threat.

In this report, we disclose the design and synthesis of a series of 2-trifluoromethyl-2H- chromene ethers as novel COX-2 inhibitors with low ulcerogenicity. Among them, 6-fluoro-3-(4-methoxyphenyl)-2-(2-(thiophen-3-yl)ethoxy)-2-(trifluoromethyl)-2H-chromene (E25) significantly suppressed LPS-induced release of NO and PGE2, expression of COX-2 and iNOS, and activation of NF-κB pathway. The inhibitory effect of E25 on human recombinant COX-2 (IC50=70.7±4.7nM) and molecular docking studies suggest that E25 functions as a COX-2 inhibitor. Moreover, the results of the cellular thermal shift assay also substantiate the interaction between E25 and COX-2. E25 manifests potent anti-inflammatory and analgesic efficacy on a par with or even superior to indomethacin in rodent models including carrageenan-induced paw edema, cotton pellet-induced granuloma, acetic acid-induced writhes, and adjuvant-induced arthritis. The possible mechanism of action of E25 might be to bind to COX-2 and suppress the NF-κB pathway as well as the expression of related proteins, thereby exerting anti-inflammatory and analgesic effects. Encouragingly, compared with indomethacin, E25 induces smaller areas and fewer ulcers, a lower level of inflammatory infiltration, a lower expression of MMP-9 and apoptosis of mucosal epithelial cells in rat gastric tissues. Overall, E25 and other analogues are promising candidates worthy of further investigation for the treatment of inflammation and pain, as well as other symptoms in which COX-2 and PGE2 play a role in their etiology.

S100A8 and S100A9–Critical Mediators of Inflammation in Arthritis

Abstract: Damage-associated molecular Patterns (DAMP) released on the onset of tissue injury interact predominantly with pattern-recognizing receptors (PRRs) and multiple receptors to trigger and mediate inflammation and play a critical role in the progression of various diseases. Among the multiple DAMPs, S100A8 (MRP-8) and S100A9 (MRP-14) are low molecular weight calcium- binding proteins primarily expressed in innate immune cells such as myeloid cells: neutrophils, monocytes, keratinocytes, and early differentiation states of macrophages giving them the name Myeloid related proteins (MRP). S100A8 and S100A9 can exist both as homo- and heterocomplexes – shown to elicit different functions in cellular physiology, chemotaxis, phagocyte migration and modulation of various macrophage functions, apoptosis in cancer, and activation of NF-κB pathway. S100A8 and S100A9 have been shown to trigger innate immunity via TLR-4 signaling, thereby increasing the production of proinflammatory cytokines and other downstream effectors of the cascade. An influx in S100A8 and S100A9 proteins has been implicated in the most prevalent arthritic conditions, such as rheumatoid arthritis (RA) and osteoarthritis (OA). Hence, the primary objective of the review is to provide a comprehensive and systematic analysis imparting insights into the role of S100A8 and S100A9 proteins as mediators of inflammation in RA and OA.

ADAMTS7 Enhances Gastric Cancer Growth and Metastasis by Triggering the NF-κB Signaling Pathway.

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of metalloproteinases plays a vital role in various biological and pathological processes, including tissue remodeling, angiogenesis, and cancer progression. Among the 19 ADAMTS family members, our research focused on ADAMTS7, which exhibited significant overexpression in gastric cancer (GC). This overexpression was strongly correlated with poor clinical outcomes, including reduced overall survival and heightened metastatic potential. To investigate the role of ADAMTS7 in GC, we employed an integrated approach encompassing bioinformatics analysis, Western blotting, immunofluorescence, as well as in vitro and in vivo functional analyses. Our results showed that silencing ADAMTS7 expression significantly inhibited the proliferation, migration, and invasion of GC cells, and furthermore, silencing ADAMTS7 significantly inhibited the growth and metastasis of tumour cells in vivo in nude mice, highlighting its critical role in driving the malignant behaviour of GC cells. Further mechanistic studies identified the NF-κB signaling pathway as a key downstream target of ADAMTS7, with ADAMTS7 silencing resulting in a notable reduction in NF-κB pathway activity. These findings establish ADAMTS7 as a significant contributor to the aggressiveness of GC and a pivotal activator of the NF-κB pathway, a major regulator of inflammation and tumor progression. Consequently, ADAMTS7 emerges as a promising therapeutic target and prognostic biomarker for GC. Our study opens new avenues for the development of targeted therapies aimed at inhibiting ADAMTS7 activity, thereby potentially improving treatment outcomes and survival rates for patients with GC.

Inhibition of lncEPS by TLR4/NF-κB pathway induces ventilator-induced lung injury by decreasing its binding to and upregulating Hspa5

Whether LincRNA erythroid prosurvival (LncEPS) reduces VILI remains unclear. A GSE200932 microarray was used to screen differentially expressed genes (DEGs). A VILI mouse model was constructed by mechanical ventilation (MV), with or without TAK242 or SN50 pretreatment. Airway transfection with adeno-associated virus (AAV) was used to overexpress lncEPS in alveolar macrophages (AMs). Lung tissues were collected to assess pathological injury and macrophage polarization. NLRP3 inflammasome, TLR4/ NF-κB pathway activation and heat shock protein family A member 5 (Hspa5) in lung tissue and AMs wre evaluated. LncEPS localization and regulatory changes were assessed using in situ hybridization and RT–PCR. Lung tissues after lncEPS overexpression were subjected to transcriptomics. Chromatin isolation and mass spectrometry (MS) were performed to identify proteins interacting with lncEPS. GSE200932 microarray showed that the DEGs were related to NF-κB pathway, Toll-like receptor pathway and NOD-like receptor pathway. TAK242 or SN50 treatment increased polarization of M2 macrophages and decreased NLRP3 inflammasome activation by inhibiting TLR4/NF-κB pathway in VILI. Inhibition of TLR4/NF-κB pathway upregulated lncEPS expression in AMs. Overexpression of lncEPS increased polarization of M2 macrophages and decreased NLRP3 inflammasome activation, eventually alleviating VILI in mice. Mechanistically, lncEPS bound to Hspa5 and downregulated its expression to inhibit inflammatory response.

Antigen B from Echinococcus granulosus regulates macrophage phagocytosis by controlling TLR4 endocytosis in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is characterized by a reduction in platelet counts, stemming from an autoimmune-mediated process where platelets are excessively cleared by macrophages. This enhanced phagocytosis is a cardinal pathogenic mechanism in ITP. Antigen B (AgB), a principal component of the Echinococcus granulosus cyst fluid, plays a pivotal role in safeguarding the parasite from host immune defenses by modulating macrophage activation. In this study, we explored the potential of AgB to regulate macrophage activation in the context of ITP. Our observations indicated a diminished presence of M1 macrophages and a reduced phagocytic capacity in patients infected with E. granulosus sensu stricto. We isolated AgB from E. granulosus cyst fluid (EgCF) and discovered that it could suppress the polarization of M1 macrophages and weaken their phagocytic activity via Fcγ receptors, consequently alleviating thrombocytopenia in an ITP mouse model. At the molecular level, AgB was found to suppress the activation of nuclear factor kappa B (NF-κB) and interferon regulatory factor 3 (IRF3) by impeding their nuclear translocation, leading to a reduction in the generation of inflammatory cytokines. Furthermore, AgB was shown to inhibit Toll-like receptor 4 (TLR4) endocytosis and the recycling of CD14. In aggregate, our findings uncover a novel immunomodulatory mechanism of AgB, which suppresses macrophage phagocytosis by regulating TLR4 endocytosis and the subsequent activation of NF-κB and IRF3 signaling pathways. These insights shed new light on the molecular intricacies of E. granulosus-induced immune evasion and suggest that AgB may serve as a promising therapeutic agent for ITP.

Dissolving microneedle synergistic rocaglamide-loaded liposome to regulate abnormal neutrophils for anti-psoriasis.

Psoriasis seriously affects the physical and mental health of patients. Rocaglamide (RocA), derived from Aglaia odorata, exhibits potent pharmacological activities. Although its efficacy in psoriasis is unclear, RocA could be a promising therapeutic drug. In this work, RocA showed a good therapeutic effect in psoriasis mice induced by imiquimod, and subsequent TMT-based proteomics analysis verified that the effect of RocA was related to IL-1 family cytokines. Furthermore, a RocA-loaded liposome (RocA@Lipo) was developed and encapsulated in the tip-layer of microneedles (MNs) to construct a MN-based nano drug delivery system (RocA@Lipo-MNs). In vitro HaCaT cell assays demonstrated that RocA@Lipo enhanced the cytotoxicity and cell uptake of RocA. In vivo, RocA@Lipo-MNs outperformed other RocA formulations in inhibiting psoriasis epidermal thickening and spleen enlargement. Immunohistochemical, ELISA, western blot, and PCR experiments further proved that RocA@Lipo-MNs could inhibit neutrophil infiltration in the skin, revealing that the anti-psoriasis mechanism of RocA was deemed to inhibit the binding of IL-1α and IL-1R1 to regulate the activation of MAPK and NF-κB pathways. Thus, the production of inflammatory factors and neutrophil chemokines was reduced, which was associated with apoptosis inhibition. Importantly, RocA@Lipo-MNs significantly improved the transdermal properties of RocA and exhibited good skin and blood safety. This work provides new ideas for the clinical application of RocA and the treatment options for psoriasis.

Genome-wide CRISPR-based screen identifies E2F transcription factor 1 as a regulator and therapeutic target of aristolochic acid-induced nephrotoxicity.

Aristolochic Acid I (AAI) is widely present in traditional Chinese medicines derived from the Aristolochia genus and is known to cause significant damage to renal tubular epithelial cells. Genome-wide screening has proven to be a powerful tool in identifying critical genes associated with the toxicity of exogenous substances. To identify undiscovered key genes involved in AAI-induced renal toxicity, a genome-wide CRISPR library screen was conducted in the human kidney-2 (HK-2) cell line. Among the altered sgRNAs, a significant enrichment of those targeting the E2F transcription factor 1 (E2F1) gene was observed in surviving HK-2 cells in the AAI-treated group. Interestingly, the role of E2F1 had not been previously explored in studies of AAI nephrotoxicity. Further investigations revealed that E2F1 promotes apoptosis by activating the p53 signaling pathway and upregulating pro-apoptotic genes, such as BAK and BAX. Additionally, using the high-throughput experiment- and reference-guided database of traditional Chinese medicine (HERB), cannabidiol (CBD) was identified as an inhibitor of E2F1 by suppressing the activity of NF-κB pathway. In vitro and in vivo models confirmed that CBD inhibits AAI-induced upregulation of E2F1, thereby suppressing p53-mediated apoptosis. In conclusion, this study highlights the crucial role of E2F1 in AAI-induced renal cell apoptosis and identifies CBD as a novel therapeutic candidate for mitigating AAI nephrotoxicity.

Senolytics Cocktail Dasatinib and Quercetin alleviate chondrocyte senescence and facet joint osteoarthritis in mice

Background contextLow back pain (LBP) is a pervasive issue, causing substantial economic burden and physical distress worldwide. Facet joint osteoarthritis (FJ OA) is believed to be a significant contributor to this problem. However, the precise role of chondrocyte senescence in FJ OA remains unclear, as does whether the clearance of chondrocyte senescence can alleviate the progression of FJ OA. PurposeThe goal of this study was to understand the potential of Dasatinib (D) and Quercetin (Q) as a treatment to clear chondrocyte senescence during the progression of FJ OA. Study designWe used a preclinical bipedal standing mice model with the administration of Dasatinib (D) (5 mg/kg) and Quercetin (Q) (50 mg/kg) after 10 weeks of bipedal standing. Materials and methodsHuman degenerative lumbar facet joint (LFJ) samples were obtained to investigate the relationship between chondrocyte cellular senescence and LFJ osteoarthritis (OA). Subsequently, we established an in vitro model of excessive mechanical stress on chondrocytes and an in vivo bipedal standing mice model to induce LFJ OA. IHC (immunohistochemistry) staining in vivo and SA-β-gal staining, qRT-PCR and Western blot analysis were applied to test the senolytic effect of the combination of Dasatinib (D) and Quercetin (Q). IHC staining and X-ray microscope were also performed to examine the contribution of D+Q to the anabolism in cartilage and subchondral bone recoupling. Immunofluorescence and Western blot analysis in vitro and IHC staining in vivo were conducted to assess the impact of D+Q on the regulation of the NF-κB pathway activation during chondrocyte senescence. ResultsWe observed that facet joint cartilage degeneration is associated with chondrocyte cellular senescence in both human and mouse degenerative samples. Following treatment with D+Q in vitro, cellular senescence was significantly reduced. Upon oral gavage administration of D+Q in the bipedal standing mice model, decreased cellular senescence and reversed chondrocyte anabolism were observed. Furthermore, administration of D+Q maintained subchondral bone remodeling homeostasis and potentially reversed the activation of the NF-κB pathway in chondrocytes of the lumbar facet joint. ConclusionsIn summary, our investigation unveiled a significant correlation between chondrocyte senescence and LFJOA. Treatment with the senolytic combination of D+Q in FJ OA yielded a notable reduction in chondrocyte senescence, along with a decrease in the release of SASP factors. Additionally, it facilitated the promotion of cartilage anabolism, maintenance of subchondral bone coupling, and amelioration of NF-κB pathway activation. Clinical significanceOur outcomes revealed that D+Q, the renowned combination used for senolytic treatment, alleviate the progression of LFJ OA. The utilization of D+Q as a senolytic demonstrates a novel and promising alternative for LFJ OA treatment.

Polydatin, a derivative of resveratrol, ameliorates busulfan-induced oligozoospermia in mice by inhibiting NF-κB pathway activation and suppressing ferroptosis

Polydatin, a derivative of resveratrol, ameliorates busulfan-induced oligozoospermia in mice by inhibiting NF-κB pathway activation and suppressing ferroptosis

Human leukocyte antigen DR alpha inhibits renal cell carcinoma progression by promoting the polarization of M2 macrophages to M1 via the NF-κB pathway

Human leukocyte antigen DR alpha (HLA-DRA) is recognized for its inhibitory effect on the progression of clear cell renal cell carcinoma (ccRCC); high HLA-DRA expression levels are positively correlated with improved prognosis in patients with ccRCC. In this study, we evaluated HLA-DRA expression in ccRCCs, its effects on tumor-associated macrophage recruitment, and the influence of polarization. Clinical cohort analyses revealed that elevated HLA-DRA expression in ccRCC cells was correlated with enhanced tumor infiltration by M1-type macrophages. In addition, ccRCC prognosis was predicted by combining HLA-DRA expression level analysis and the M1/M2 macrophage ratio. In vitro studies demonstrated that ccRCC cells with increased HLA-DRA expression promoted THP-1 cell migration and induced macrophage polarization toward the M1 phenotype. The effect was further substantiated in a mouse xenograft model in which an increase in M1 macrophages was observed. In addition, co-culturing macrophages with the supernatant from cells overexpressing HLA-DRA induced the expression of proteins associated with both M1 and M2 macrophage polarization. HLA-DRA was intricately linked to the expression and secretion of chemokines, including CCL2, CCL5, MIP-1ɑ, and CXCL-10. Moreover, the NF-κB pathway activation promoted polarization to M1 macrophages. This study shows that HLA-DRA and the M1/M2 ratio are indicators of favorable prognosis in patients with ccRCC. HLA-DRA promotes M1-like polarization by regulating NF-κB, which can be used as a therapeutic target to enhance anti-tumor immunity.

Plumbagin alleviates muscle atrophy in female mice through inhibiting the DANCR/NF-κB axis

BackgroundMuscle atrophy is a condition of the skeletal muscular system closely related to inflammation and significantly affects a person's quality of life and physical activity. It is characterized primarily by the progressive loss of muscle mass, strength, and function. Plumbagin (PB), the main bioactive component of the traditional Chinese medicine Plumbago zeylanica L., has bFeen shown to treat various inflammatory diseases, such as osteoporosis, osteoarthritis, and sepsis. Furthermore, many biological processes, including inflammation, involve differentiation antagonistic nonprotein-coding RNA (DANCR). However, their role and clinical importance in myogenesis and amyotrophy are not well understood. PurposeThis study aimed to explore the role of DANCR and the inflammatory response in the anti-muscle atrophy effects of PB. MethodsThe expression of DANCR in muscle atrophic mice and during myogenic differentiation was examined using quantitative reverse transcription PCR (RT‒qPCR). The mechanism of DANCR in muscle atrophy was confirmed through gene knockdown, RNA sequencing (RNA-seq), RNA pull-down, RNA immunoprecipitation (RIP), immunofluorescence (IF), and luciferase reporter gene assays. Bioinformatics was utilized to investigate the mechanism by which PB treatment affects muscle atrophy. The relationship between PB and DANCR was verified by surface plasmon resonance (SPR) and RT‒qPCR. Additionally, the role of PB in muscle atrophy was explored through its control of DANCR-mediated regulation of the NF-κB pathway. Finally, the effect of PB on the myogenic differentiation of human skeletal muscle cells (HsKMCs) was investigated. ResultsDANCR expression was upregulated in the muscle tissues of mice with muscle atrophy and downregulated during myogenic differentiation. Knockout of DANCR promoted myogenic differentiation and significantly alleviated the loss of muscle mass, strength, and function in mice with muscle atrophy. The primary mechanism involved DANCR directly binding to the p65 protein to regulate NF-κB pathway activity. Experiments revealed that PB could target the degradation of DANCR, reduce the nuclear entry of p65, and inhibit the activation of the NF-κB pathway. Consequently, PB significantly inhibited myotube atrophy and the inflammatory response in HsKMCs and promoted their myogenic differentiation by regulating the NF-κB pathway. ConclusionsOur results suggest that PB regulates myogenesis and prevents amyotrophy by targeting the degradation of DANCR and inhibiting the activation of the NF-κB pathway. This study reveals the crucial role of DANCR in maintaining muscle physiology during muscle atrophy and identifies PB as an effective drug that can target DANCR degradation to alleviate muscle atrophy.

Epigenetic Inhibitors Differentially Impact TGF-β1 Signaling Cascades in COPD Airway Smooth Muscle Cells.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by progressive and incurable airflow obstruction and chronic inflammation. Both TGF-β1 and CXCL8 have been well described as fundamental to COPD progression. DNA methylation and histone acetylation, which are well-understood epigenetic mechanisms regulating gene expression, are associated with COPD progression. However, a deeper understanding of the complex mechanisms associated with DNA methylation, histone post-translational changes and RNA methylation in the context of regulatory pathways remains to be elucidated. We here report on how DNA methylation and histone acetylation inhibition differentially affect CXCL8 signaling in primary human non-COPD and COPD airway cells. Methods: Airway smooth muscle (ASM) cells, a pivotal cell type in COPD, were isolated from the small airways of heavy smokers with and without COPD. Histone acetylation and DNA methylation were inhibited before the TGF-β1 stimulation of cells. Subsequently, CXCL8 production and the abundance and activation of pertinent transcription regulatory proteins (NF-κB, p38 MAPK and JNK) were analyzed. Results: TGF-β1-stimulated CXCL8 release from ASM cells from 'healthy' smoker subjects was significantly modulated by DNA methylation (56.32 pg/mL and 56.60 pg/mL) and acetylation inhibitors (27.50 pg/mL and 48.85 pg/mL) at 24 and 48 h, respectively. However, modulation via the inhibition of DNA methylation (34.06 pg/mL and 43.18 pg/mL) and acetylation (23.14 pg/mL and 27.18 pg/mL) was observed to a lesser extent in COPD ASM cells. These changes were associated with differences in the TGF-β1 activation of NF-κB and MAPK pathways at 10 and 20 min. Conclusions: Our findings offer insight into differential epigenetics in controlling COPD ASM cells and provide a foundation warranting future studies on epigenetic differences associated with COPD diagnosis. This would provide a scope for developing therapeutic interventions targeting signaling and epigenetic pathways to improve patient outcomes.

EXPRESSION OF CONCERN: Dose-Dependent Biphasic Leptin-Induced Proliferation is Caused by Non-Specific IL-6/NF-κB Pathway Activation in Human Myometrial Cells.

EXPRESSION OF CONCERN: Dose-Dependent Biphasic Leptin-Induced Proliferation is Caused by Non-Specific IL-6/NF-κB Pathway Activation in Human Myometrial Cells.

Porcine Epidemic Diarrhea Virus Infection of Porcine Intestinal Epithelial Cells Causes Mitochondrial DNA Release and the Activation of the NLRP3 Inflammasome to Mediate Interleukin-1β Secretion.

Porcine epidemic diarrhea virus (PEDV) induces enteritis and diarrhea in piglets. Mitochondrial DNA (mtDNA) contributes to virus-induced inflammatory responses; however, the involvement of inflammasomes in PEDV infection responses remains unclear. We investigated the mechanism underlying inflammasome-mediated interleukin (IL)-1β secretion during the PEDV infection of porcine intestinal epithelial (IPEC-J2) cells. IL-1β production and caspase-1 activity were assessed by quantitative PCR and enzyme-linked immunosorbent assay. NLRP3 inflammasome activation was assessed using immunoprecipitation experiments. Mitochondrial damage was evaluated by analyzing the mitochondrial membrane potential and ATP levels and by the flow cytometry examination of mitochondrial reactive oxygen species (mtROS). Mitochondria and mtDNA localization were observed using immunofluorescence. The inhibition of mtROS and mtDNA production allowed NLRP3 inflammasome and IL-1β expression detection and the evaluation of the pathway underlying NLRP3 inflammasome activation in PEDV-infected IPEC-J2 cells. IPEC-J2 cells upregulated IL-1β upon PEDV infection, where mature IL-1β secretion depended on caspase-1 activity, triggered NLRP3 inflammasome expression and assembly, and caused mitochondrial dysfunction, leading to mtDNA release and NLRP3 inflammasome activation, while mtROS contributed to NF-κB pathway activation, enhancing IL-1β secretion. This is the first demonstration of the mechanism underlying mtDNA release and NLRP3 inflammasome activation facilitating IL-1β secretion from PEDV-infected IPEC-J2 cells. These data enhance our understanding of the inflammatory mechanisms triggered by PEDV.

P72 The role of the transcription factor Bcl11b in IL-17, IL-23 and/or TNF-induced activation of the canonical and non-canonical NF-κB pathways in psoriatic keratinocytes

Abstract Bcl11b (CTIP2) is a crucial transcription factor involved in the differentiation processes across various cell types, including T cells and keratinocytes. It functions both as a transcriptional repressor and activator, interacting with complexes such as NuRD and proteins like p300. Bcl11b is significantly expressed in skin and immune cells suggesting its potential involvement in diseases like psoriasis, which is characterized by hyperproliferation and aberrant differentiation of keratinocytes alongside immune cell infiltration and cytokine release. However, the mechanism underlying the role of Bcl11b in the pathogenesis of psoriasis remains unidentified. To elucidate Bcl11b’s role in psoriasis by analysing its expression and correlated pathways through meta-analysis of sequencing-based transcriptome profiles and by functional assays in normal and genetically engineered keratinocytes under inflammatory conditions. RNA-sequencing datasets from psoriatic and healthy skin samples were processed and analysed for differential expression of transcription factors, with a focus on Bcl11b. Pearson correlation analysis identified genes correlated with Bcl11b expression. Enrichment and pathway analyses were conducted using Enrichr. Functional studies involved Bcl11b knockdown in HaCaT keratinocytes via CRISPRi, followed by cytokine stimulation including IL-17A, IL-23, TNF-α and the combinations. The activation of canonical and non-canonical NF-κB pathways was assessed. Downregulation of Bcl11b in psoriatic lesions was confirmed. Correlated genes were enriched in pathways involving IL-17A, IL-23 and TNF-α signalling. Knockdown of Bcl11b in keratinocytes resulted in morphological changes indicative of incomplete differentiation and reduced proliferation. Under cytokine-driven inflammatory conditions, canonical NF-κB pathway activation was impaired in Bcl11b KD cells, while non-canonical NF-κB pathway activation was dependent on IL-17A and the combination of IL-23/TNF-α in these KD cells. These data highlight the pivotal role of Bcl11b in the proinflammatory cytokine-induced regulation of the NF-κB signalling pathways. Absence of Bcl11b results in abnormal keratinocyte behaviour associated with psoriasis. Our data suggest that key proinflammatory cytokines in psoriasis act differently on Bcl11b associated activation of the NF-kB pathways.