NF-κB Pathway Research Articles

Xuefu Zhuyu Decoction improves hyperlipidemia through the MAPK/NF-κB and MAPK/PPARα/CPT-1A signaling pathway.

Xuefu Zhuyu Decoction (XZD) is widely used in the treatment of cardiovascular diseases. The purpose of this study was to explore the pharmacological effects and molecular mechanisms of XZD in improving hyperlipidemia and to provide a theoretical framework for clinical application. In this study, the signaling pathways regulated by XZD in improving hyperlipidemia were predicted by network pharmacology. Molecular docking was used to verify the affinity between the components in XZD and the target. Furthermore, a hyperlipidemic model in rats was constructed through feeding a high-fat diet. The effect of XZD on hyperlipidemia was verified by histopathological staining, Elisa, and western blot. The results found that the XZD improved dyslipidemia and inflammatory factor disorders, and inhibited liver function damage, pathological damage, and oxidative stress damage in hyperlipidemic rats. The findings from molecular docking and network pharmacology suggested that the mechanism of XZD improving hyperlipidemia may be closely related to the MAPK, NF-κB, and PPAR pathways. This study demonstrated that the XZD inhibited liver lipid metabolism disorder and inflammatory response by regulating the MAPK/NF-κB and MAPK/PPARα/CPT-1A pathway, significantly improved liver histopathological damage and oxidative stress injury, and played a protective role in hyperlipidemic rats.

An Aligned-to-Random PLGA/Col1-PLGA/nHA Bilayer Electrospun Nanofiber Membrane Enhances Tendon-to-Bone Healing in a Murine Model.

The challenge of achieving effective tendon-to-bone healing remains a significant concern in sports medicine, necessitating further exploration. Biomimetic electrospun nanomaterials present promising avenues for improving this critical healing process. To investigate the biological efficacy of a novel aligned-to-random PLGA/Col1-PLGA/nHA bilayer electrospun nanofiber membrane in facilitating tendon-to-bone healing. Controlled laboratory study. The bilayer membrane's composition, combining PLGA/Col1 for tendon attachment and PLGA/nHA for bone integration, was examined using scanning electron microscopy, Fourier transform infrared spectroscopy, and mechanical testing. Positioned between the Achilles tendon and bone, its design aimed for harmonious integration with both types of tissue. In vitro, biocompatibility, cell adhesion, and proliferation of the biomaterial were evaluated using live/dead staining and the CCK-8 assay. Collagen secretion and mineralization were measured for 2 cell types. In vivo, tendon-to-bone insertion samples harvested from mice were analyzed: micro-computed tomography assessed bone formation; histological staining evaluated chondrogenesis, tendinogenesis, and the 4-layer structure of the insertion; and biomechanical testing measured insertion strength. Real-time polymerase chain reaction identified genes involved in tendon-to-bone healing, and transcriptome analysis elucidated the underlying cellular and molecular mechanisms. The optimal composition was determined as 10% 3:1 for aligned PLGA/Col1 and 9% 5:1 for PLGA/nHA. Coculture showed minimal cell death, firm cell adherence, and steady proliferation, with PLGA/Col1 enhancing collagen secretion. In vivo, the material promoted bone and cartilage formation and improved tendon-to-bone interface strength. Transcriptome analysis indicated links to TNF and NF-κB pathways and to genes IL-1β, ADAM8, and EGR2. The novel aligned-to-random PLGA/Col1-PLGA/nHA bilayer nanofiber membrane outperformed other materials in both in vitro and in vivo evaluations, significantly enhancing tendon-to-bone healing. It notably improved cartilage and bone formation, tendon maturation, and biomechanical strength at the surgical interface. These effects may be associated with the TNF and NF-κB pathways and with the genes IL-1β, ADAM8, and EGR2. This study introduces a biomimetic nanofiber membrane enhancing tendon-to-bone healing, which is crucial for sports medicine. Its efficacy in improving healing outcomes, including bone and cartilage formation and biomechanical strength, could significantly lower failure rates in surgical procedures such as rotator cuff repair and anterior cruciate ligament reconstruction. This advancement offers promising implications for patient recovery and the effectiveness of surgical interventions in tendon-to-bone injuries.

The circulating plasma microRNA signature in human visceral leishmaniasis.

Visceral leishmaniasis (VL) is a vector-borne disease caused by the obligate intracellular protozoan Leishmania donovani in India. VL can be complicated by post-kala-azar dermal leishmaniasis (PKDL), a macular or nodular rash that develops in 10%-20% of patients after treatment of VL in India. Patients with PKDL are infectious to sand flies, promoting further transmission of the parasite. MicroRNAs (miRNAs) are 18-25 nt, non-coding RNAs that simultaneously regulate the expression of several or many target transcripts. This study was based on the hypothesis that the host response to L. donovani is modified by distinct sets of miRNAs in VL or PKDL and that these might differ from healthy controls. We investigated this hypothesis using a NanoString panel to profile the miRNAs expressed in the plasma of patients with VL or PKDL diagnosed at a hospital in Bihar, India. We compared these to plasma microRNAs of healthy control individuals from the same endemic villages. miRNAs hsa-miR-223-3p, hsa-miR-191-5p, hsa-miR-23a-3p, and hsa-1285-5p were significantly higher in the plasma samples from patients with VL compared to either PKDL or endemic controls. Prediction programs highlighted potential mRNA targeted by these miRNAs, among which we verified the down-modulation of several transcripts belonging to the NFκB and NLRP3 inflammasome pathways in circulating leukocytes of VL patients. By contrast, miRNA patterns in subjects with PKDL were similar to control subjects, possibly suggesting that the pathogenic immune response during PKDL is primarily localized in the skin.IMPORTANCEInfection of humans with the protozoan Leishmania donovani can be asymptomatic or it can cause fatal visceral leishmaniasis (VL), sometimes followed by the cutaneous complication PKDL. Parasites are spread through sand fly bites in endemic regions, and parasites in post-kala-azar dermal leishmaniasis (PKDL) skin lesions are a source of prolonged parasite transmission to sand flies, compromising disease eradication efforts. Since microRNAs can simultaneously modify the expression of multiple genes, we examined microRNAs in the blood that might be partial determinants of pathogenic responses leading to VL or PKDL. Our studies revealed several miRNAs expressed that are elevated in the plasma of patients with VL, which suppress some of the inflammatory responses that promote parasite killing. However, miRNA profiles were very similar between PKDL patients and controls, raising the possibility that major factors that lead to prolonged retention of parasites in the skin during PKDL are not systemic but are localized in the skin.

SPHK1 enhances olaparib resistance in ovarian cancer through the NFκB/NRF2/ferroptosis pathway

PARPis resistance is a challenge in the treatment of ovarian cancer. To investigate the potential mechanism involved in olaparib resistance of ovarian cancer, high-throughput sequencing was performed on olaparib-resistant SKOV3 cell line named SK/Ola. SPHK1 was upregulated in SK/Ola cells and was related to the PFS and OS in ovarian cancer patients. However, the effect and mechanism of SPHK1 on olaparib sensitivity in ovarian cancer were obscure. In this study, we found that SPHK1 promoted olaparib resistance. While, SPHK1 knockdown and SPHK1 inhibitor (PF-543 hydrochloride, named PF-543 in this article) enhanced the effect of olaparib on ovarian cancer cells. In mechanism, SPHK1 activated the NF-κB pathway through promoting p-IκBα degradation. Moreover, SPHK1 inhibited, but PF-543 activated ferroptosis in OC cells. Further investigation revealed that SPHK1 activated NF-κB p65, which in turn transcriptionally regulated NRF2 to inhibit ferroptosis in OC cells. Functionally, NF-κB p65 attenuated the PF-543-induced ferroptosis, and this effect was rescued by ferroptosis inducer erastin and RSL3. We conclude that SPHK1 inhibition triggers ferroptosis by restricting NF-κB-activated NRF2 transcription, thereby enhancing olaparib sensitivity in ovarian cancer. In vivo experiments also confirmed that the SPHK1 inhibitor increased olaparib sensitivity. A combination of SPHK1 inhibitors and olaparib may provide a therapeutic strategy for ovarian cancer.

The cellular response of lipopolysaccharide-induced inflammation in keratoconus human corneal fibroblasts to RB-PDT: Insights into cytokines, chemokines and related signaling pathways.

Rose Bengal Photodynamic Therapy (RB-PDT) offers dual therapeutic benefits by enhancing corneal stiffness and providing antibacterial activity, presenting significant potential for patients with keratoconus complicated by keratitis. Our purpose was to assess the effect of rose bengal photodynamic therapy (RB-PDT) on the expression of pro-inflammatory cytokines and chemokines, as well as on extracellular matrix (ECM)-related molecules, in lipopolysaccharide (LPS)-induced inflammation of keratoconus human corneal fibroblasts (KC-HCFs). Additionally, the involvement of the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways which are downstream of the Toll-like receptor 4 (TLR4) pathway were examined. KC-HCFs were stimulated with varying concentrations of LPS (0-10 μg/ml), which was followed by RB-PDT. The expression levels of interleukin-1β (IL-1β), IL-6, IL-8, interferon alpha 2 (IFNA2), IFNB1, intercellular adhesion molecule 1 (ICAM-1), chemokine (C-C motif) ligand 4 (CCL-4), collagen I, collagen V, lysyl oxidase (LOX), transforming growth factor β 1(TGF-β1) were measured using qPCR, ELISA, or western blot. The activation of the NF-κB and MAPK pathways was assessed using qPCR and western blot. In LPS-induced inflammation of KC-HCFs, the expression of IL-6 was further amplified by the treatment with RB-PDT (p = 0.001). However, the activation of the MAPK and NF-κB pathways did not increase following RB-PDT. Additionally, RB-PDT reduced the transcription of collagen I and collagen V (p≤0.03), while the transcription of LOX and TGF-β1 secretion remained unchanged in KC-HCFs exposed to LPS. In LPS-induced inflammation of KC-HCFs treated with RB-PDT, despite the increased expression of pro-inflammatory cytokines, the activation of the TLR4 signaling pathways is lacking. RB-PDT may have no adverse effects on corneal scar formation of keratoconus corneas in the short term.

Angiotensin 1-7 Attenuates the Development of Ischemia-Reperfusion-Induced Arrhythmia in Rats: Electrophysiology, Molecular, and Immunohistochemical Study.

Arrhythmia is a common and serious global health problem, contributing to cardiovascular morbidity and mortality. The cardiac muscle is susceptible to ischemia-reperfusion (I/R) injury, which can lead to fatal arrhythmias during open-heart surgery. We investigated the potential prophylactic effect of angiotensin 1-7 (Ang 1-7) using an invivo rat model of I/R injury and examined the underlying mechanisms. Rats were treated with Ang 1-7 (1 mg/kg, IP) 30 min before the surgical procedures. Twenty-four rats were equally divided into four groups: sham control, sham-treated with Ang 1-7, I/R injury group, and I/R injury group treated with Ang 1-7. Invivo I/R injury was induced by clamping the left coronary artery for 30 min, followed by 1 hour of reperfusion. The I/R group showed abnormal electrophysiological changes and arrhythmic episodes during electrocardiography (ECG) recording, increased oxidative stress, downregulation of peroxisome proliferator-activated receptor gamma (PPAR-γ), and upregulation of C-X-C motif chemokine ligand 16 (CXCL16) expression in cardiac tissue, which increased cardiac NF-kB expression and IL-17 levels. Moreover, I/R injury caused significant histological disruption and increased cyclooxygenase 2 (COX-2) and heat shock protein 90 (HSP90) immunoreactions, correlating with the extent of cardiac damage. However, preoperative Ang 1-7 administration significantly improved the electrophysiological, biochemical, and histopathological changes induced by I/R injury. This study demonstrated that Ang 1-7 exerted protective anti-arrhythmic, anti-inflammatory, and pro-healing effects by upregulating PPAR-γ and downregulating CXCL16, IL-17, and NF-kB pathways, suggesting it is a promising cardioprotective agent for preventing arrhythmias induced by I/R injury.

Antioxidant and anti-inflammatory function of walnut green husk aqueous extract (WNGH-AE) on human hepatocellular carcinoma cells (HepG2) treated with t-BHP.

Oxidative damage, oxidative inflammation, and a range of downstream diseases represent significant threats to human health. The application of natural antioxidants and anti-inflammatory agents can help prevent and mitigate these associated diseases. In this study, we aimed to investigate the effectiveness of walnut green husk (WNGH) as an antioxidant and anti-inflammatory agent in an in vitro setting. HepG2 cells were treated with tert-butyl hydroperoxide (t-BHP) to establish a cellular model of oxidative damage and inflammation. We assessed the biocompatibility of walnut green husk aqueous extract (WNGH-AE) on HepG2 cells using MTT and LDH assays (WNGH-AE concentration: 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 mg/mL). Additionally, we measured intracellular oxidative stress indicators, such as ROS and 8-OHdG, along with inflammatory factors TNF-α and IL-1β through ELISA to evaluate the antioxidant and anti-inflammatory capacity of WNGH-AE (concentration: 0.025, 0.05, 0.1 mg/mL) in HepG2 cells. We also determined the free radical scavenging capacity of various extracts of WNGH using DPPH and ·OH methods. The total phenols, total polysaccharides, and total flavonoids in WNGH-AE were analyzed using the Folin-Ciocalteu's reagent, the phenol-sulfuric acid method, and the spectrophotometry, respectively. The bioactive components of WNGH-AE were analyzed using LC-MS/MS. Our results demonstrated that WNGH-AE was highly biocompatible with HepG2 cells. The antioxidant effect of WNGH-AE involved the scavenging of intracellular ROS, while its anti-inflammatory effect was linked to the down-regulation of the NF-κB pathway. Compared to other extractants (ethyl acetate, n-butanol, 75% ethanol, and petroleum ether), WNGH-AE exhibited the strongest free radical scavenging ability. Through LC-MS/MS analysis, we identified 403 compounds in WNGH-AE, with gentisic acid being the most abundant and possessing high antioxidant capacity, suggesting it may be a key active component contributing to the antioxidant activity of WNGH-AE. In conclusion, our findings indicate that WNGH-AE is a natural, high-quality antioxidant and anti-inflammatory biomaterial deserving further research and development, with significant potential applications in healthcare. (311 words).

Fish Oil's Preventive Effect on Two-Stage Skin Carcinogenesis in Swiss Albino Mice: Involvement of NF-ҝB Pathways and Oxidative Stress in a Dose- and Route Dependent Manner.

This study investigated the chemopreventive mechanisms of fish oil (FO) at different doses and administration routes in skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and croton oil (CO) in Swiss albino mice. Seventy mice were divided into 10 groups, including controls and those receiving FO either orally or topically, with or without the carcinogenesis protocol. Warts were morphologically analyzed. Anatomopathological analysis, qRT-PCR of nuclear factor kappa B (NF-қB) subunits' gene expression, and evaluation of oxidative parameters were conducted. Anatomopathological analysis revealed a presence of invasive squamous cell carcinoma (SCC) in DMBA group. Both oral (500mg/kg/day) and topical FO treatment showed no signs of cancer, while oral administration at 50mg/kg/day had no therapeutic effect, and 250mg/kg/day resulted in low-grade malignancy. Both oral (250 and 500mg/kg/day) and topical FO significantly reduced NF-кB1 gene expression, alleviated oxidative stress markers, and restored antioxidant enzyme activities compared to the DMBA group. FO shows dose-dependent chemopreventive effects, with oral administration potentially as effective as topical application when using an appropriate dosage. The development of SCC is linked to the stress status and the upregulation of the canonical NF-κB pathway, while FO's chemoprotective effects likely result from its downregulation.

Modeling the response to interleukin-21 to inform natural killer cell immunotherapy.

Natural killer (NK) cells are emerging agents for cancer therapy. Several different cytokines are used to generate NK cells for adoptive immunotherapy including interleukin (IL)-2, IL-12, IL-15 and IL-18 in solution, and membrane-bound IL-21. These cytokines drive NK cell activation through the integration of signal transducers and activators of transcription (STAT) and nuclear factor-kappa B (NF-κB) pathways, which overlap and synergize, making it challenging to predict optimal cytokine combinations for both proliferation and cytotoxicity. We integrated functional assays for NK cells cultured in a variety of cytokine combinations with mathematical modeling using feature selection and mechanistic regression models. Our regression model successfully predicts NK cell proliferation for different cytokine combinations and indicates synergy of activated STATs and NF-κB transcription factors between priming and post-priming phases. The use of IL-21 in solution in the priming of NK cell culture resulted in an improved NK cell proliferation, without compromising cytotoxicity potential or interferon gamma secretion against hepatocellular carcinoma cell lines. Our work provides an integrative framework for interrogating NK cell proliferation and activation for cancer immunotherapy.

The role of celastrol in inflammation and diseases.

Celastrol is one of the main active ingredients extracted from the plant Tripterygium wilfordii Hook F. A growing number of studies have shown that celastrol has various pharmacological effects, including anti-inflammation, anti-rheumatism, treatment of neurodegenerative diseases, and anti-tumor. This article systematically summarized the mechanism and role of celastrol in lipid metabolism and obesity, rheumatoid arthritis (RA), osteoarthritis (OA), gouty arthritis, inflammatory bowel disease, neurodegenerative diseases, and cancer and other diseases (such as diabetes, respiratory-related diseases, atherosclerosis, psoriasis, hearing loss, etc.). The celastrol played roles in inflammation response, cell apoptosis, autophagy, ferroptosis, and lipid metabolism mainly by acting on chondrocytes, macrophages, mitochondria, and endoplasmic reticulum (ER) through NF-κB, STAT, MAPK, TLR, PI3K-AKT-mTOR, and other signal pathways. This review could provide a reference for the clinical application and further development and utilization of celastrol.

Integrative Transcriptome-Wide Association Study With Expression Quantitative Trait Loci Colocalization Identifies a Causal VAMP8 Variant for Nasopharyngeal Carcinoma Susceptibility.

Nasopharyngeal carcinoma (NPC) is an Asia-prevalent malignancy, yet its genetic underpinnings remain incompletely understood. Here, a transcriptome-wide association study (TWAS) is conducted on NPC, leveraging gene expression prediction models based on epithelial tissues and genome-wide association study (GWAS) summary statistics from 1577 NPC cases and 6359 controls of southern Chinese descent. The TWAS identifies VAMP8 on chromosome 2p11.2 as a novel susceptibility gene for NPC. Further fine-mapping analyses pinpoint rs1058588, located within VAMP8, as a causal variant through eQTL colocalization, and GWAS analyses across multiple cohorts, achieving GWAS significance (OR=1.18, P=3.09 × 10-10). Functional assays demonstrate that VAMP8 exerts a tumorigenic role in NPC, enhancing cell proliferation, migration, and tumor growth. Mechanically, it is uncovered that rs1058588 modulates VAMP8 expression by altering its binding affinity to miR-185. Furthermore, the results show that VAMP8 interacts with DHX9 to facilitate the nuclear recruitment of p65, activating the NF-κB pathway. Collectively, the findings shed light on the genetic predisposition to NPC and underscore the critical role of the functional axis involving miR-185, VAMP8, DHX9, and the NF-κB pathway in NPC pathogenesis.

Unraveling the immunomodulatory and metabolic effects of bioactive glass S53P4 on macrophages in vitro

Macrophage metabolism is closely linked to their phenotype and function, which is why there is growing interest in studying the metabolic reprogramming of macrophages. Bioactive glass (BG) S53P4 is a bioactive material used especially in bone applications. Additionally, BG S53P4 has been shown to affect macrophages, but the mechanisms through which the possible immunomodulatory effects are conveyed remain unclear. According to the results presented here, the lipopolysaccharide (LPS) induced suppression in oxidative phosphorylation is rescued in macrophages cultured with BG S53P4 before the inflammatory stimulus. Additionally, BG S53P4-exposed macrophages expressed lower mRNA levels of inflammatory cytokines Il6 and Il1b, as well as demonstrated decreased activation of inflammatory interferon regulatory factor (IRF) and NF-κB pathways and nitrogen oxide secretion in response to LPS. These results did not rely on cells being in direct contact with the material as similar effects were observed in the presence of BG S53P4-conditioned medium. Our findings link the immunomodulatory properties of BG S53P4 and macrophage metabolism, which improves our understanding of the mechanisms underlying the clinical efficacy of bioactive glasses.Graphical

Fucoidan alleviated colitis aggravated by fiber deficiency through protecting the gut barrier, suppressing the MAPK/NF-κB pathway, and modulating gut microbiota and metabolites

Insufficient dietary fiber intake has become a global public health issue, affecting the development and management of various diseases, including intestinal diseases and obesity. This study showed that dietary fiber deficiency enhanced the susceptibility of mice to colitis, which could be attributed to the disruption of the gut barrier integrity, activation of the NF-κB pathway, and oxidative stress. Undaria pinnatifida fucoidan (UPF) alleviated colitis symptoms in mice that fed with a fiber deficient diet (FD), characterized by increased weight gain and reduced disease activity index, liver and spleen indexes, and histological score. The protective effect of UPF against FD-exacerbated colitis can be attributed to the alleviation of oxidative stress, the preservation of the gut barrier integrity, and inhibition of the MAPK/NF-κB pathway. UPF ameliorated the gut microbiota composition, leading to increased microbiota richness, as well as increased levels of Muribaculaceae, Lactobacillaceae, and Bifidobacterium and reduced levels of Proteobacteria, Bacteroidetes, and Bacteroides. Metabolomics analysis revealed that UPF improved the profile of microbiota metabolites, with increased levels of carnitine and taurine and decreased levels of tyrosine and deoxycholic acid. This study suggests that UPF has the potential to be developed as a novel prebiotic agent to enhance human health.

Dichloroacetate: A metabolic game-changer in alleviating macrophage inflammation and enhancing recovery after myocardial infarction.

Dichloroacetate (DCA) has shown potential in modulating cellular metabolism and inflammation, particularly in cardiac conditions. This study investigates DCA's protective effects in a mouse model of myocardial infarction (MI), focusing on its ability to enhance cardiac function, reduce inflammation, and shift macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. An acute MI model was created using left anterior descending coronary artery ligation. Mice were assigned to four groups: normal control, MI control, MI + 50 mM DCA, and MI + 100 mM DCA. Cardiac fibrosis and injury were assessed through H&E staining. Cardiac function was evaluated via echocardiography, and serum levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were measured. Inflammation and apoptosis were analyzed through immunohistochemistry, ELISA, western blotting, and flow cytometry in heart tissue and RAW264.7 cells. Additionally, macrophage polarization and relevant signaling pathways were examined. DCA significantly improved cardiac function in MI mice, evidenced by reduced myocardial injury and lower CK-MB and LDH levels. It also decreased inflammatory cytokines (TNF-α, IL-6 and IL-1β) and facilitated macrophage polarization from M1 to M2. Western blotting revealed that DCA inhibited iNOS and COX2 while enhancing Arg1 expression, alongside improved mitochondrial function and reduced apoptosis. Additionally, by injecting AAV-PDHK4 (pyruvate dehydrogenase kinase) into MI mice, we found that DCA effectively inhibited the progression of MI through the suppression of PDHK4. DCA protects against myocardial infarction by enhancing cardiac function, reducing inflammation, and promoting macrophage polarization, likely through inhibition of PDHK4 and NF-κB pathways, positioning it as a potential therapeutic strategy for cardiac repair post-MI.

Erianin alleviates autoimmune myocarditis by suppressing the M1 polarization of macrophages via the NF-κB/NLRP3 signaling pathway.

Myocarditis tends to lead to a poor prognosis, but there are no satisfactory preventive or therapeutic strategies. Erianin, a natural benzene compound, has been found to have antioxidant and anti-inflammatory effects. However, the effects of erianin on myocarditis remain unclear. This study aimed to investigate the effects of erianin on myocarditis and the involved mechanisms. We developed a mouse model of experimental autoimmune myocarditis (EAM) and assessed the effects of erianin using echocardiography and pathological staining. In vitro experiments, flow cytometry, immunofluorescence and western blotting were performed to determine whether erianin inhibits macrophage activation. Erianin ameliorated cardiac fibrosis and cardiac dysfunction and reduced the percentage of M1-type macrophages and the secretion of inflammatory factors. Mechanistically, erianin inhibits the activation of the NLRP3 inflammasome by inhibiting the NF-κB pathway. Erianin could be a natural therapeutic drug for EAM by inhibiting NLRP3 activation and M1 macrophage-mediated inflammation through the IκBα/NF-κB pathway.

P1293 Novel mutations in SASH3 cause very early onset IBD by impairing the NF-kB and mTORC1 pathways

Abstract Background Human SAM and SH3 domain-containing 3 (SASH3) variants globally affect TCR signaling including its downstream mTORC1 and NF-kB signaling. These 3 pathys play great roles in the pathogenesis of very early-onset inflammatory bowel diseases (VEO-IBD). Here, we report a now 4-year-old patient (P1) who was ultimately diagnosed with SASH3 deficiency in the nuclear localization signal 1(NLS1) of the protein presenting with VEO-IBD along with his adult uncle clinically asymptomatic (P2) who carries the identical variant. Methods a 3.2-year-old boy suspected VEO-IBD was evaluated in our clinic. Tissues were obtained by endoscopy. Genomic DNA, extracted from the peripheral blood mononuclear cells of the child and parents, were subjected to whole-exome sequencing(WES) and sanger sequencing. After mutations in the SASH3 were identified, sanger sequencing were performed for their relatives,the immunological features of our patients were analyzed including the cell proliferation assay by flow cytometry. Western blot(WB) was performed to study the protein level of SASH3 and the NF-kB and mTORC1 pathways in patients. Results P1 was diagnosed with Crohn’s disease according the images of endoscopy and biopsies (Figure A). WES and sanger sequencing identical a variant at exon1 in SASH3 (c.10 C>T/p.R4C ) in P1, his mother was a heterozygous carrier (Figure B). Sanger sequencing confirmed that P2 who was clinically asymptomatic to date, carries the identical variant at exon1 in SASH3 (c.10 C>T/p.R4C ) which inherited from P1’ grandmother(Figure C,D). The SASH3 variants identified in them is located in the NLS1 of the protein(Figure E), it caused the protein is exclusively located in the cytoplasm and no protein was found inside the nucleus which were proved by WB( Figure F). The immune phenotype were not seriously damaged in our 2 patients at homeostatic conditions, but upon stimulation with anti-CD3 and anti-CD28, both CD4+ and CD8+ T cells from P1, demonstrated impaired expression of CD71 and GLUT1 (FigureG, H), indicating defective cell proliferation and activation. The WB demonstrated that both the level of p-NFkB-p65 and p-S6 was markedly reduced in P1 (Figure I). We used Infliximab to treat P1, one year later the endoscopy showed mucosal healing and the pathology showed improved in inflammation and absent in granulation tissue. (Figure J) Conclusion We identified a novel mutations in SASH3 located in the NLS1 of the protein which cause VEO-IBD by impairing the NF-kB and mTORC1 pathways.

P0132 ACSS2 inhibition alleviates inflammatory signaling and improves epithelial integrity in Inflammatory Bowel Disease in vitro models

Abstract Background Inflammatory bowel disease (IBD), encompassing Ulcerative Colitis (UC) and Crohn’s Disease (CD), is marked by chronic gastrointestinal inflammation stemming from genetic, environmental, immunological, and microbiota factors1. Acetyl-CoA Synthetase Short Chain Family Member 2 (ACSS2) converts acetate into acetyl-CoA and is primarily cytosolic, but translocates to the nucleus under metabolic stress, where it regulates gene transcription and cellular processes2. Although the gastrointestinal microbiota influences acetate levels, the role of ACSS2 in colitis and intestinal inflammation remains poorly understood3. Methods To evaluate ACSS2's role in IBD, we employed in vitro IBD models using Caco-2 and HT-29 intestinal epithelial cell lines. ACSS2 activity was inhibited with an ACSS2 inhibitor, followed by inflammatory stimulation with 100 ng/ml Tumor Necrosis Factor-α (TNF-α) for 30 minutes or 1% Dextran Sodium Sulfate (DSS) for 6 hours. Protein expression levels were assessed via Western blot and immunohistochemistry (IHC) on paraffin-embedded samples from UC patients. The effects of ACSS2 inhibition on inflammatory signaling pathways and cell integrity were assessed using RT-qPCR, immunoblotting, and transepithelial electrical resistance (TEER) measurements. Results Treatment of Caco-2 and HT-29 cells with DSS or TNF-α induced upregulation of ACSS2 protein levels compared to controls. IHC analysis of 30 paired paraffin-embedded colon samples from UC patients revealed higher ACSS2 expression and nuclear localization in inflamed sections compared to non-inflamed regions. Inhibition of ACSS2 activity in stimulated Caco-2 and HT29 cells led to a significant decrease in the gene expression levels of pro-inflammatory cytokines such as Interleukin 8 (IL-8), IL-1β and TNF-α. Additionally, immunoblotting demonstrated reduced phosphorylation of p65 and ERK/MEK, indicating suppression of NF-κB and MAPK inflammatory signaling pathways. To assess the effect of ACSS2 inhibition on cell integrity, TEER assays revealed that ACSS2 inhibition preserved cell integrity in DSS-treated monolayers, accompanied by increased expression of tight junction proteins Occludin and ZO-1, suggesting improved epithelial integrity in the inflammatory context. Conclusion Our findings demonstrate a pro-inflammatory role for ACSS2 in IBD, mediated through its involvement in inflammatory signaling and epithelial integrity. These results highlight ACSS2 as a potential therapeutic target for IBD and warrant further investigations in more advanced models, including human-derived intestinal organoids and in vivo systems, to validate its role in disease pathogenesis and therapeutic potential.

A Novel 167-Amino Acid Protein Encoded by CircPCSK6 Inhibits Intrahepatic Cholangiocarcinoma Progression via IKBα Ubiquitination.

Intrahepatic cholangiocarcinoma (ICC), a formidable challenge in oncology, demands innovative biomarkers and therapeutic targets. This research highlights the importance of the circular RNA (circRNA) circPCSK6 and its peptide derivative circPCSK6-167aa in ICC. CircPCSK6 is significantly downregulated in both ICC patients and mouse primary ICC models, and its lower expression is linked to adverse prognosis, highlighting its pivotal role in ICC pathogenesis. Functionally, this study elucidates the regulatory effect of circPCSK6-167aa on IκBα ubiquitination within the NF-κB pathway, which is mediated by its competitive binding to the E3 ligase RBBP6. This complex interaction leads to reduced activation of the NF-κB pathway, thereby curbing tumor cell proliferation, migration, invasion, stemness, and hepatic-lung metastasis in vivo. This groundbreaking discovery expands the understanding of circRNA-driven tumorigenesis through atypical signaling pathways. Additionally, this investigation identified EIF4A3 as a detrimental regulator of circPCSK6, exacerbating ICC malignancy. Importantly, by leveraging patient-derived xenograft (PDX), organoids, and organoid-derived PDX models, higher levels of circPCSK6-167aa enhance sensitivity to gemcitabine, indicating its potential to improve the effectiveness of chemotherapy. These insights emphasize the therapeutic promise of targeting circPCSK6-167aa, offering vital biological insights and clinical directions for developing cutting-edge therapeutic approaches, thus revealing innovative strategies and targets for future treatments.

Antagonism of CD28 blocks allergic responses in the ovalbumin-induced asthmatic model mice.

Allergen-reactive T helper (Th) 2 cells play a pivotal role in initiating asthma pathogenesis. The absence or interruption of CD28 signaling causes significant consequences for T-cell activation, leading to reduced cell proliferation and interleukin (IL)-2 production. A novel compound, Cyn-1324, exhibits a higher binding affinity to CD28 than CD80. Thus, targeting the CD28-CD80 interaction emerges as a promising therapeutic approach for allergic asthma. However, the impact of CD28 antagonists on allergen-induced asthma remains unreported. In this study, we explored the effects of intranasally administered Cyn-1324 on airway inflammation in the ovalbumin (OVA)-induced murine allergic model. The results revealed a significant reduction in airway hyper-responsiveness (AHR), eosinophil recruitment, and cell infiltration in lung tissues, as well as decreased OVA-specific IgE in serum and Th2 cytokine levels in OVA-stimulated lymphocyte cultures. Additionally, we demonstrated the immunosuppressive effects of Cyn-1324 in vitro, including decreased T-cell proliferation and IL-2 secretion, together with increased p27kip1 expression via inhibiting the PI3K signaling pathway. Notably, Cyn-1324 not only inhibited the NF-κB pathway, but also appeared to suppress p38 activation, which is downstream of CD3 signaling, and reduced calcium-induced NFAT protein expression. These findings suggest that Cyn-1324 alleviates allergic responses by inhibiting the CD28-CD80 interaction and holds promise as an immunosuppressive agent for allergic patients.

RAGE-mediated intestinal pro-inflammatory responses triggered by Giardia duodenalis.

Giardia duodenalis is a waterborne zoonotic protozoan that causes gastrointestinal inflammation. Giardiasis and metabolic illnesses share features such as chronic inflammation and intestinal symptoms. Receptor for advanced glycation end products (RAGE) signaling plays a role in metabolic illnesses and intestinal inflammatory responses. The presence of protozoan viruses can influence host immunological responses triggered by protozoa. However, these effects of G. duodenalis remain unknown. In this study, mice treated with the RAGE inhibitor FPS-ZM1 showed more severe intestinal damage, including increased intestinal permeability and lesions, compared to that of the untreated group. Next, we found that G. duodenalis infection activated RAGE, leading to increased secretion of pro-inflammatory cytokines, including IL-1 β, IL-6, IL-12, TNF-α and IFN-γ in mouse intestinal epithelial cells. Notably, these pro-inflammatory responses were significantly higher in Giardiavirus (GLV)-free Giardia than those of GLV-containing Giardia, except for IFN-γ. Additionally, lactate dehydrogenase (LDH) release, GSDMD-N cleavage, and the morphological observation of pyroptosis were significantly higher in cells induced by GLV-free Giardia than those infected with GLV-carrying Giardia. Differences were also observed in the MAPK (p-JNK, p-38, p-ERK) and NF-κB pathway activation, as well as reactive oxygen species (ROS) levels, with higher activation in cells infected by GLV-free Giardia, and the ROS was involved in the regulation of p38 MAPK and JNK activation. These findings reveal the potential of RAGE as a target for developing vaccines or drugs, suggesting the differences in the regulation of host immune responses induced by GLV-free Giardia or GLV-containing Giardia, providing new insights for the prevention and treatment of Giardia.