P65 Subunit Research Articles

Selective PPARδ Agonist GW501516 Protects Against LPS-Induced Macrophage Inflammation and Acute Liver Failure in Mice via Suppressing Inflammatory Mediators.

Inflammation is critical in the development of acute liver failure (ALF). Peroxisome proliferator-activated receptor delta (PPARδ) regulates anti-inflammatory responses and is protective in several diseases such as obesity and cancer. However, the beneficial effects and underlying mechanisms of PPARδ agonist GW501516 in ALF remain unclear. This study investigated the molecular mechanisms underlying the anti-inflammatory effects of GW501516 in macrophages and assessed its protective potential against lipopolysaccharide (LPS)/galactosamine (GalN)-induced ALF. In vivo administration of GW501516 significantly reduced LPS/GalN-induced hepatotoxicity, as evidenced by lower mortality, decreased liver damage, and attenuated secretion of IL-1β, IL-6, and TNF-α. GW501516 treatment also decreased LPS-induced nitric oxide synthase 2 (NOS2) expression and nitric oxide (NO) production in RAW264.7 cells, an effect reversed by PPARδ siRNA. Additionally, GW501516 inhibited LPS-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK), suggesting that inactivation of these MAPKs contributes to its effects. The secretion of IL-6, TNF-α, and NF-κB DNA-binding activity were also suppressed by GW501516, while the nuclear translocation of the NF-κB p65 subunit was unaffected. In conclusion, our findings suggest that GW501516 exerts protective effects in ALF by inhibiting the production of inflammatory mediators. Therefore, GW501516 may act as a potential agent for developing anti-inflammatory therapies for ALF.

EZH2 Activates HTLV-1 bZIP Factor-Mediated TGF-β Signaling in Adult T-Cell Leukemia.

Adult T-cell leukemia (ATL) is an aggressive malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection. Enhancer of zeste homolog 2 (EZH2) has been implicated in the development and progression of multiple cancers, including virus-induced malignancies. However, the potential function of EZH2 in HTLV-1-induced oncogenesis has not been clearly elucidated. In the present study, we showed that EZH2 was overexpressed and activated in HTLV-1-infected cell lines, potentially due to the activation of EZH2 promoter by HTLV-1 Tax and NF-κB p65 subunit. In addition, we found that EZH2 enhanced the HBZ-induced activation of TGF-β signaling in a histone methyltransferase-independent manner. As a mechanism for these actions, we found that EZH2 targeted Smad3/Smad4 to form a ternary complex, and the association between Smad3 and Smad4 was markedly enhanced in the presence of EZH2. Knockdown of EZH2 in ATL cells indeed repressed the expressions of the TGF-β target genes. In particular, EZH2 synergistically enhanced the HBZ/TGF-β-induced Foxp3 expression. Treatment of 3-Deazaneplanocin A, a specific inhibitor of EZH2 significantly inhibited the Foxp3 expression. Taken together, our results suggest that EZH2 may be involved in the differentiation of regulatory T cells through activating the HBZ-Smad3-TGF-β signaling axis, which is considered to be a key strategy for viral persistence.

Expression of key molecules of the classical inflammation activation pathway in blood leukocytes of autistic children

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders, whose causes are currently not fully understood. Research suggests that inflammation and changes in immune functions may play an important role in the development of autism. Increased levels of proinflammatory cytokines in the brains of autistic children lead to negative regulation of synaptic plasticity, as well as impaired proliferation and differentiation of neurons through activation of the nuclear factor kappa B (NF-κB) signaling pathway. The purpose of the work is to analyze the levels of mRNA: TLR2, TLR4, MyD88, IκBα, NF-κB p50, NF-κB p65 in peripheral blood leukocytes of children in comparison with the severity of autism spectrum disorders. The study included 126 children aged from 3 to 13 years (the ratio of boys to girls was 4:1): 45 children with typical neurodevelopment, and 81 children with a clinically confirmed diagnosis of autism. According to the Childhood Autism Rating Scale, 51 children had mild to moderate ASD (CARS score: 29-36), and 30 children had severe autism (CARS score: 36-60). The expression of inflammatory signal transduction pathway molecules was determined in peripheral blood leukocytes using real-time polymerase chain reaction with SYBRGreen. To compare the samples, one-way ANOVA and Tukey’s test were used. It was found that in leukocytes of children with severe ASD, the expression of the adapter protein MyD88 and the p65 subunit of the nuclear transcription factor NF-κB was significantly reduced, and the expression of the NF-κB inhibitor, IκBα, was significantly increased, compared to the control group. In leukocytes of children with mild ASD, a decrease in NF-κB p65 expression was found at a trend level. When comparing groups of children with different severity of autism symptoms (mild/severe), no significant differences were found in the levels of mRNA of key signaling molecules of the classical inflammation activation pathway in blood leukocytes. Thus, in the blood leukocytes of children with severe ASD, suppression of the expression of key molecules of the classical inflammation activation pathway (NF-κB) is observed, which leads to a decrease in the expression of pro-inflammatory cytokines: IL-1β, IL-18 and IL-2, against the background of increased expression of key cytokine of Th1 cells – IFNγ.

Preventive effects of matrine on LPS-induced inflammation in RAW 264.7 cells and intestinal damage in mice through the TLR4/NF-κB/MAPK pathway

BackgroundMatrine is a tetracyclic quinolizidine alkaloid with diverse bioactive properties, including anti-inflammatory and neuroprotective properties. However, the underlying anti-inflammatory mechanisms remain unclear. PurposeThis study aimed to explore how matrine reduces Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and to assess its protective effects against LPS-induced intestinal damage. MethodsThe effect of matrine on cell viability was assessed using the cell counting kit-8 (CCK-8) assay. Additionally, its impact on inflammatory cytokines and macrophage polarization was assessed using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The effects on intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), nitric oxide (NO) production, and oxidative stress were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate staining and JC-1 and Griess assays. Immunofluorescence staining was used to observe the translocation of the NF-κB p65 subunit. Western blotting (WB) and qRT-PCR were employed to analyze the expression levels of proteins related to the toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. An LPS-induced mouse model was established to study the intestinal inflammation and barrier injury. Mouse feces characteristics, colon length, and disease activity index (DAI) were recorded. Hematoxylin-eosin (H&E) and alcian blue/periodic acid schiff (AB/PAS) staining were used to observe morphological changes and barrier damage in the duodenum, jejunum, ileum, and colon and to measure villus length, crypt depth, goblet cell count, and positive areas in the duodenum, jejunum, and ileum. The content of short-chain fatty acids (SCFAs) in the colon was determined using gas chromatography (GC). ResultsMatrine inhibited LPS-induced inflammatory cytokine levels, suppressed macrophage M1 polarization, and promoted M2 macrophage polarization. Matrine reduced LPS-induced increases in ROS and NO levels and regulates oxidative stress. Additionally, matrine inhibited the nuclear translocation of the NF-κB p65 subunit and exerted anti-inflammatory effects by suppressing the activation of the TLR4/NF-κB/MAPK pathway. In vivo experiments indicated that matrine significantly alleviated LPS-induced diarrhea, increased DAI, and shortened the colon. Matrine reduced the production of the pro-inflammatory cytokine interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α and the pro-inflammatory mediator NO in mouse intestinal tissues while promoting the content of the anti-inflammatory cytokine IL-10. Furthermore, it improved intestinal tissue structure and alleviated LPS-induced intestinal barrier damage. Finally, matrine increased the SCFA levels in the intestine. ConclusionMatrine exerted its anti-inflammatory effects and protects against intestinal injury through the TLR4/NF-κB/MAPK signaling pathway.

The Generation of ROS by Exposure to Trihalomethanes Promotes the IκBα/NF-κB/p65 Complex Dissociation in Human Lung Fibroblast.

Background: Disinfection by-products used to obtain drinking water, including halomethanes (HMs) such as CH2Cl2, CHCl3, and BrCHCl2, induce cytotoxicity and hyperproliferation in human lung fibroblasts (MRC-5). Enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) modulate these damages through their biotransformation processes, potentially generating toxic metabolites. However, the role of the oxidative stress response in cellular hyperproliferation, modulated by nuclear factor-kappa B (NF-κB), remains unclear. Methods: In this study, MRC-5 cells were treated with these compounds to evaluate reactive oxygen species (ROS) production, lipid peroxidation, phospho-NF-κB/p65 (Ser536) levels, and the activities of SOD, CAT, and GPx. Additionally, the interactions between HMs and ROS with the IκBα/NF-κB/p65 complex were analyzed using molecular docking. Results: Correlation analysis among biomarkers revealed positive relationships between pro-oxidant damage and antioxidant responses, particularly in cells treated with CH2Cl2 and BrCHCl2. Conversely, negative relationships were observed between ROS levels and NF-κB/p65 levels in cells treated with CH2Cl2 and CHCl3. The estimated relative free energy of binding using thermodynamic integration with the p65 subunit of NF-κB was -3.3 kcal/mol for BrCHCl2, -3.5 kcal/mol for both CHCl3 and O2•, and -3.6 kcal/mol for H2O2. Conclusions: Chloride and bromide atoms were found in close contact with IPT domain residues, particularly in the RHD region involved in DNA binding. Ser281 is located within this domain, facilitating the phosphorylation of this protein. Similarly, both ROS interacted with the IPT domain in the RHD region, with H2O2 forming a side-chain oxygen interaction with Leu280 adjacent to the phosphorylation site of p65. However, the negative correlation between ROS and phospho-NF-κB/p65 suggests that steric hindrance by ROS on the C-terminal domain of NF-κB/p65 may play a role in the antioxidant response.

SERPINA1 promotes the invasion, metastasis, and proliferation of pancreatic ductal adenocarcinoma via the PI3K/Akt/NF-κB pathway

Serpin peptidase inhibitor clade A member 1 (SERPINA1) is highly expressed in a variety of solid tumors. However, its role in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here, we report evidence that SERPINA1 acts as a potent oncogene to drive its extremely malignant character. We found that elevated SERPINA1 expression in primary tumors was associated with lymph node metastasis and shorter survival in PDAC patients. Mechanistic investigations revealed that overexpression of SERPINA1 induced nuclear translocation and phosphorylation of the p65 subunit through the PI3K/Akt/NF-κB pathway, thereby promoting the invasion, metastasis and proliferation of PDAC cells in vitro and in vivo. Conversely, the knockdown of SERPINA1 attenuated this signaling pathway and restored the phenotype of PDAC cells overexpressing SERPINA1. Overall, our study reveals that SERPINA1 affects the properties of PDAC through the PI3K/Akt/NF-κB pathway, and its activation confers the clinical features of epithelial-mesenchymal transition and proliferation in the disease.

Type I Diabetes Mellitus impairs cytotoxic immunity through CEACAM5 upregulation in colorectal cancer : Exploring the intersection of autoimmune dysfunction and cancer progression: the role of NF-κB p65 in colorectal cancer.

Type 1 diabetes (T1D) is characterized by an autoimmune-mediated destruction of pancreatic beta cells and a chronic inflammatory state, which may influence the progression of colorectal cancer (CRC) through immune system dysregulation and enhanced tumor immune evasion. This study aims to elucidate the role of p65 in modulating the tumor microenvironment in CRC within the context of T1D and to determine how this modulation affects tumor growth, immune cell infiltration, and the expression of immune evasion molecules such as CEACAM5. NOD mice, which model T1D, were inoculated with MC38 colon carcinoma cells engineered to knock down p65. Tumor growth was monitored, and the tumor microenvironment was analyzed using flow cytometry to assess the infiltration of immune cells. The expression of Ki-67 and CEACAM5 in tumor cells was also evaluated. Additionally, in vitro assays were conducted to study the proliferation and activation of T cells co-cultured with tumor cells. Knockdown of p65 in tumor cells significantly inhibited tumor growth in NOD mice. This was accompanied by an increased infiltration of cytotoxic CD8+ T cells and no significant change in CD4+ or Foxp3 + T regulatory cells within the tumor microenvironment. There was a notable reduction in the expression of Ki-67 and CEACAM5, indicating decreased proliferation and potential immune evasion capabilities of the tumor cells. Our findings demonstrate that the NF-κB p65 subunit plays a crucial role in promoting tumor growth and modulating the immune microenvironment in CRC, particularly in the context of T1D. Knocking down p65 not only reduces tumor progression but also enhances the anti-tumor immune response by decreasing immune evasion mechanisms. These results suggest that targeting the NF-κB pathway may be a viable strategy to improve the efficacy of cancer immunotherapy, especially in patients with autoimmune diseases like T1D. Physical activity enhances the effect of immune checkpoint blockade by inhibiting the intratumoral HIF1-α/CEACAM5 axis.

The β2-adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner.

Adrenergic receptors regulate metabolic, cardiovascular, and immunological functions in response to the sympathetic nervous system. The effect of β2-adrenergic receptor (AR) as a high expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional β2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells, highlighting its potential for IL-17A suppression via a non-canonical β2-AR cell signaling pathway. The effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with anti-CD3/anti-CD28/anti-CD2 ImmunoCult reagent. IFN-γ, IL-4, and IL-17A, which are primarily derived from Th1, Th2, and Th17 cells, respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated serine133-CREB and phosphorylated NF-κB p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. Nebivolol treatment decreased IL-17A and IFN-γ secretion by activated memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the β2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells toward the Th17 phenotype. IL-10 and the proliferation index remained unchanged. Nebivolol-treated β2-knockout memory Th cells showed significant inhibition of β2-AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-κB p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. The findings demonstrate that nebivolol acts through a β2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing the phosphorylation of NF-κB. This highlights nebivolol's potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.

The long non-coding RNA GAS5 contributes to the suppression of inflammatory responses by inhibiting NF-κB activity.

Nuclear factor kappa B (NF-κB) is a key regulator of immune and inflammatory responses. Glucocorticoid drugs (GC) act through the glucocorticoid receptor (GR) as immunosuppressant also in pediatric patients inhibiting NF-κB activity. The long non-coding RNA GAS5 interacts with the GR, influencing GC activity. No data on the role of GAS5 on GR-dependent inhibition of NF-κB activity have been published. This study investigated the impact of GAS5 on NF-κB activity in HeLa cells overexpressing GAS5, both under basal conditions and during GC treatment. The study used EMSA, RNA-immunoprecipitation (RIP), Western blotting, and bioinformatic analyses to assess NF-κB DNA binding, GAS5-p65 interaction, and NF-κB signaling pathway modulation. GAS5 overexpression increased NF-κB DNA binding activity in untreated cells. RNA-IP confirmed a direct interaction between GAS5 and the NF-κB subunit p65, suggesting a potential regulatory mechanism. GAS5 overexpression led to downregulation of NF-κB target genes, TNF-α, and NR3C1. GC treatment reduced NF-κB DNA binding activity in GAS5-overexpressing cells, indicating a potential synergistic effect. Furthermore, GAS5 overexpression increased IκB levels and reduced p-p65/pan-p65 levels during GC treatment. GAS5 appears to modulate NF-κB activity in a complex manner, influencing both basal and GC-induced signaling. The interaction between GAS5, GCs, and NF-κB is multi-faceted, and further research is needed to fully elucidate the underlying mechanisms. These findings suggest that GAS5 could be a potential target for personalized therapy, particularly in pediatric patients with inflammatory conditions.

Comparative proteomic analysis of regenerative mechanisms in mouse retina to identify markers for neuro-regeneration in glaucoma

The retina is part of the central nervous system (CNS). Neurons in the CNS and retinal ganglion cells lack the ability to regenerate axons spontaneously after injury. The intrinsic axonal growth regulators, their interaction and roles that enable or inhibit axon growth are still largely unknown. This study endeavored to characterize the molecular characteristics under neurodegenerative and regenerative conditions. Data-Independent Acquisition Mass Spectrometry was used to map the comprehensive proteome of the regenerative retina from 14-day-old mice (Reg-P14) and adult mice after lens injury (Reg-LI) both showing regrowing axons in vitro, untreated adult mice, and retina from adult mice subjected to two weeks of elevated intraocular pressure showing degeneration. A total of 5750 proteins were identified (false discovery rate < 1%). Proteins identified in both Reg-P14 and Reg-LI groups were correlated to thyroid hormone, Notch, Wnt, and VEGF signaling pathways. Common interactors comprising E1A binding protein P300 (EP300), CREB binding protein (CBP), calcium/calmodulin dependent protein kinase II alpha (CaMKIIα) and sirtuin 1 (SIRT1) were found in both Reg-P14 and Reg-LI retinas. Proteins identified in both regenerating and degenerative groups were correlated to thyroid hormone, Notch, mRNA surveillance and measles signaling pathways, along with PD-L1 expression and the PD-1 checkpoint pathway. Common interactors across regenerative and degenerative retinas comprising NF-kappa-B p65 subunit (RELA), RNA-binding protein with serine-rich domain 1 (RNPS1), EP300 and SIN3 transcription regulator family member A (SIN3A). The findings from our study provide the first mapping of regenerative mechanisms across postnatal, mature and degenerative mouse retinas, revealing potential biomarkers that could facilitate neuro-regeneration in glaucoma.

Huaiyu pill alleviates inflammatory bowel disease in mice blocking toll like receptor 4/ myeloid differentiation primary response gene 88/ nuclear factor kappa B subunit 1 pathway.

To investigate the therapeutic effects of Huaiyu pill (, HYP) on inflammatory bowel disease (IBD) and the underlying mechanisms have not been elucidated. To establish the IBD model, mice were administered with dextran sulfate sodium (DSS). Mice were intragastrically pre-treated with sulfasalazine (SASP) and HYP. Disease activity index (DAI) and colon length were monitored, and the colonic tissues were subjected to hematoxylin-eosin staining. Pro-inflammatory factors and vascular inflammation-related proteins were determined using enzyme-linked immunosorbent assay (ELISA). The potential mechanisms of HYP were examined using network pharmacology analysis.The expressions of zona occludens 1 (ZO-1), occludin, toll like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MYD88), and nuclear factor kappa B p65 subunit (NF-κB p65) in colon tissues were examined using Western blotting or immunohistochemical analyses. Pre-treatment with HYP enhanced the colon length, decreased DAI scores, and mitigated histopathological alterations in DSS-treated mice. HYP alleviated intestinal inflammation by downregulating the levels of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin 17 (IL-17). Additionally, HYP suppressed the disruption of the gut barrier by upregulating the ZO-1, occludin, and mucin 2 (MUC2) levels and downregulating the endothelin 1 (ET-1) and erythropoietin (EPO) levels. Network pharmacological analysis and experimental results revealed that HYP downregulated the colonic tissue levels of TLR4, MYD88, and NF-κB p65 in DSS-treated mice. This study investigated the in vivotherapeutic effects of HYP on IBD and the underlying molecular mechanisms. These findings provide an experimental foundation for the clinical application of HYP.

Investigating the Anticancer Potential of Biochanin A in KB Oral Cancer Cells Through the NFκB Pathway.

Squamous cell carcinoma (SCC) is a malignancy primarily affecting squamous cells. Its development is linked to multiple risk factors, such as alcohol and tobacco consumption, human papillomavirus (HPV) infection, and Epstein-Barr Virus (EBV) infection. Biochanin A (BCA), a phytoestrogen extracted from red clover, has been extensively researched for its therapeutic properties. It spans antioxidant activity, anti-inflammatory effects, neuroprotection, cardioprotection, and anticancer potential in different bodily systems. However, its impact on oral cancer remains unexplored. Therefore, this investigation aims to assess the potential anticancer effects of BCA, specifically on KB oral cancer cells. This study utilized KB cells to evaluate the impact of BCA on various cellular parameters, including cell viability, apoptosis, intracellular ROS production, mitochondrial membrane potential, and cell migration. BCA treatment induced several notable effects on KB cells, including reduced cell viability, altered morphology suggestive of apoptosis, heightened oxidative stress, and alterations in mitochondrial membrane potential. Moreover, BCA treatment demonstrated an inhibitory effect on cell migration. The study further investigated the impact of BCA on antioxidant enzyme activities and lipid peroxidation, revealing decreased antioxidant enzyme activities and increased lipid peroxidation across different BCA concentrations (IC50 and IC90). Immunocytochemistry and qRT-PCR analyses unveiled that BCA treatment at varying doses (IC50 and IC90) downregulated the expression of nuclear factor-κB (NF-κB) subunits p50 and p65, pivotal players in cancer progression. In summary, this study sheds light on the promising potential of BCA as an anticancer therapeutic agent for treating oral cancer. Its demonstrated ability to induce apoptosis, perturb cellular functions, and modulate gene expression within cancer cells underscores its significance. Nonetheless, further research, particularly following animal studies, is imperative to comprehensively grasp the breadth of BCA's effects and its viability for clinical applications.

Artemisitene induces apoptosis of breast cancer cells by targeting FDFT1 and inhibits the growth of breast cancer patient-derived organoids

Artemisitene (ATT) is a natural bioactive compound with anti-breast cancer activity. However, the direct target and clinical efficacy of ATT on breast cancer are still unclear. The current study aimed to identify the target protein and underlying mechanism of ATT in anti-breast cancer. Moreover, patient-derived organoids (PDOs) were employed to assess the clinical efficacy of ATT on breast cancer. Herein, molecular docking, molecular dynamics simulation, cellular thermal shift assay (CETSA) combined with Western blot, surface plasmon resonance (SPR) were applied to confirm the interactional target of ATT in breast cancer cells. Bioinformatics analysis, Western blot, flow cytometry, plasmid construction and lentivirus infection, chromatin immunoprecipitation (ChIP) assay, and quantitative real-time PCR (RT-qPCR) were performed to reveal the potential mechanism of ATT in treating breast cancer. PDOs were established to evaluate the clinical therapeutic efficiency of ATT on breast cancer. We found that ATT interacted with Farnesyl-diphosphate farnesyltransferase 1 (FDFT1) in breast cancer cells. Knockdown of FDFT1 induced NEDD4 expression and apoptosis in breast cancer cells. Overexpression of FDFT1 could rescue ATT-induced apoptosis, while interfering with FDFT1 expression decreased the level of RelA (NF-κB p65 subunit) in the nucleus in breast cancer cells. Knockdown of FDFT1 induced NEDD4 expression by regulating TNFR1/NF-κB pathway. Overexpression of FDFT1 could reverse the activation of ATT-induced TNFR1/NF-κB/NEDD4 pathway in breast cancer cells. Interestingly, the ChIP assay revealed that p65 could regulate NEDD4 transcription. Furthermore, ATT exhibited a broad-spectrum inhibitory effect on the growth of breast cancer PDOs with different pathological subtypes, and showed an excellent safety profile in comparison with that of conventional chemotherapy drugs. In summary, this work demonstrated that ATT targets FDFT1 to induce apoptosis of breast cancer cells through regulating TNFR1/NF-κB/NEDD4 pathway and suppresses breast cancer PDOs growth, which supported ATT as an effective and potential drug candidate for breast cancer treatment.

Luteolin Mitigates Dopaminergic Neuron Degeneration and Restrains Microglial M1 Polarization by Inhibiting Toll Like Receptor 4.

Luteolin is a natural flavonoid and its neuroprotective and anti-inflammatory effects have been confirmed to mitigate neurodegeneration. Despite these findings, the underlying mechanisms responsible for these effects remain unclear. Toll-like receptor 4 (TLR4) is widely distributed in microglia and plays a pivotal role in neuroinflammation and neurodegeneration. Here studies are outlined that aimed at determining the mechanisms responsible for the anti-inflammatory and neuroprotective actions of luteolin using a rodent model of Parkinson's disease (PD) and specifically focusing on the role of TLR4 in this process. The mouse model of PD used in this experiment was established through a single injection of lipopolysaccharide (LPS). Mice were then subsequently randomly allocated to either the luteolin or vehicle-treated group, then motor performance and dopaminergic neuronal injury were evaluated. BV2 microglial cells were treated with luteolin or vehicle saline prior to LPS challenge. MRNA expression of microglial specific marker ionized calcium-binding adapter molecule 1 (IBA-1) and M1/M2 polarization markers, as well as the abundance of indicated pro-inflammatory cytokines in the mesencephalic tissue and BV2 were quantified by real time-polymerase chain reaction (RT-PCR) and Enzyme-linked Immunosorbent Assay (ELISA), respectively. Cell viability and apoptosis of neuron-like PC12 cell line co-cultured with BV2 were detected. TLR4 RNA transcript and protein abundance in mesencephalic tissue and BV2 cells were detected. Nuclear factor kappa-gene binding (NF-κB) p65 subunit phosphorylation both in vitro and in vivo was evaluated by immunoblotting. Luteolin treatment induced functional improvements and alleviated dopaminergic neuronal loss in the PD model. Luteolin inhibited apoptosis and promoted cell survival in PC12 cells. Luteolin treatment shifted microglial M1/M2 polarization towards an anti-inflammatory M2 phenotype both in vitro and in vivo. Finally, it was found that luteolin treatment significantly downregulated both TLR4 mRNA and protein expression as well as restraining NF-κB p65 subunit phosphorylation. Luteolin restrained dopaminergic degeneration in vitro and in vivo by blocking TLR4-mediated neuroinflammation.

MiR-155 induces sepsis-associated damage to the intestinal mucosal barrier via sirtuin 1/nuclear factor-κB-mediated intestinal pyroptosis.

Sepsis is a life-threatening state of organ dysfunction caused by systemic inflammation and a dysfunctional response to host infections that can induce severe intestinal mucosal damage. Pyroptosis is mediated by the activated NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome after stimulation by various inflammatory factors during sepsis. The inflammatory response is a major driver of intestinal damage during sepsis. Intestinal mucosal barrier dysfunction in sepsis is associated with pyroptosis, a type of programmed inflammatory cell death. Several studies have confirmed the role of miR-155 in sepsis and other diseases. However, the effect of miR-155 on intestinal pyroptosis in the context of intestinal mucosal barrier dysfunction during sepsis remains unclear. Thus, a model of sepsis in Sprague-Dawley rats is established using cecal ligation and puncture (CLP), and a series of molecular biological methods are used in this study. The results show that the expression of miR-155 is increased and that of sirtuin 1 (SIRT1) is decreased in the intestinal tissues of patients with sepsis. miR-155 expression is negatively correlated with SIRT1 expression. Increased miR-155 expression significantly inhibits SIRT1 activity and upregulates the expressions of NOD-like receptor family pyrin domain-containing 3 (NLRP3), caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC), interleukin-1β (IL-1β) and interleukin-18 (IL-18) to promote pyroptosis. The inhibition of miR-155 expression is associated with increased SIRT1 expression, promotes the deacetylation of p65, and significantly downregulates p65 acetylation. Herein, we propose that miR-155 induces pyroptosis in the intestine partly by regulating SIRT1, thereby reducing the deacetylation of the nuclear factor (NF)-κB subunit p65 and increasing NF-κB signaling activity in sepsis, leading to intestinal barrier damage.

Lactobacillus delbrueckii alleviates lipopolysaccharide-induced muscle inflammation and atrophy in weaned piglets associated with inhibition of endoplasmic reticulum stress and protein degradation.

Pro-inflammatory cytokines in muscle play a pivotal role in physiological responses and in the pathophysiology of inflammatory disease and muscle atrophy. Lactobacillus delbrueckii (LD), as a kind of probiotics, has inhibitory effects on pro-inflammatory cytokines associated with various inflammatory diseases. This study was conducted to explore the effect of dietary LD on the lipopolysaccharide (LPS)-induced muscle inflammation and atrophy in piglets and to elucidate the underlying mechanism. A total of 36 weaned piglets (Duroc × Landrace × Large Yorkshire) were allotted into three groups with six replicates (pens) of two piglets: (1) Nonchallenged control; (2) LPS-challenged (LPS); (3) 0.2% LD diet and LPS-challenged (LD+LPS). On d 29, the piglets were injected intraperitoneally with LPS or sterilized saline, respectively. All piglets were slaughtered at 4 h after LPS or saline injection, the blood and muscle samples were collected for further analysis. Our results showed that dietary supplementation of LD significantly attenuated LPS-induced production of pro-inflammatory cytokines IL-6 and TNF-α in both serum and muscle of the piglets. Concomitantly, pretreating the piglets with LD also clearly inhibited LPS-induced nuclear translocation of NF-κB p65 subunits in the muscle, which correlated with the anti-inflammatory effects of LD on the muscle of piglets. Meanwhile, LPS-induced muscle atrophy, indicated by a higher expression of muscle atrophy F-box, muscle RING finger protein (MuRF1), forkhead box O 1, and autophagy-related protein 5 (ATG5) at the transcriptional level, whereas pretreatment with LD led to inhibition of these upregulations, particularly genes for MuRF1 and ATG5. Moreover, LPS-induced mRNA expression of endoplasmic reticulum stress markers, such as eukaryotic translational initiation factor 2α (eIF-2α) was suppressed by pretreatment with LD, which was accompanied by a decrease in the protein expression levels of IRE1α and GRP78. Additionally, LD significantly prevented muscle cell apoptotic death induced by LPS. Taken together, our data indicate that the anti-inflammatory effect of LD supply on muscle atrophy of piglets could be likely regulated by inhibiting the secretion of pro-inflammatory cytokines through the inactivation of the ER stress/NF-κB singling pathway, along with the reduction in protein degradation.

MiR-21 attenuated inflammation targeting MyD88 in human chondrocytes stimulated with Hyaluronan oligosaccharides

Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes.The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p-ERK1/2, p-AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE2 levels have been assayed by colorimetric and ELISA kits, respectively.HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action.Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88.

MicroRNA-146a-5p protects retinal ganglion cells through reducing neuroinflammation in experimental glaucoma.

Neuroinflammation plays important roles in retinal ganglion cell (RGC) degeneration in glaucoma. MicroRNA-146 (miR-146) has been shown to regulate inflammatory response in neurodegenerative diseases. In this study, whether and how miR-146 could affect RGC injury in chronic ocular hypertension (COH) experimental glaucoma were investigated. We showed that in the members of miR-146 family only miR-146a-5p expression was upregulated in COH retinas. The upregulation of miR-146a-5p was observed in the activated microglia and Müller cells both in primary cultured conditions and in COH retinas, but mainly occurred in microglia. Overexpression of miR-146a-5p in COH retinas reduced the levels pro-inflammatory cytokines and upregulated the levels of anti-inflammatory cytokines, which were further confirmed in the activated primary cultured microglia. Transfection of miR-146a-5p mimic increased the percentage of anti-inflammatory phenotype in the activated BV2 microglia, while transfection of miR-146a-5p inhibitor resulted in the opposite effects. Transfection of miR-146a-5p mimic/agomir inhibited the levels of interleukin-1 receptor associated kinase (IRAK1) and TNF receptor associated factor 6 (TRAF6) and phosphorylated NF-κB subunit p65. Dual luciferase reporter gene assay confirmed that miR-146a-5p could directly target IRAK1 and TRAF6. Moreover, downregulation of IRAK1 and TRAF6 by siRNA techniques or blocking NF-κB by SN50 in cultured microglia reversed the miR-146a-5p inhibitor-induced changes of inflammatory cytokines. In COH retinas, overexpression of miR-146a-5p reduced RGC apoptosis, increased RGC survival, and partially rescued the amplitudes of photopic negative response. Our results demonstrate that overexpression of miR-146a-5p attenuates RGC injury in glaucoma by reducing neuroinflammation through downregulating IRAK1/TRAF6/NF-κB signaling pathway in microglia.

Bacterial peptidoglycan signalling in microglia: Activation by MDP via the NF-κB/MAPK pathway

Bacterial peptidoglycan (PGN) fragments are commonly studied in the context of bacterial infections. However, PGN fragments recently gained recognition as signalling molecules from the commensal gut microbiota in the healthy host. Here we focus on the minimal bioactive PGN motif muramyl dipeptide (MDP), found in both Gram-positive and Gram-negative commensal bacteria, which signals through the Nod2 receptor. MDP from the gut microbiota translocates to the brain and is associated with changes in neurodevelopment and behaviour, yet there is limited knowledge about the underlying mechanisms. In this study we demonstrate that physiologically relevant doses of MDP induce rapid changes in microglial gene expression and lead to cytokine and chemokine secretion. In immortalised microglial (IMG) cells, C–C Motif Chemokine Ligand 5 (CCL5/RANTES) expression is acutely sensitive to the lowest physiologically prevalent dose (0.1 µg/ml) of MDP. As CCL5 plays an important role in memory formation and synaptic plasticity, microglial CCL5 might be the missing link in elucidating MDP-induced alterations in synaptic gene expression. We observed that a higher physiological dose of MDP elevates the expression of cytokines TNF-α and IL-1β, indicating a transition toward a pro-inflammatory phenotype in IMG cells, which was validated in primary microglial cultures. Furthermore, MDP induces the translocation of NF-κB subunit p65 into the nucleus, which is blocked by MAPK p38 inhibitor SB202190, suggesting that an interplay of both the NF-κB and MAPK pathways is responsible for the MDP-specific microglial phenotype. These findings underscore the significance of different MDP levels in shaping microglial function in the CNS and indicate MDP as a potential mediator for early inflammatory processes in the brain. It also positions microglia as an important target in the gut microbiota-brain-axis pathway through PGN signalling.

Structural and biochemical analyses of the nuclear IκBζ protein in complex with the NF-κB p50 homodimer.

As part of the efforts to understand nuclear IκB function in NF-κB-dependent gene expression, we report an X-ray crystal structure of the IκBζ ankyrin repeat domain in complex with the dimerization domain of the NF-κB p50 homodimer. IκBζ possesses an N-terminal α helix that conveys domain folding stability. Affinity and specificity of the complex depend on a small portion of p50 at the nuclear localization signal. The model suggests that only one p50 subunit supports binding with IκBζ, and biochemical experiments confirm that IκBζ associates with DNA-bound NF-κB p50:RelA heterodimers. Comparisons of IκBζ:p50 and p50:κB DNA complex crystallographic models indicate that structural rearrangement is necessary for ternary complex formation of IκBζ and p50 with DNA.