TRAF6 Research Articles

Regnase-1 regulates inflammation in T cells of ankylosing spondylitis through the TRAF6.

The study aimed to investigate the regulatory role of Regnase-1 in ankylosing spondylitis (AS) inflammation. We collected 10ml peripheral venous blood and epidemiological data from 45 AS patients and 45 healthy controls and performed enzyme-linked immunosorbent assay (ELISA) experiments to measure the levels of inflammatory cytokines. Then CD3 + T lymphocytes were isolated by magnetic bead sorting method, and the transcriptional levels of Regnase-1 and TNF receptor-associated factor 6 (TRAF6) were detected by real-time quantitative PCR (qRT-PCR). The regnase-1 knockdown human T lymphocyte leukemia cell (Jurkat T) model was constructed by small interfering RNA (siRNA) technology. Then PCR and Western blot were used to detect the transcription level and protein level of downstream genes. Co-immunoprecipitation was used to verify the interaction between Regnase-1 and TRAF6. Regnase-1 and TRAF6 transcription levels were down-regulated and positively correlated with each other in T cells from AS patients. The ROC curve analysis indicates that both Regnase-1 and TRAF6 possess diagnostic capabilities, with Regnase-1 demonstrating a particularly high area under the curve (AUC) of 0.876 (95% CI: 0.789-0.936). Subgroup analysis shows NSAIDs boost Regnase-1 and TRAF6 transcription while reducing IL23 and IL17 levels. The results of cell experiments showed that si-Regnase-1 significantly reduced the mRNA and protein levels of TRAF6 in Jurkat T cells and increased the expression level of inflammatory gene TNF-α. Co-immunoprecipitation assay further verified the binding between the two proteins. Regnase-1 may participate in the chronic inflammatory process of AS by regulating TNF-α through TRAF6.

MiR-146a-5p downregulated TRAF6/NF-κB p65 pathway to attenuate the injury of HT-22 cells induced by oxygen-glucose deprivation/reoxygenation.

MicroRNA-146a-5p (miR-146a-5p) actively participates in the process of cerebral ischemia-reperfusion (CI/R) injury. Dysregulation of the tumor necrosis factor receptor-associated factor 6 (TRAF6)/nuclear factor kappa-B (NF-κB) p65 axis is closely associated with inflammatory response. This study aimed to investigate the potential involvement of miR-146a-5p and TRAF6/NF-κB p65 in mediating CI/R progression in vitro. HT-22 cells were challenged with oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate CI/R in vitro. HT-22 cells were transfected with miR-146a-5p mimics or TRAF6 overexpression constructs. The impact of miR-146a-5p on apoptosis, inflammation, and TRAF6/NF-κB p65 activation were investigated. OGD/R inhibited HT-22 cell viability, induced apoptosis, reduced miR-146a-5p levels and activated the TRAF6/NF-κB p65 pathway. MiR-146a-5p mimics reduced pro-inflammatory factor release, limited apoptosis-related protein expression, and inactivated the TRAF6/NF-κB p65 pathway in OGD/R-challenged HT-22 cells. Mechanistically, miR-146a-5p was verified to bind to TRAF6 3'UTR. TRAF6 overexpression reversed the beneficial effects of miR-146a-5p mimics on apoptosis, inflammation, and TRAF6/NF-κB p65 activation. This work revealed that miR-146a-5p targeted TRAF6 and suppressed the TRAF6/NF-κB p65 pathway, thereby reducing OGD/R-induced inflammation and apoptosis in HT-22 cells. These findings suggest the potential of the miR-146a-5p/TRAF6/NF-κB p65 axis in the treatment of CI/R.

Natural lung-tropic TH9 cells: a sharp weapon for established lung metastases

BackgroundLung metastasis remains the primary cause of tumor-related mortality, with limited treatment options and unsatisfactory efficacy. In preclinical studies, T helper 9 (TH9) cells have shown promise in treating solid...

USP33 promotes caerulein-induced apoptotic, oxidative and inflammatory injuries in acute pancreatitis by deubiquitinating TRAF3.

Background: Tumor necrosis factor receptor associated factor 3 (TRAF3) and deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) have been identified to play important roles in inflammatory diseases, including acute pancreatitis (AP). Here, we aimed to explore whether USP33 affected AP progression by affecting TRAF3 expression through deubiquitination.Methods: Caerulein-treated HPDE6-C7 cells were used to mimic AP conditions in vitro. Levels of mRNAs and proteins were examined by qRT-PCR and Western blot. Cell proliferation and apoptosis were evaluated using CCK-8 assay, EdU assay and flow cytometry. Cell oxidative stress was assessed by detecting the production of superoxide dismutase and malonaldehyde. ELISA analysis detected IL-6 and TNF-α levels. Macrophage M1 polarization was evaluated by flow cytometry. Cellular ubiquitination analyzed the ubiquitination effect on TRAF3. Protein interaction between USP33 and TRAF3 was identified by immunofluorescence staining.Results: Caerulein dose-dependently induced apoptosis, oxidative stress, and inflammatory response in HPDE6-C7 cells and promoted macrophage M1 polarization to enhance inflammation (P < 0.05). TRAF3 was highly expressed in AP patients (3.5±1.10 vs. 1.0 ±0.74, P < 0.05) and caerulein-induced HPDE6-C7 cells (3.3 ±0.34 vs. 1.0 ±0.10, P < 0.05). Knockdown of TRAF3 protected HPDE6-C7 cells from caerulein-induced apoptotic, oxidative and inflammatory injuries. Mechanistically, USP33 interacted with TRAF3 and induced TRAF3 deubiquitination to up-regulate its expression (P < 0.05). Further analyses showed that USP33 knockdown reversed caerulein-induced apoptosis, oxidative stress and inflammation in HPDE6-C7 cells by TRAF3 (P < 0.05). Moreover, USP33-TRAF3 activated the NF-κB pathway (P < 0.05).Conclusion: USP33 promoted caerulein-induced apoptosis, oxidative stress and inflammation in pancreatic ductal cells by deubiquitinating TRAF3, indicating a novel insight into the pathogenesis of AP.

Silenced long non-coding RNA RMST ameliorates cardiac dysfunction and inflammatory response in doxorubicin-induced heart failure in C57BL/6 mice via the modulation of the microRNA-10b-5p/TRAF6 axis.

Long non-coding RNA rhabdomyosarcoma 2-associated transcript (RMST) has been found to exert effects on cardiovascular diseases. However, the research for probing its role in heart failure (HF) is limited. Our study intends to unravel the regulatory effects of RMST on HF via the microRNA (miR)-10b-5p/tumor necrosis factor receptor-associated factor 6 (TRAF6) axis. The mouse model of HF was induced by doxorubicin. The expression levels of RMST, miR-10b-5p and TRAF6 were detected. The virus carrying RMST, miR-10b-5p or TRAF6 vectors were injected into doxorubicin-induced HF mice to examine the cardiac function, inflammatory response, pathological changes and cell apoptosis in doxorubicin-induced HF mice. The target relationships among RMST, miR-10b-5p and TRAF6 were confirmed. RMST and TRAF6 were elevated and miR-10b-5p was reduced in doxorubicin-induced HF mice. RMST or TRAF6 silencing or miR-10b-5p overexpression could improve doxorubicin-induced cardiac dysfunction, and inflammatory response, and reduce cardiomyocyte apoptosis. Down-regulation of miR-10b-5p or overexpression of TRAF6 were both able to inverse the therapeutic effect of silencing RMST on doxorubicin-induced HF mice. RMST bound to miR-10b-5p that targeted TRAF6. RMST silencing could attenuate inflammatory response and cardiomyocyte apoptosis and upregulate cardiac function in mice with doxorubicin-induced HF by modulating the miR-10b-5p/TRAF6 axis. The study provides novel therapeutic targets for HF treatment.

Analysis of damaging non-synonymous SNPs in GPx1 gene associated with the progression of diverse cancers through a comprehensive in silico approach

Glutathione Peroxidase 1 (GPx1) gene has been reported for its role in cellular redox homeostasis, and the dysregulation of its expression is linked with the progression of diverse cancers. Non-synonymous single nucleotide polymorphism (nsSNPs) have been emerged as the crucial factors, playing their role in GPx1 overexpression. To understand the deleterious mutational effects on the structure and function of GPx1 enzyme, we delved deeper into the exploration of possibly damaging nsSNPs using in-silico based approaches. Eight widely utilized computational tools were employed to roughly shortlist the deleterious nsSNPs. Their damaging effects on structure and function of the genes were evaluated by using different bioinformatics tools. Subsequently, the three final proposed deleterious mutants including mutations rs373838463, rs2107818892, and rs763687242, were docked with their reported binder, TNF receptor-associated factor 2 (TRAF2). The lowest binding affinity and stability of the docked mutant complexes as compared to the wild type GPx1 were validated by molecular dynamic simulation. Finally, the comparison of RMSD, RMSF, RoG and hydrogen bond analyses between wild-type and mutant’s complexes validated the deleterious effects of proposed nsSNPs. This study successfully identified and verified the possibly damaging nsSNPs in GPx1 enzyme, which may be linked the progression of various types of cancer. Our findings underscore the value of in-silico approaches in mutational analysis and encourage further preclinical and clinical trials.

The Role of TNF Receptor-Associated Factor 5 in the Formation of Germinal Centers by B Cells During the Primary Phase of the Immune Response in Mice.

TNF receptor-associated factors (TRAFs) function as intracellular adaptor proteins utilized by members of the TNF receptor superfamily, such as CD40. Among the TRAF family proteins, TRAF5 has been identified as a potential regulator of CD40. However, it remains unclear whether TRAF5 regulates the generation of germinal center (GC) B cells and antigen-specific antibody production in the T-dependent (TD) immune response. TRAF5-deficient (Traf5-/-) and TRAF5-sufficient (Traf5+/+) mice were immunized in the footpad with 2,4,6-trinitrophenol-conjugated keyhole limpet hemocyanin (TNP-KLH) and complete Freund's adjuvant (CFA). We found that GC B cell generation and antigen-specific IgM and IgG1 production were significantly impaired in Traf5-/- mice compared to Traf5+/+ mice. The expression levels of CD40-target genes Fas and Lta, which are involved in GC formation, were significantly decreased in B220+ cells isolated from immunized Traf5-/- mice. Traf5-/- B cells showed decreased antibody production, proliferation, and induction of CD40-target genes Tnfaip3, Tnfsf4, and Cd80 in response to agonistic Fc-CD40L protein in vitro. Furthermore, administration of TNP-KLH and Fc-CD40L to Traf5-/- mice resulted in a severe loss of GC B cell development. These results highlight the crucial role of TRAF5 in driving CD40-mediated TD immune response in vivo.

Nuclear receptor subfamily 4 group a member 1 eases angiotensin II-arose oxidative stress in vascular smooth muscle cell by boosting nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 transcription

Objective: Hypertension significantly contributes to morbidity and mortality. Nuclear receptor subfamily 4 group a member 1 (Nur77) participates in regulating oxidative stress, but the mechanism in hypertension remains unclear. This study aimed to explore the function of Nur77 in oxidative stress induced by Angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) in hypertension. Material and Methods: First, models of VSMC with Nur77, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 (NLRC3) and tumor necrosis factor receptor-associated factor 6 (TRAF6) knockdown or overexpression were constructed using Short Hairpin RNA (Nur77) or pcDNA3.1 vector, respectively. Next, the putative-binding motifs between Nur77 and NLRC3 promoters were detected by dual luciferase assay. We conducted reverse transcription quantitative polymerase chain reaction (qPCR) and Western blot (WB) analysis to detect Nur77, NLRC3, and TRAF6 levels in VSMCs. Then, cell counting kit-8 assay, 5-ethynyl-2’-deoxyuridine assay, wound-healing assay, enzyme-linked immunosorbent assay, and 2’,7’-dichlorofluorescin diacetate were employed to examine the impact of the knockdown or overexpression of Nur77, NLRC3, and TRAF6 on VSMCs treated with Ang II. The assays measured cell viability and proliferation, cell migration, malondialdehyde levels, and reactive oxygen species levels. Results: The overexpression of Nur77 repressed cell growth (P &lt; 0.001), migration (P &lt; 0.01), and oxidative stress (P &lt; 0.01) induced by Ang II in VSMCs. Nur77 transcriptionally promoted the expression of NLRC3 (P &lt; 0.001), and the upregulation of NLRC3 suppressed cell proliferation (P &lt; 0.05) and oxidative stress (P &lt; 0.001) mediated by Ang II. Furthermore, NLRC3 negatively regulated the TRAF6/nuclear factor-kappa B (NF-κB) axis activated by Ang II, which resulted in the repression of hyperproliferation of VSMCs (P &lt; 0.01) and oxidative stress (P &lt; 0.001). Conclusion: Nur77 suppressed growth and oxidative stress induced by Ang II in VSMCs by promoting NLRC3 transcription, which, further, repressed the TRAF6/NF-κB axis. This understanding provides novel insights into the pathogenesis of hypertension.

MicroRNA expression profiling in the adult offspring of rats with periodontal disease

ObjectiveThe present study investigated the relationship between maternal periodontal disease, insulin resistance, activation of inflammatory pathways and epigenetic modifications in adult offspring. DesignTherefore, female Wistar rats were divided into control and experimental groups. Seven days after the induction of periodontal disease, female rats from both groups were mated with healthy male rats. After weaning, male offspring were divided into control offspring (CN-o) and periodontal disease offspring (PED-o) groups. Body weight was measured at 0–75 days of age. At day 75, the following were measured in the offspring: insulin resistance by the HOMA-IR index; global miRNAs by microtranscriptome array; validation of the selected miRNAs by quantitative real-time PCR expression; interleukin 1 receptor associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) content in the gastrocnemius muscle tissue (GSM) by western blotting. ResultsMaternal periodontal disease leads to low birth weight (LBW) in the offspring and insulin resistance in adulthood; changes in global miRNA expression (5 miRNAs upregulated and 6 downregulated); and increased protein expression of IRAK1 and TRAF6 in GSM. ConclusionsThese findings demonstrate that maternal periodontal disease causes LBW, insulin resistance, activation of inflammatory pathways, and changes in global miRNA expression.

LncRNA DANCR suppresses acute myocardial infarction in mice via mediating p-RXRA/TRAF2/NIK/IKK/NF-κB signaling pathway.

This study aimed to investigate the role of LncRNA differentiation antagonizing non-protein coding RNA (DANCR) in acute myocardial infarction (AMI). A mouse model of AMI was established, and the cardiac contractile function was detected. Next, cardiomyocytes treated with oxygen-glucose deprivation (OGD) were used for gain- and loss-function experiments in vitro. RIP analysis was used to verify the binding of DANCR and Retinoid X receptor alpha (RXRA), and Co-IP assay was used to measure the binding of phosphorylated RXRA to TNF receptor associated factor 2 (TRAF2). The expression of DANCR in myocardial tissues of AMI mice were significantly downregulated. Overexpression of DANCR decreased myocardial infarct size and enhanced cardiac contractile function in AMI mice. Moreover, overexpression of DANCR promoted proliferation and inhibited apoptosis in OGD-induced cardiomyocytes. Mechanism studies demonstrated that DANCR interacted with RXRA and promoted glycogen synthase kinase 3beta (GSK3β)-mediated phosphorylation of RXRA, and phosphorylated RXRA interacted with TRAF2 protein to downregulate TRAF2 protein level. Bexarotene (Bex), an activator of RXRA, inhibited TRAF2 protein expression, while RXRA overexpression had no effect on TRAF2 protein expression. Bex treatment or silencing TRAF2 promoted proliferation and inhibited apoptosis in cardiomyocytes. In addition, silencing DANCR inhibited cardiomyocyte proliferation and induced apoptosis by activating the NIK/IKK/NF-κB pathway, while B022, an inhibitor of NIK, counteracted the effects of DANCR silencing on cardiomyocytes. Studies demonstrated that DANCR suppressed AMI in mice by mediating p-RXRA/TRAF2/NIK/IKK/NF-κB pathway.

Embryonic Lethal Abnormal Visual-Like Protein 1 Aggravates Caerulein-Induced AR42J Cell Injury and Macrophage M1 Polarization to Accelerate Acute Pancreatitis by Upregulating TRAF6.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been found to promote the progression of acute pancreatitis (AP). However, its underlying molecular mechanisms in AP need to be further revealed. Caerulein-induced AR42J cells were used to construct AP cell models. Cell viability and apoptosis were measured by Cell Counting Kit 8 assay and flow cytometry. Levels of inflammatory factors and oxidative stress-related markers were assessed. The medium of AR42J cells was collected for coculturing RAW264.7 cells. Macrophage marker CD86+ cell rates were checked with flow cytometry. The levels of TRAF6, embryonic lethal abnormal visual-like protein 1 (ELAVL1), and inducible nitric oxide synthase (iNOS) were examined by Western blot or quantitative real-time polymerase chain reaction. RNA immunoprecipitation assay was performed to evaluate the interaction between ELAVL1 and TRAF6. TRAF6 mRNA stability was tested using actinomycin D treatment. Caerulein treatment suppressed viability, induced AR42J cell apoptosis, inflammation, oxidative stress, and accelerated macrophage M1 polarization. TRAF6 downregulation could alleviate caerulein-induced AR42J cell injury and macrophage M1 polarization. ELAVL1 interacted with TRAF6 to stabilize its expression. Meanwhile, ELAVL1 knockdown relieved caerulein-induced AR42J cell injury and macrophage M1 polarization, while these effects were abolished by TRAF6 overexpression. TRAF6, stabilized by ELAVL1, promoted caerulein-induced AR42J cell injury and macrophage M1 polarization, suggesting that it might accelerate AP9 progression.

CSIG-01. DECREASING EXPRESSION OF TRAF PROTEINS WITH INCREASING MALIGNANCY WITH LOWEST EXPRESSION IN IDH-WILDTYP GLIOBLASTOMA

Abstract The tumor necrosis factor receptor-associated factors (TRAF) are cytoplasmatic adapter proteins that can interact directly with the intracellular domains of cell surface receptors, such as the TNF receptor superfamily. TRAFs are thus involved in cellular processes like proliferation or apoptosis. Seven members are known; all expressed in the cytosol of various cell and tissue types. Increased levels of TRAF4 in GBM tissue are related to cell proliferation and migration. About 25 % of grade I meningioma show mutations in TRAF7, which are associated with a lower risk of malignant transformation. Therefore expression patterns of all seven TRAF members are evaluated in different glioma grades. Following tumor samples were obtained during neurosurgery and shock frozen in liquid nitrogen: peritumoral tissue, astrocytoma and oligodendroglioma (G2, G3) and glioblastoma (primary and recurrent). Transcription rate of TRAF1-7 was measured via qPCR. Correlation of TRAF expression level and patient overall survival was evaluated using TCGA datasets of the TCGA-LGG” and “TCGA-GBM” projects. With the exception of TRAF5, a decreased mRNA level with increasing malignancy could be found for all TRAFs (TRAF1 p=n.s.; TRAF2 p=0.0026; TRAF3 p&amp;lt;0.0001; TRAF4 p=0.0021; TRAF6 p&amp;lt;0.0001; TRAF7 p&amp;lt;0.0001). IDHwt glioblastoma showed significant lower expression level compared to IDHmut glioblstoma in all TRAFs except for TRAF1 (TRAF2 p&amp;lt;0.0001; TRAF3 p=0.0346; TRAF4 p&amp;lt;0.0001; TRAF6 p=0.0447; TRAF7 p=0.0081). TRAF5 again showed a reverse effect (p=0.0065). Correlation of TRAF expression level in low grade glioma and patient survival revealed longer overall survival with low TRAF1,2,4 and TRAF5 expression (p&amp;lt;0.0001, p= 0.0178, p=0.0307, p&amp;lt;0.0001). No difference in survival rate in high grade glioma could be found.

Petunidin attenuates vinclozolin instigated testicular toxicity in albino rats via regulating TLR4/MyD88/TRAF6 and Nrf-2/Keap-1 pathway: A pharmacodynamic and molecular simulation approach

Vinclozolin (VZN) is a widely used fungicide which exerts deleterious impacts on various organs including testis. Petunidin (PDN) is a polyphenolic compound that demonstrates a broad range of pharmacological activities. Thirty-two rats were divided into 4 groups including the control, VZN (100 mg/kg), VZN (100 mg/kg) + PDN (4 mg/kg) and PDN (4 mg/kg) treated group. The activities of antioxidant enzymes were assessed by using previously documented protocols. The gene expressions were determined by using qRT-PCR. The levels of hepatic function and apoptotic markers were evaluated by using standard ELISA technique. The histological analysis was carried out as per the standard protocol of histology. It was revealed that VZN disrupted the Nrf-2/Keap-1 pathway. Moreover, the activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), heme-oxygenase-1 (HO-1) and glutathione reductase (GSR) were reduced whereas levels of reactive oxygen species (ROS) & malondialdehyde (MDA) were promoted following the VZN intoxication. Furthermore, VZN intoxication reduced total sperm count, viability, motility as well as luteinizing hormone (LH), follicle stimulating hormone (FSH), and plasma testosterone. Besides, administration of VZN decreased the expressions of 3β-Hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR) and 17β-Hydroxysteroid dehydrogenase (17β-HSD). Moreover, VZN exposure escalated the expressions of Bcl-2–associated X protein (Bax) and cysteine–aspartic acid protease-3 (Caspase-3) while reducing the expressions of B-cell lymphoma-2 (Bcl-2). Additionally, VZN administration increased the gene expression of toll-like receptor 4 (TLR4), tumor necrosis factor receptor-associated factor 6 (TRAF-6) and myeloid differentiation primary response 88 (MyD88). The levels of interleukin-6 (IL-6), nuclear factor kappa-B (NF-κB), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and the activity of cyclooxygenase-2 (COX-2) were promoted following the VZN administration. Furthermore, VZN intoxication disrupted the normal histology of testicular tissues. However, VZN + PDN treatment ameliorated testicular damage via regulating aforementioned dysregulations owing to its anti-inflammatory, antioxidative as well as anti-apoptotic potentials. Lastly, molecular docking (MD) was performed to assess the effectiveness of PDN as a curative compound by analyzing its binding affinity with the targeted proteins (Keap1, TLR4 and StAR). Our in-silico evaluations confirmed that PDN possesses the potential to interact with binding pockets of these proteins, emphasizing its capability as a curative compound to mitigate VZN-prompted reproductive damage.

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.

Zhuangyao Jianshen Wan ameliorates senile osteoporosis in SAMP6 mice through Modulation of the GCN5L1-mediated PI3K/Akt/wnt signaling pathway

BackgroundSenile osteoporosis (SOP) is a systemic bone disease characterized by increased susceptibility to fractures. However, there is currently no effective treatment for SOP. The Zhuangyao Jianshen Wan (ZYJSW) pill is traditionally believed to possess kidney-nourishing and bone-strengthening effects, demonstrating efficacy in treating fractures. Despite this, its effectiveness and mechanism in SOP remain unclear. This study aims to investigate the therapeutic potential of ZYJSW in treating SOP in senescence accelerated mouse prone 6 (SAMP6, P6) mice, and elucidate the underlying mechanisms. MethodsFour-month-old SAMP6 mice were categorized into six groups: the model group (SAMP6), low, medium, and high-dose ZYJSW treatment groups, calcitriol treatment (positive control 1) group, and metformin treatment (positive control 2) group. Gastric administration was carried out for 15 weeks, and a normal control group comprising four-month-old Senescence-Accelerated Mouse Resistant 1 (SAMR1) mice. Changes in body weight, liver and kidney function, bone protective effects, and muscle quality were evaluated using various assays, including H&E staining, Goldner staining, bone tissue morphology analysis, Micro-CT imaging, and biomechanical testing. Qualitative analysis and quality control of ZYJSW were performed via LC-MS/MS analysis. To explore mechanisms, network pharmacology and proteomics were employed, and the identified proteins were validated by Western blotting. ResultsOral administration of ZYJSW to P6 mice exerted preventive efficacy against osteopenia, impaired bone microstructure, and poor bone and muscle quality. ZYJSW attenuated the imbalance in bone metabolism by promoting bone formation, as evidenced by the upregulation of key factors such as Runt-related transcription factor 2 (RUNX2), Bone Morphogenetic Protein (BMP2), Osteoprotegerin (OPG) and Osteocalcin (OCN), while simultaneously inhibiting bone resorption through the downregulation of TNF receptor associated factor 6 (TRAF6), Tartrate resistant acid phosphatase (TRAP), Receptor activator for nuclear factor-κB ligand (RANKL) and Cathepsin K (CTSK). Additionally, ZYJSW enhanced muscle structure and function by counteracting the elevation of Ubiquitin (Ub), Muscle RING-finger protein-1 (Murf-1), F-Box Protein 32 (FBOX32), and Myogenin (Myog). Network pharmacology predictions, proteomics analysis corroborated by published literature demonstrated the role of ZYJSW involving in safeguarding mitochondrial biogenesis. This was achieved by suppressing GCN5L1 expression, contributing to the heightened expression of TFAM, PGC-1α, and nuclear respiratory factor-1 (NRF-1) proteins. ZYJSW also positively modulated Wnt signaling pathways responsible for bone formation, due to regulating expressions of key components like β-catenin, GSK-3β, and LRP5. In addition, ZYJSW causes the downregulation of the PI3K/Akt pathway by inhibiting the phosphorylation of both PI3K and Akt. ConclusionsThe study highlights the significance of ZYJSW in preserving the health of both bone and muscle in P6 mice, potentially through the regulation of the GCN5L1-mediated PI3K/Akt/Wnt signaling pathway. The translational potential of this articleOur research provides evidence and a mechanistic rationale for ZYJSW as a candidate for SOP treatment, offering insights for further exploration and strategy development.

Adaptor protein 3BP2 regulates gene expression in addition to the ubiquitination and proteolytic activity of MALT1 in dectin-1-stimulated cells.

Dectin-1, a C-type lectin, plays important roles in the induction of antifungal immunity. Caspase recruitment domain-containing protein 9 (CARD9) is essential for the dectin-1-induced production of cytokines through the activation of NF-κB. However, the molecular mechanisms underlying the dectin-1-mediated activation of CARD9 have not been fully elucidated. Recently, we reported that the adaptor protein SH3 domain-binding protein 2 (3BP2) is required for the dectin-1-induced production of cytokines and activation of NF-κB, although the relationship between 3BP2 and CARD9 in dectin-1-mediated signaling remains unclear. Here, we report that 3BP2 is required for dectin-1-induced expression of several genes that may contribute to antifungal immunity in bone marrow-derived dendritic cells (BMDCs). The results of reporter assays using HEK-293T cells indicate that 3BP2 induces CARD9-mediated activation of NF-κB through B-cell leukemia/lymphoma 10, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and TNF receptor-associated factor 6-dependent mechanisms. In addition, we show that 3BP2 induces CARD9-mediated ubiquitination of cellular proteins and that MALT1 cleaves 3BP2 in a CARD9-dependent manner. Furthermore, we show that 3BP2 is required for the ubiquitination, in addition to the activation, of MALT1, which leads to MALT1-depenedent cleavage of 3BP2 in dectin-1-stimulated BMDCs. Finally, we identified hematopoietic cell-specific Lyn substrate 1 as a target of 3BP2, which is essential for dectin-1-induced expression of interleukin 10 in BMDCs. These results indicate that 3BP2 regulates gene expression and functions of MALT1 in dectin-1-stimulated cells and that 3BP2 plays an important role in the dectin-1-mediated antifungal immunity.

Alleviating effects of Gracilaria verrucosa supplement on non-specific immunity, antioxidant capacity and immune-related genes of pacific white shrimp (Litopenaeus vannamei) provoked with white spot syndrome virus

Our work evaluated the possible underlying roles of dietary dried seaweed (Gracilaria verrucosa; GV) on the inherent immune response, antioxidant capacity, immune-related gene expression, and protection of whiteleg shrimp (Litopenaeus vannamei) contra white spot syndrome virus (WSSV). Three hundred and sixty healthy L. vannamei (15.26 g ± 1.29 g) were graded into four supplemental groups ( Triplicate/group) and fed with diets including 0 (control), 2, 4, and 8 g GV (kg diet) −1 for 21 days. Following the feeding period, each group of shrimp received an intramuscular WSSV injection (1.4 × 106 copies/ml). Hemolymph and gills samples were collected before and after the challenge with WSSV. Notably, the administration of dietary GV significantly enhanced the innate immune parameters of pacific white shrimp including total hemocyte count (THC), phagocytosis, phenoloxidase activity, reactive oxygen species (ROS) production, and lysozyme activity before and after challenge with WSSV. Additionally, dietary supplementation of 4, and 8 g of GV (kg diet)−1 remarkably elevated ACP, AKP, SOD, GPx, and catalase activities along with a decrease in the MDA level in gills of shrimp before and post-WSSV challenge. In response to the GV supplement, significant upregulation of expression of ALF1, CRU1, PEN4, and CTL with downregulation of TRAF6, STAT, TLR1, and NOS genes was recorded in the gills tissue before and post-challenge with WSSV, especially at a dose of 8.0 GV g kg − 1. Dietary inoculated shrimp with GV revealed notably higher survival percentages after being challenged with WSSV. Conclusively, these data indicate that Gracilaria verrucosa can be recommended as a valuable supplemented seaweed to stimulate the innate immunity and enhance the health of Litopenaeus vannamei against viral infection.

TRAF4 promotes lung cancer development by activating tyrosine kinase of EGFR

Objective: To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion. Methods: Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay. Results: The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin (r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4-/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size (P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4-/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4-/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions: TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

Astragaloside promotes the secretion of MSC-derived exosomal miR-146a-5p by regulating TRAF6/NF-κB pathway to attenuate inflammation in high glucose-impaired endothelial cells.

This study aimed to explore the potential of using mesenchymal stem cell (MSC)-derived exosomes (MSC-Exos) pre-treated with Astragaloside IV (ASIV) to alleviate inflammation in high glucose (HG)-damaged endothelial cells. MSC-Exos were isolated from untreated MSCs and ASIV-pre-treated MSCs, and their characteristics were assessed. The expression of miR-146a-5p in MSC-Exos was determined, and it was found that ASIV treatment enhanced its expression. In order to assess the impact of highly miR-146a-5p-expressing MSC-Exos on HG-injured endothelial cells, we established a model of HG-induced inflammation using human umbilical vein endothelial cells (HUVECs). The study measured cell viability, apoptosis, tube formation, and levels of inflammatory cytokines among the different treatment groups. It was found that transferring MSC-Exos with high miR-146a-5p expression to HG-damaged HUVECs increased cell viability and tube formation ability while reducing the number of apoptotic cells. Additionally, changes in inflammatory factors indicated a reduction in the inflammatory response. Further investigation demonstrated that miR-146a-5p inhibited the expression of TNF receptor associated factor 6 (TRAF6) and phosphorylated NF-κB, which are involved in the inflammatory response. This resulted in the alleviation of inflammation in HG-damaged endothelial cells. In summary, our findings indicate that ASIV treatment stimulated the secretion of MSC-Exos that exhibited increased levels of miR-146a-5p. These exosomes, in turn, regulated the TRAF6/NF-κB pathway. As a result of this modulation, the inflammatory response in HG-damaged endothelial cells was alleviated. These findings offer a fresh approach to addressing vascular complications associated with diabetes, which could lead to novel treatment strategies in the field.

USP33 promotes pulmonary microvascular endothelial cell pyroptosis by stabilizing TRAF2 through deubiquitination.

Inhibiting the pyroptosis of human pulmonary microvascular endothelial cells (HPMECs) is a promising therapeutic modality for acute lung injury (ALI). Given the undefined effect of ubiquitin-specific protease 33 (USP33) and tumor necrosis factor receptor-associated factor 2 (TRAF2) on pyroptosis in lung injury, this study investigates their roles in the pyroptosis of HPMECs during ALI. The hypoxia/reoxygenation (H/R)-induced model was constructed in HPMECs. Cell viability, cytotoxicity, and cell death were determined by the cell counting kit-8 (CCK-8), Lactate dehydrogenase (LDH), and Hoechst-PI staining, respectively. Western blot and qRT-PCR were used to detect protein and gene expression levels of pyroptosis-related markers, respectively. The TRAF2 ubiquitination level was measured via immunoprecipitation. USP33 and TRAF2 expressions were elevated in H/R-induced HPMECs. Knockdown of USP33 increased cell viability and inhibited cellular pyroptosis, accompanied by decreases in IL-1β, IL-18, and Caspase-1. USP33 stabilized TRAF2 by deubiquitination. TRAF2 overexpression reversed the effect of USP33 silencing on suppressing HPMEC pyroptosis. USP33 stabilizes TRAF2 by deubiquitination to promote HPMEC pyroptosis during ALI.