LXA4 protected mice from renal ischemia/reperfusion injury by promoting IRG1/Nrf2 and IRAK-M-TRAF6 signal pathways.

Excessive inflammatory response and increased oxidative stress play an essential role in the pathophysiology of ischemia/reperfusion (I/R)-induced acute kidney injury (IRI-AKI). Emerging evidence suggests that lipoxin A4 (LXA4), as an endogenous negative regulator in inflammation, can ameliorate several I/R injuries. However, the mechanisms and effects of LXA4 on IRI-AKI remain unknown. In this study, A bilateral renal I/R mouse model was used to evaluate the role of LXA4 in wild-type, IRG1 knockout, and IRAK-M knockout mice. Our results showed that LXA4, as well as 5-LOX and ALXR, were quickly induced, and subsequently decreased by renal I/R. LXA4 pretreatment improved renal I/R-induced renal function impairment and renal damage and inhibited inflammatory responses and oxidative stresses in mice kidneys. Notably, LXA4 inhibited I/R-induced the activation of TLR4 signal pathway including decreased phosphorylation of TAK1, p36, and p65, but did not affect TLR4 and p-IRAK-1. The analysis of transcriptomic sequencing data and immunoblotting suggested that innate immune signal molecules interleukin-1 receptor-associated kinase-M (IRAK-M) and immunoresponsive gene 1 (IRG1) might be the key targets of LXA4. Further, the knockout of IRG1 or IRAK-M abolished the beneficial effects of LXA4 on IRI-AKI. In addition, IRG1 deficiency reversed the up-regulation of IRAK-M by LXA4, while IRAK-M knockout had no impact on the IRG1 expression, indicating that IRAK-M is a downstream molecule of IRG1. Mechanistically, we found that LXA4-promoted IRG1-itaconate not only enhanced Nrf2 activation and increased HO-1 and NQO1, but also upregulated IRAK-M, which interacted with TRAF6 by competing with IRAK-1, resulting in deactivation of TLR4 downstream signal in IRI-AKI. These data suggested that LXA4 protected against IRI-AKI via promoting IRG1/Itaconate-Nrf2 and IRAK-M-TRAF6 signaling pathways, providing the rationale for a novel strategy for preventing and treating IRI-AKI.

MiR-146b-5p enriched bioinspired exosomes derived from fucoidan-directed induction mesenchymal stem cells protect chondrocytes in osteoarthritis by targeting TRAF6.

Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive cartilage degradation and inflammation. In recent years, mesenchymal stem cells (MSCs) derived exosomes (MSCs-Exo) have attracted widespread attention for their potential role in modulating OA pathology. However, the unpredictable therapeutic effects of exosomes have been a significant barrier to their extensive clinical application. In this study, we investigated whether fucoidan-pretreated MSC-derived exosomes (F-MSCs-Exo) could better protect chondrocytes in osteoarthritic joints and elucidate its underlying mechanisms. In order to evaluate the role of F-MSCs-Exo in osteoarthritis, both in vitro and in vivo studies were conducted. MiRNA sequencing was employed to analyze MSCs-Exo and F-MSCs-Exo, enabling the identification of differentially expressed genes and the exploration of the underlying mechanisms behind the protective effects of F-MSCs-Exo in osteoarthritis. Compared to MSCs-Exo, F-MSCs-Exo demonstrated superior effectiveness in inhibiting inflammatory responses and extracellular matrix degradation in rat chondrocytes. Moreover, F-MSCs-Exo exhibited enhanced activation of autophagy in chondrocytes. MiRNA sequencing of both MSCs-Exo and F-MSCs-Exo revealed that miR-146b-5p emerged as a promising candidate mediator for the chondroprotective function of F-MSCs-Exo, with TRAF6 identified as its downstream target. In conclusion, our research results demonstrate that miR-146b-5p encapsulated in F-MSCs-Exo effectively inhibits TRAF6 activation, thereby suppressing inflammatory responses and extracellular matrix degradation, while promoting chondrocyte autophagy for the protection of osteoarthritic cartilage cells. Consequently, the development of a therapeutic approach combining fucoidan with MSC-derived exosomes provides a promising strategy for the clinical treatment of osteoarthritis.

Bone Marrow Mesenchymal Stem Cell Exosomal miR-345-3p Ameliorates Cerebral Ischemia-reperfusion Injury by Targeting TRAF6.

INTRODUCTION: The purpose of this study was to investigate the effects of bone marrow mesenchymal stem cells (BMSCs) exosomal miR-345-3p and tumor necrosis factor receptorassociated factor 6 (TRAF6) on cerebral ischemia reperfusion (CIR) injury. Exosomes (Exos) derived from BMSCs were isolated and identified. PC12 (rat pheochromocytoma) cells were used to establish an oxygen and glucose deprivation/reoxygenation (OGD/R) model. METHODS: Cell counting kit-8, TUNEL staining, lactate dehydrogenase staining, RT-qPCR, and western blotting were utilized for analyzing the functions of miR-345-3p about PC12 cells. Dualluciferase reporter experiment was then to confirm the link between miR-345-3p and TRAF6. Finally, using male SD rats, the middle cerebral artery occlusion (MCAO) model was constructed. Regulation of I/R damage in MCAO rats of miR-345-3p and TRAF6 were further explored in the changes of modified neurological severity score, cerebral infarction pictures, relative infarct volume, and histopathological changes. After OGD/R treatment, neuronal apoptosis was dramatically increased. After treatment with exosomal miR-345-3p, OGD/R-induced neuroapoptosis was dramatically inhibited. Exosomal miR-345-3p inhibited OGD/R-induced neuroapoptosis by downregulating the expression of TRAF6. However, the miR-345-3p inhibitor aggravated the changes caused by OGD/R. RESULTS: The corresponding regulations of miR-345-3p were reversed with TRAF6 overexpression. The animal experiments in vivo further verified that miR-345-3p ameliorated brain I/R injury in MCAO rats by targeting TRAF6. CONCLUSION: This study found that BMSCs-exosomal miR-345-3p protected against CIR injury by decreasing TRAF6.

[Effect of circRASA2 targeting miR-543/TRAF6 axis on LPS-induced periodontal ligament cell proliferation and osteogenic differentiation].

PURPOSE: To investigate the possible role of circRASA2 in periodontitis and its potential regulatory mechanism. METHODS: Periodontitis cell model was established by lipopolysaccharide(LPS)-induced periodontal ligament cells(PDLCs). Cell proliferation activity was detected by CCK-8 assay, cell migration ability was detected by Transwell chamber assay, and the expression of osteogenic differentiation-related proteins in cells was detected by Western blot. The target miRNA of circRASA2 and its downstream target genes were predicted using the databases circinteractome and starBase, respectively, and the targeting relationship between the target genes was verified by dual-luciferase reporter gene experiment. GraphPad Prism 8.0 software package was used to analyze the data. RESULTS: circRASA2 was highly expressed in LPS-treated PDLCs cells. LPS-induced PDLCs cell proliferation activity, migration ability and osteogenic differentiation ability decreased, while knockdown of circRASA2 promoted proliferation, migration and osteogenic differentiation ability of PDLCs under LPS treatment. circRASA2 targeted and negatively regulated the expression of miR-543, and overexpression of miR-543 promoted proliferation, migration and osteogenic differentiation of PDLCs under LPS treatment. TRAF6 was a downstream target gene of miR-543, knockdown of circRASA2 down-regulated the expression of TRAF6 through the sponge action of miR-543. Overexpression of TRAF6 reversed the promotion of circRASA2 knockdown on proliferation, migration and osteogenic differentiation of PDLCs. CONCLUSIONS: circRASA2 accelerated the pathological process of periodontitis in vitro through miR-543/TRAF6 axis, and might improve periodontitis by targeting down the expression of circRASA2.

The Natural Alkaloid (-)-N-Hydroxyapiosporamide Suppresses Colorectal Tumor Progression as an NF-κB Pathway Inhibitor by Targeting the TAK1-TRAF6 Complex.

Colorectal cancer (CRC) is an exceptionally deadly disease, whereas effective therapeutic drugs for CRC have declined over the past few decades. Natural products have become a reliable source of anticancer drugs. Previously we isolated an alkaloid named (-)-N-hydroxyapiosporamide (NHAP), which exerts potent antitumor effects, but its effect and mechanism in CRC remain unclear. This study aimed to reveal the antitumor target of NHAP and identify NHAP as a promising lead compound for CRC. Various biochemical methods and animal models were used to investigate the antitumor effect and molecular mechanism for NHAP. These results showed that NHAP exhibited potent cytotoxicity, induced both apoptosis and autophagic cell death of CRC cells, and inhibited the NF-κB signaling pathway by blocking the interaction of the TAK1-TRAF6 complex. NHAP also markedly inhibited CRC tumor growth in vivo without obvious toxicities and possessed good pharmacokinetic characteristics. These findings identify, for the first time, that NHAP is an NF-κB inhibitor with potent antitumor activity in vitro and in vivo. This study clarifies the antitumor target of NHAP against CRC, which will contribute to the future development of NHAP as a novel therapeutic lead compound for CRC.

Resveratrol suppresses lipopolysaccharide-mediated activation of osteoclast precursor RAW 264.7 cells by increasing miR-181a-5p expression.

Resveratrol (Res) has anti-inflammation and antiosteoporosis functions. We evaluated the effect of Res on osteoclast differentiation by releasing inflammatory cytokines from osteoclast precursor RAW 264.7 cells stimulated by lipopolysaccharide (LPS). In the study, LPS (1 ng/L) was used to induce the Raw 264.7 inflammatory injury model in vitro. A total of 25 ng/mL M-CSF + 30 ng/mL RANKL or plus 1 µg/L LPS was used to induce osteoclastogenesis in the experiments. We utilized the Cell Counting Kit-8 assay to measure the relative cell survival of RAW 264.7 cells. Then, enzyme-linked immunosorbent assays were utilized to measure the abundance of inflammatory markers, such as interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and IL-6. Subsequently, Western blot analysis was applied to assess the abundance of phosphorylated transforming growth factor beta-activated kinase 1 (P-TAK1) protein, TNF receptor-associated factor 6 (TRAF6), nuclear factor-κB inhibitor protein (IκB), phosphorylated IκB-α (P-IκB-α), and nuclear factor κB65 (NF-κB65). mRNA expression levels of miR-181a-5p, TRAF6, specific gene calcitonin receptor (CTR), activated T nuclear factor 1 (NFATC1), cathepsin K (CTSK), and matrix metalloproteinase (MMP)-9 were determined via a real-time polymerase chain reaction. Osteoclast bone resorption function was determined. Finally, tartrate-resistant acid phosphatase (TRAP) staining was performed.The results found that Compared with the model group, the degrees of expressions of supernatant inflammatory factors TNF-α, IL-1ß, and IL-6 were substantially attenuated in the Res treatment group (p < 0.05). Furthermore, the extent of miR-181a-5p expression in the RAW 264.7 cells significantly increased, whereas P-IκB-α, P-TAK1, NF-κB65, and TRAF6 expressions significantly decreased in the Res treatment group as opposed to the model group (p < 0.05). The CTR, NFATC1, MMP-9, CTSK, and TRAP mRNA expression levels were substantially reduced during osteoclast differentiation and bone resorption in the Res treatment group.The results suggest that Res can reduce the RAW 264.7 cell differentiation into osteoclasts and relieve LPS-stimulated osteoporosis, and the underlying mechanism may be associated with the Res-inhibited activity of the TRAF6/TAK1 pathway through the increased miR-181a-5p expression.

Effects of Co-exposure to Fluoride and Arsenic on TRAF-6 Signaling and NF-κB Pathway of Bone Metabolism.

Little is known about the combined effect of fluoride (F) and arsenic (As) on bone metabolism. This study aims to explore the effect of co-exposure to F and As on the expressions of TNF receptor-associated factor 6 (TRAF-6), nuclear factor-kappa B (NF-κB), and the related factors in cell and animal experiments. With the rats exposed to different doses of F, As, and combined F-As, we found that F exposure doses were positively correlated with the protein expression of receptor activator of nuclear factor-kappa B ligand (RANKL), receptor activator of nuclear factor-kappa B (RANK), TRAF-6, NF-κB, and nuclear factor of activated T cells (NFAT-c1) (P < 0.001). As exposure doses were negatively correlated with RANK, TRAF-6, NF-κB, and NFAT-c1 (P < 0.001). The effect of F and As interaction on the protein expression of RANKL, TRAF-6, NF-κB, and NFAT-c1 was significant in bone tissue (P < 0.05). In the cellular experiment, F could promote the mRNA expression of RANK, TRAF-6, and NFAT-c1. A higher concentration of As could inhibit the mRNA expression of Tartrate-resistant acid phosphatase (TRAP), RANK, TRAF-6, and NFAT-c1. The effect of F and As interaction on the mRNA expression of TRAP, RANK, TRAF-6, and NFATc1 in osteoclasts was significant (P < 0.001). In conclusion, the expression of TRAF-6 and NF-κB pathway was affected by F and As co-exposure in osteogenic differentiation, and As could antagonize the promoting effect of F on the expression of TRAF-6, TRAP, RANKL, RANK, NF-κB, and NFAT-c1 in these exposure levels. These results could provide a scientific basis for understanding the interaction of F and As in bone formation.

MCPIP1 Suppresses the NF-κB Signaling Pathway Through Negative Regulation of K63-Linked Ubiquitylation of TRAF6 in Colorectal Cancer.

The abnormal activation of the nuclear factor-kappa B (NF-κB) signaling pathway is an important precipitating factor for the inception and development of colorectal cancer (CRC), one of the most common tumors worldwide. As a pro-apoptotic transcription factor, monocyte chemotactic protein-induced protein 1 (MCPIP1) has been closely associated with many tumor types. In the present study, the expression of MCPIP1 was firstly discovered reduced in CRC tissues and correlated with poor patient prognosis. The decreased expression was caused by promoter hypermethylation. Overexpressed MCPIP1 was found to inhibit the proliferative and migratory abilities of CRC cells, whereas knockdown of MCPIP1 produced the opposite result. The subsequent investigation demonstrated that MCPIP1 exerted its "anti-cancer" effect by suppression of the NF-κB signaling pathway through negative regulation of K63-linked ubiquitylation of TNF receptor associated factor 6 (TRAF6). Therefore, our results indicate a prognostic marker for CRC and a theoretical basis for MCPIP1 as a treatment.

Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response in Fusarium solani-Induced Keratitis via the NF-κB Signaling Pathway.

Purpose: The purpose of this study was to elucidate the effect of methyltransferase-like enzyme 3 (METTL3) on inflammation and the NF-κB signaling pathway in fungal keratitis (FK). Methods: We established corneal stromal cell models and FK mouse models by incubation with Fusarium solani. The overall RNA N6-methyladenosine (m6A) level was determined using an m6A RNA methylation assay kit. The expression of METTL3 was quantified via real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Subsequently, the level of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) was identified by Western blotting and immunofluorescence. Moreover, we assessed the effect of METTL3 by transfecting cells with siRNA (in vitro) or adeno-associated virus (in vivo). Hematoxylin and eosin (H&E) staining and slit-lamp biomicroscopy were performed to evaluate corneal damage. Furthermore, the state of NF-κB signaling pathway activation was examined by Western blotting. In addition, RT-PCR and enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate levels of the pro-inflammatory factors interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and TNF-ɑ. Results: Our data demonstrated that the levels of the RNA m6A methylation and METTL3 were dramatically increased and that the NF-κB signaling pathway was activated in Fusarium solani-induced keratitis. Inhibition of METTL3 decreased the level of TRAF6, downregulated the phospho-p65(p-p65)/p65 and phospho-IκB(p-IκB)/IκB protein ratios, simultaneously attenuating the inflammatory response and fungal burden in FK. Conclusions: Our research suggests that the m6A methyltransferase METTL3 regulates the inflammatory response in FK by modulating the NF-κB signaling pathway.

Environmental concentrations of 2, 4-DTBP cause immunotoxicity in zebrafish (Danio rerio) and may elicit ecological risk to wildlife.

Synthetic phenolic antioxidant 2,4-di-tert-butylphenol (2,4-DTBP) has gained growing concerns due to relatively high concentrations in aquatic ecosystems. There are, however, significant knowledge gaps regarding its potential toxicity to aquatic organisms. In this study, zebrafish (Danio rerio) larvae were exposed to 0.01, 0.1, or 1 µM 2,4-DTBP for 6 d. Transcriptomic analysis of larvae revealed that biological processes related to anti-inflammatory function of macrophage M2 lineage were inhibited by 0.01 µM 2,4-DTBP. Decreases of transcripts related to the IL1B-MYD88-NF-κB pathway (i.e., il1b, il1rl1, myd88, irak4, irak1, traf6, ikbkg, nfkbia, nfkb) and protein levels of NF-κB in larvae intestine confirmed anti-inflammatory effects of 2,4-DTBP. Subsequently, larvae exposed to 2,4-DTBP were challenged with E. coli and showed higher survival rate, suggesting sustained activation of inflammation via LPS can be attenuated by 2,4-DTBP. Moreover, histological examination revealed that intestine barrier was compromised and there was an imbalance of intestine macrophage homeostasis. Food intake was also reduced following exposure to 0.1 and 1 µM 2,4-DTBP. In addition, a risk assessment revealed that 2,4-DTBP in surface water pose low to high ecological risks to aquatic organisms. Taken together, exposure to environmentally relevant concentrations of 2,4-DTBP could negatively affect immune response in zebrafish and may elicit ecological risk in fish population.

CCL3 Promotes Proliferation of Colorectal Cancer Related with TRAF6/NF-κB Molecular Pathway.

Chemokine C-C motif chemokine ligand 3 (CCL3) plays an important role in the invasion and metastasis of malignant tumors. For developing new therapeutic targets and antitumor drugs, the effect of chemokine CCL3 and the related cytokine network on colorectal cancer should be investigated. This study used cell, tissue, and animal experiments to prove that CCL3 is highly expressed in colorectal cancer and confirmed that CCL3 can promote the proliferation of cancer cells, and its expression is closely related to TRAF6/NF-κB molecular pathway. In addition, protein chip technology was used to examine colorectal cancer tissue samples and identify the key factors of chemokine CCL3 and the toll-like receptors/nuclear factor-κB (TLR/NF-κB) pathway in cancer and metastatic lymph nodes. Furthermore, the lentiviral vector technology was employed for transfection to construct interference and overexpression cell lines. The experimental results reveal the mechanism of CCL3 and TNF receptor-associated factor 6 (TRAF6)/NF-κB pathway-related factors and their effects on the proliferation of colon cancer cells. Finally, the expression and significance of CCL3 in colorectal cancer tissues and its correlation with clinical pathology were studied by immunohistochemistry. Also, the results confirmed that CCL3 and C-C motif chemokine receptor 5 (CCR5) were expressed in adjacent tissues, colorectal cancer tissues, and metastatic cancer. The expression level was correlated with the clinical stage and nerve invasion. The expression of chemokine CCL3 and receptor CCR5 was positively correlated with the expression of TRAF6 and NF-κB and could promote the proliferation, invasion, and migration of colorectal cancer cells through TRAF6 and NF-κB.

Asiaticoside reverses M2 phenotype macrophage polarization-evoked osteosarcoma cell malignant behaviour by TRAF6/NF-κB inhibition.

CONTEXT: M2 phenotype macrophage polarization is an attractive target for therapeutic intervention. Asiaticoside (ATS) has multiple pharmacological functions. OBJECTIVE: This study investigates the effect of ATS on M2 phenotype macrophage polarization in osteosarcoma. MATERIALS AND METHODS: The differentiation of human THP-1 monocytes into M0 phenotype macrophages was induced by 100 nM phorbol myristate acetate for 24 h, and treated with 20 ng/mL IL-4 and 20 ng/mL IL-13 for 48 h to obtain M2 phenotype macrophages. The function of ATS on the growth and invasion was investigated by cell counting kit-8, transwell, and western blot under the co-culture of M2 phenotype macrophages and osteosarcoma cells for 24 h. The mechanism of ATS on osteosarcoma was assessed using molecular experiments. RESULTS: ATS reduced the THP-1 cell viability with an IC50 of 128.67 µM. Also, ATS repressed the M2 phenotype macrophage polarization induced by IL-4/IL-13, and the effect was most notably at a 40 µM dose. ATS (40 µM) restrained the growth and invasion of osteosarcoma cells induced by M2 phenotype macrophages. In addition, ATS reduced the tumour necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB activity in osteosarcoma cells and the TRAF6 knockdown reduced the growth and invasion of osteosarcoma cells induced by M2 phenotype macrophages. TRAF6 (2 µg/mL) attenuated the inhibitory effect of ATS on the growth and invasion of osteosarcoma cells caused by M2 phenotype macrophages. In vivo studies further confirmed ATS (2.5, 5, or 10 mg/kg) repressed osteosarcoma tumour growth. DISCUSSION AND CONCLUSIONS: ATS reversed M2 phenotype macrophage polarization-evoked osteosarcoma cell malignant behaviour by reducing TRAF6/NF-κB activity, suggesting ATS might be a promising drug for the clinical treatment of osteosarcoma.

CD40×HER2 bispecific antibody overcomes the CCL2-induced trastuzumab resistance in HER2-positive gastric cancer.

BACKGROUND: There was much hard work to study the trastuzumab resistance in HER2-positive gastric cancer (GC), but the information which would reveal this abstruse mechanism is little. In this study, we aimed to investigate the roles of tumor cell-derived CCL2 on trastuzumab resistance and overcome the resistance by treatment with the anti-CD40-scFv-linked anti-HER2 (CD40 ×HER2) bispecific antibody (bsAb). METHODS: We measured the levels of CCL2 expression in HER2-positive GC tissues, and revealed biological functions of tumor cell-derived CCL2 on tumor-associated macrophages (TAMs) and the trastuzumab resistance. Then, we developed CD40 ×HER2 bsAb, and examined the targeting roles on HER2 and CD40, to overcome the trastuzumab resistance without systemic toxicity. RESULTS: We found the level of CCL2 expression in HER2-postive GC was correlated with infiltration of TAMs, polarization status of infiltrated TAMs, trastuzumab resistance and survival outcomes of GC patients. On exposure to CCL2, TAMs decreased the M1-like phenotype, thereby eliciting the trastuzumab resistance. CCL2 activated the transcription of ZC3H12A, which increased K63-linked deubiquitination and K48-linked auto-ubiquitination of TRAF6/3 to inactivate NF-κB signaling in TAMs. CD40 ×HER2 bsAb, which targeted the CD40 to restore the ubiquitination level of TRAF6/3, increased the M1-like phenotypic transformation of TAMs, and overcame trastuzumab resistance without immune-related adversary effects (irAEs). CONCLUSIONS: We revealed a novel mechanism of trastuzumab resistance in HER2-positive GC via the CCL2-ZC3H12A-TRAF6/3 signaling axis, and presented a CD40 ×HER2 bsAb which showed great antitumor efficacy with few irAEs.

Gonadal hormone trigger the dynamic microglial alterations through Traf6/TAK1 axis that correlate with depressive behaviors.

Gonadal hormone deficiency is associated with the development of depression, but what mediates this association is unclear. To test the possibility that it reflects neuroimmune and neuroinflammatory processes, we analyzed how gonadal hormone deficiency and replacement affect microglial activation and inflammatory response during the development of depressive symptomatology in gonadectomized male mice. Testosterone level and the ratio of testosterone to estradiol in the serum and brain tissue of mice exposed to 3-35 days of chronic unpredictable stress were much lower than in control animals. Gonadal hormone sustained deficiency in gonadectomized mice and subsequent led to acute inflammation at day 7 following castration. Activating microglia in mice exposed to 7 days of castration subsequently suppressed the proliferation of microglia, such that their numbers in hippocampus and cortex were lower than the numbers in sham-operated mice after 30 days of castration. Here, we showed that gonadal hormone deficiency induces Traf6-mediated microglia activation, a type of inflammatory mediator. Microglia treated in this way for long time showed down-regulation of activation markers, abnormal morphology and depressive-like behaviors. Restoration and maintenance of a fixed ratio of testosterone to estradiol significantly suppressed microglial activation, neuronal necroptosis, dramatically inducing hippocampal neurogenesis and reducing depressive behaviors via the suppression of Traf6/TAK1 pathway. These findings suggest that activated or immunoreactive microglia contribute to gonadal hormone deficiency-induced depression, as well as testosterone and estradiol exert synergistic anti-depressant effects via suppressing microglial activaton in gonadectomized male mice, possibly through Traf6 signaling.

Role of Cardiomyocyte-Derived Exosomal MicroRNA-146a-5p in Macrophage Polarization and Activation.

Myocardial infarction arises from an excessive or prolonged inflammatory response, leading to ventricular remodeling or impaired cardiac function. Macrophages exhibit different polarization types associated with inflammation both at steady state and after myocardial infarction. Exosomal miR-146a-5p has been identified as an important molecule in the cardiovascular field in recent years. However, the effect of cardiomyocyte-derived exosomal miR-146a-5p on macrophages has not yet been elucidated. Initially, we found that exosomes with low expression of miR-146a-5p derived from myocardial infarction tissues modulated macrophage polarization. To determine whether cardiomyocyte-derived exosomal miR-146a-5p mediated macrophage polarization, we treated macrophages with exosomes rich in miR-146a-5p collected from neonatal mouse cardiomyocytes. The effects of exosomal miR-146a-5p on macrophage polarization were measured using RT-qPCR, transwell assays, and western blotting. The results showed that the increased expression of miR-146a-5p promoted M1 macrophage polarization, inhibited M2 macrophage polarization, and increased the expression of VEGFA. However, the decreased expression of exosomalmiR-146a-5p showed the opposite trends. Interestingly, in contrast to treatment with the solitary miR-146a-5p mimic, exosomal miR-146a-5p derived from neonatal mouse cardiomyocytes reduced TNFα and iNOS expression. In addition, when macrophages were activated by the miR-146a-5p mimic or exosomal miR-146a-5p, the expression of TNF receptor-associated factor 6 (TRAF6), a target gene of miR-146a-5p, was reduced significantly. Taken together, these findings indicate that exosomal miR-146a-5p derived from cardiomyocytes could stimulate M1 macrophage polarization to induce an inflammatory reaction, while targeting TRAF6, exerting an anti-inflammatory effect. Exosomal miR-146a-5p plays important roles in macrophages, illuminating a novel potential therapeutic target in myocardial infarction.

Hyperoside suppresses osteoclasts differentiation and function through downregulating TRAF6/p38 MAPK signaling pathway.

Hyperoside (HP), as a natural product, can promote proliferation and differentiation of osteoblasts and presents a protective effect on ovariectomized (OVX) mice. However, the inhibitory effect of HP on osteoclasts (OCs) and the potential mechanism remain to be elucidated. In this study, it was found that HP could effectively inhibit the differentiation and bone resorption of OCs, and its intrinsic molecular mechanism was related to the inhibition of TRAF6/p38 MAPK signaling pathway. Therefore, HP could be a promising natural compound for lytic bone diseases.

Notch1 signaling contributes to TLR4-triggered NF-κB activation in macrophages.

Macrophages substantially influence the development, progression, and complications of inflammation-driven diseases. Although numerous studies support the critical role of Notch signaling in most inflammatory diseases, there is limited data on the role of Notch signaling in TLR4-induced macrophage activation and interaction of Notch signaling with other signaling pathways in macrophages during inflammation, such as the NF-κB pathway. This study confirmed that stimulation with lipopolysaccharide (LPS), a TLR4 ligand, upregulated Notch1 expression in monocyte/macrophage-like RAW264.7 cells and bone marrow-derived macrophages (BMDMs). LPS also induced increased mRNA expression of Notch target genes Notch1 and Hes1 in macrophages, suggesting that TLR4 signaling enhances activation of the Notch pathway. The upregulation of Notch1, Notch1 intracellular domain (NICD), and Hes1 proteins by LPS treatment was inhibited by DAPT, a Notch1 inhibitor. Additionally, the increased TNF-α, IL-6, and IL-1ß expression induced by LPS was inhibited by DAPT and rescued by jagged1, a Notch1 ligand. Furthermore, suppression of Notch signaling by DAPT upregulated Cylindromatosis (CYLD) expression but downregulated TRAF6 expression, IκB kinase (IKK) α/ß phosphorylation, and subsequently, phosphorylation and degradation of IκB-α, indicating that DAPT inhibited NF-κB activation triggered by TLR-4. Interestingly, DAPT did not inhibit the increased MyD88 expression induced by LPS. Our study findings demonstrate that macrophage stimulation via the TLR4 signaling cascade triggers activation of Notch1 signaling, which regulates the expression patterns of genes involved in pro-inflammatory responses by activating NF-κB. This effect may be dependent on the CYLD-TRAF6-IKK pathway. Thus, Notch1 signaling may provide a therapeutic target against infectious and inflammation-driven diseases.

miR-146a contributes to atherosclerotic plaque stability by regulating the expression of TRAF6 and IRAK-1.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease. The vulnerable plaque of atherosclerotic can lead to the development of many diseases including acute coronary syndrome and coronary heart disease. It is well known that miR-146a is the key brake miRNA of the inflammatory signal transduction pathway. However, the effect of miR-146a on the stability of atherosclerotic plaque remains to be elucidated. METHODS AND RESULTS: We constructed animal models of atherosclerosis and foam cell models, and overexpressed and knocked-down miR-146a in models. After staining with Hematoxylin-Eosin (HE), Oil Red O, immunocytochemistry (IHC) and Sirius Red, we used the proportion of (Lipids area + Macrophage area) and (SMCs area + collagen area) to evaluate atherosclerotic plaque stability. TUNEL and flow cytometry were performed to detect the apoptosis level of macrophages. Levels of inflammatory factors were detected via ELISA assay. The results showed that miR-146a, IRAK1 and TRAF6 were abnormally expressed in plaques of atherosclerotic animals. Overexpression of miR-146a contributed to the stability of plaques that inhibited plaque formation, macrophage apoptosis and levels of pro-inflammatory factors. The Dual-luciferase reporter gene assay, IF and FISH were used to verify the regulatory mechanism of miR-146a on IRAK1 and TRAF6. We found that IRAK1 and TRAF6 promoted lipid uptake, apoptosis, and release of pro-inflammatory factors of RAW264.7 macrophages, whereas miR-146a restored RAW264.7 macrophages phenotype by inhibiting IRAK1 and TRAF6 expression. CONCLUSIONS: We display for the first time that miR-146a inhibits the formation of foam cells, RAW264.7 macrophage apoptosis and pro-inflammatory reaction through negative regulation of IRAK1 and TRAF6 expression, thereby enhancing the stability of atherosclerotic plaques.

Variations within Toll-like receptor (TLR) and TLR signaling pathway-related genes and their synergistic effects on the risk of Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is the commonest post-infectious polyradiculopathy. Although genetic background of the host seems to play an important role in the susceptibility to GBS, genes conferring major risk are not yet known. Dysregulation of Toll-like receptor (TLR) molecules exacerbates immune-inflammatory responses and the genetic variations within TLR pathway-related genes contribute to differential risk to infection. The aim of this study was to delineate the impact of genetic variations within TLR2, TLR3, and TLR4 genes as well as TLR signaling pathway-related genes such as MyD88, TRIF, TRAF3, TRAF6, IRF3, NFκß1, and IκBα on risk of developing GBS. Fourteen polymorphisms located within TLR2 (rs3804099, rs111200466), TLR3 (rs3775290, rs3775291), TLR4 (rs1927911, rs11536891), MyD88 (rs7744, rs4988453), TRIF (rs8120), TRAF3 (rs12147254), TRAF6 (rs4755453), IRF3 (rs2304204), NFκß1 (rs28362491), and IκBα (rs696) genes were genotyped in 150 GBS patients and 150 healthy subjects either by PCR-RFLP or TaqMan Allelic Discrimination Assay. Genotypes of two polymorphic variants, Del/Del of rs111200466 insertion and deletion (INDEL) polymorphism of TLR2 gene and TT of rs3775290 single nucleotide polymorphism (SNP) of TLR3 gene had significantly higher frequencies among GBS patients, while the frequencies of TT genotype of rs3804099 SNP of TLR2 gene and TT genotype of rs11536891 SNP of TLR4 gene were significantly higher in controls. Gene-gene interaction study by Multifactor Dimensionality Reduction analysis also suggested a significant combined effect of TLR2, and NFκß1 genes on the risk of GBS. The SNPs in the IκBα and IRF3 genes correlated with severity of GBS. The genes encoding TLRs and TLR signaling pathway-related molecules could serve as crucial genetic markers of susceptibility and severity of GBS.

LncRNA NORAD Mediates the Proliferation and Apoptosis of Diffuse Large-B-Cell Lymphoma via Regulation of miR-345-3p/TRAF6 Axis.

BACKGROUND: Diffuse large-B-cell lymphoma (DLBCL), as the most common subtype of B Cell Non-Hodgkin lymphoma (B-NHL), is one of the lymphoid malignancies with poor prognosis worldwide. Non-coding RNA activated by the DNA damage (NORAD), a novel identified lncRNA involved in the DNA repairment process, is reportedly to participate in carcinogenesis, and it is predicted to sponge miR-345-3p. However, LncRNA NORAD has never been investigated in DLBCL. AIM OF THE STUDY: To investigate the role of LncRNA NORAD and miR-345-3p in DLBCL cells and explore the underlying mechanisms. METHODS: LncRNA NORAD and miR-345-3p levels were determined in the blood samples from patients with B-NHL. Human DLBCL cell lines DB and SU-DHL-4 were transfected with LncRNA NORAD small interfering RNA, miRNA-345-3p mimics, or miRNA-345-3p inhibitor using Lipofectamine RNAiMAX Reagent. Cell cycle, proliferation, and apoptosis were assessed in the transfected cells. RESULTS: Silencing of lncRNA NORAD and overexpression of miR-345-3p both inhibited cell proliferation, induced cell cycle arrest, and triggered apoptosis in DLBCL cells. Inhibition of miR-345-3p counteracted the suppression effects of LncRNA NORAD silencing on DLBCL progression. In addition, LncRNA NORAD shared the regulatory binding sites of miR-345-3p with TNF receptor associated factor 6 (TRAF6). Knockdown of LncRNA NORAD decreased the levels of TRAF6, simultaneously, resulted in deactivation of PI3K/Akt pathway in DLBCL cells. CONCLUSION: LncRNA NORAD regulated DLBCL cell growth and apoptosis via miR-345-3p/TRAF6/PI3K/Akt axis.