Inhibition of EZH2 (Enhancer of Zeste Homolog 2) Attenuates Neuroinflammation via H3k27me3/SOCS3/TRAF6/NF-κB (Trimethylation of Histone 3 Lysine 27/Suppressor of Cytokine Signaling 3/Tumor Necrosis Factor Receptor Family 6/Nuclear Factor-κB) in a Rat Model of Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Neuroinflammation has been proven to play an important role in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). EZH2 (enhancer of zeste homolog 2)-mediated H3K27Me3 (trimethylation of histone 3 lysine 27) has been recognized to play a critical role in multiple inflammatory diseases. However, there is still a lack of evidence to address the effect of EZH2 on the immune response of SAH. Therefore, the aim of this study was to determine the role of EZH2 in SAH-induced neuroinflammation and explore the effect of EZH2 inhibition with its specific inhibitor EPZ6438. METHODS: The endovascular perforation method was performed on rats to induce subarachnoid hemorrhage. EPZ6438, a specific EZH2 inhibitor, was administered intraperitoneally at 1 hour after SAH. SOCS3 (Suppressor of cytokine signaling 3) siRNA and H3K27me3 CRISPR were administered intracerebroventricularly at 48 hours before SAH to explore potential mechanisms. The SAH grade, short-term and long-term neurobehavioral tests, immunofluorescence staining, and western blots were performed after SAH. RESULTS: The expression of EZH2 and H3K27me3 peaked at 24 hours after SAH. In addition, inhibition of EZH2 with EPZ6438 significantly improved neurological deficits both in short-term and long-term outcome studies. Moreover, EPZ6438 treatment significantly decreased the levels of EZH2, H3K27Me3, pathway-related proteins TRAF6 (TNF [tumor necrosis factor] receptor family 6), NF-κB (nuclear factor-κB) p65, proinflammatory cytokines TNF-α, IL (interleukin)-6, IL-1ß, but increased the expression levels of SOCS3 and anti-inflammatory cytokine IL-10. Furthermore, administration of SOCS3 siRNA and H3k27me3-activating CRISPR partly abolished the neuroprotective effect of EPZ6438, which indicated that the neuroprotective effect of EPZ6438 acted, at least partly, through activation of SOCS3. CONCLUSIONS: In summary, the inhibition of EZH2 by EPZ6438 attenuated neuroinflammation via H3K27me3/SOCS3/TRAF6/NF-κB signaling pathway after SAH in rats. By targeting EZH2, this study may provide an innovative method to ameliorate early brain injury after SAH.

[The effects of silica dust on the expression of MyD88 and TRAF6 mRNA of lung macrophages in rats].

Objective: To investigate the effects of silica dust on the expression of Myeloid differentiation factor 88 (MyD88) mRNA and tumor necrosis factor receptor-associated factor (TRAF6) mRNA of lung macrophages in rats. Methods: Selecting 40 SPF-class Wistar rats with average weight (200±20) g randomly divided into control group and 30 d, 60 d, 120 d experimental groups with 10 rats in each group according to body weight. The experimental groups rats were injected with 1 ml of SiO(2) (100 mg/ml) suspension through the trachea into lung only once, then they were respectively killed after 30, 60, 120 days. The control group rats were injected with 1 ml of saline into lung, and killed after 120 days. The lungs of the rats were taken for pathological observation. Lung macrophages were extracted and counted, and their activity was detected by MTT. RT-qPCR was used to assess the relative contents of MyD88 mRNA and TRAF6 mRNA. Results: Silica dust inhalation led to infiltration of lung tissue cells, thickening the alveolar wall and destruction of alveolar structure. The longer the exposure to dust, the more obvious the results were. The number of macrophages in all experimental groups and activity in the 30 d, 60 d groups were significantly higher than that in the control group (P<0.05) . Among them, 30 d group had the largest number and the highest activity. Compared with the control group, the expression of MyD88 mRNA and TRAF6 mRNA of lung macrophages in rats increased in the experimental groups (P<0.05) , especially in the 60 d group. Conclusion: Silica dust inhalation can increase the expression of MyD88 and TRAF6 in macrophages, suggesting that silica dust can induce silicosis fibrosis by activating TLR/NF-κB signal pathway.

TRAF6 mediates high glucose-induced endothelial dysfunction.

To investigate the role of tumor necrosis factor-associated factor 6 (TRAF6) in high glucose-induced endothelial cell dysfunction. Human aortic endothelial cells (HAECs) were cultured in high glucose medium, and TRAF6 expression was assayed by quantitative real-time Polymerase Chain Reaction (PCR) and western blotting. The effect of TRAF6 on in vitro endothelial cell viability, apoptosis, migration, and endothelial-monocyte adhesion was investigated by gene knockdown. The expression of TRAF6 and related adhesion molecules was assayed in a mouse streptozotocin-induced type I diabetes model. The signaling pathways associated with TRAF6 effects on endothelial cells were investigated in high glucose HAEC cultures. Culture of HAECs in high glucose medium significantly increased TRAF6 mRNA and protein expression in a time dependent manner. High glucose markedly reduced HAEC viability, apoptosis, and migration, and these effects was significantly reversed by TRAF6 knockdown. High glucose significantly increased intercellular adhesion of THP-1 monocytic cells and HAECs via upregulation of ICAM-1 and VCAM-1 expression, and TRAF6 knockdown attenuated the effect on THP-1 cell adhesion. TRAF6, ICAM-1, and VCAM-1 expression were increased in aorta tissue of mice with streptozotocin-induced diabetes. The free radical scavenger N-acetyl-L-cysteine attenuated TRAF6 expression in HAECs cultured in high glucose medium, and TRAF6 knockdown inhibited high glucose-induced IκB-α degradation and JNK phosphorylation. TRAF6 mediated high glucose-induced endothelial dysfunction via NF-κB- and AP-1-dependent signaling. Targeting TRAF6 may delay progression of vascular diseases during diabetes mellitus and atherosclerosis.

Association of microRNA 146 with middle ear hyperplasia in pediatric otitis media.

OBJECTIVE: Toll-like receptor signaling activated by bacterial otitis media pathogens in the middle ear has been shown to play a key role in OM susceptibility, pathogenesis and recovery. Recent studies implicate microRNA 146 (miR-146) in regulation of inflammation via negative feedback of toll-like receptor signaling (TLR) in a wide variety of tissues, however its involvement in otitis media is unknown. METHODS: Human middle ear epithelial cells were stimulated with proinflammatory cytokines, interleukin 1 beta or tumor necrosis factor alpha, for two to twenty-four hours. Middle ear biopsies were collected from children with otitis media with effusion (n = 20), recurrent otitis media (n = 9), and control subjects undergoing cochlear implantation (n = 10). miR-146a, miR-146b expression was assayed by quantitative PCR (qPCR). Expression of miR-146 targets involved in TLR signaling, IRAK1 and TRAF6, was assayed by qPCR in middle ear biopsies. Middle ear biopsies were cryosectioned and epithelial thickness measured by a certified pathologist. RESULTS: Proinflammatory cytokines induced expression of miR-146 in middle ear epithelial cells in vitro. Middle ear miR-146a and miR-146b expression was elevated in otitis media patients relative to control subjects and correlated with middle ear epithelial thickness. A trend towards inverse correlation was observed between miR-146 and TRAF6 expression in the clinical population. CONCLUSIONS: This report is the first to assess miRNA expression in a clinical population with OM. Findings herein suggest miR-146 may play a role in OM.

Regulation of Transforming Growth Factor ß-Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K(63) -Linked Autoubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6.

OBJECTIVE: Transforming growth factor ß-activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1ß (IL-1ß), tumor necrosis factor (TNF), and Toll-like receptor signaling. This study was undertaken to examine the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA). METHODS: The effect of TAK1, IL-1 receptor-associated kinase 1 (IRAK-1), and TNF receptor-associated factor 6 (TRAF6) inhibition was evaluated in IL-1ß-stimulated human RA synovial fibroblasts (RASFs). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RASFs. The efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating these processes in RASFs was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were confirmed using a rat model of adjuvant-induced arthritis (AIA). RESULTS: Inhibition of TAK1, but not IRAK-1 or TRAF6, completely abrogated IL-1ß-induced IL-6 and IL-8 synthesis in RASFs. EGCG inhibited TAK1 phosphorylation at Thr(184/187) and occupied the C(174) position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K(63) -linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K(124) position on TRAF6. Furthermore, EGCG enhanced proteasome-associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses of joint homogenates from rats with AIA showed a significant increase in K(48) -linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to naive rats. Administration of EGCG (50 mg/kg/day) for 10 days ameliorated AIA in rats by reducing TAK1 phosphorylation and K(48) -linked polyubiquitination. CONCLUSION: Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.

Histone deacetylase inhibitors increase microRNA-146a expression and enhance negative regulation of interleukin-1ß signaling in osteoarthritis fibroblast-like synoviocytes.

OBJECTIVE: MiR-146a exerts negative control on inflammatory responses by suppressing cytokine-induced expression of interleukin-1 receptor-associated kinase-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) by impairing NF-κB activity and inhibiting the expression of target genes. Recent study suggests that histone deacetylases (HDACs) are involved in the regulation of microRNA (miRNA) expression. Therefore, we determined whether HDAC inhibitors can increase miR-146a expression, thereby inhibiting interleukin-1ß (IL-1ß)-induced signaling in osteoarthritis fibroblast-like synoviocytes (OA-FLS). METHOD: MiRNA expression was analyzed using real-time PCR. IL-1ß-induced downstream signals and cytokine expression were evaluated using Western blotting and ELISA. Transcription factors regulating promoter activation were identified using chromatin immunoprecipitation assays. RESULTS: IL-1ß treatment of OA-FLS induced a mild (1.7-fold) increase in miR-146a expression that was unable to appropriately downregulate IRAK1 and TRAF6 expression. HDAC inhibitors, SAHA (vorinostat), and LBH589 (panobinostat) significantly (6.1- and 5.4-fold) elevated miR-146a expression by increasing the binding of the transcription factor NF-κB to the miR-146a promoter, and negatively regulated IL-1ß-induced IKK/IκB/p65 phosphorylation signaling and IL-6 secretion. The increase in miR-146a expression induced by the HDAC inhibitors was prevented by transfection of miR-146a inhibitor or HDAC1 (class I HDAC), HDAC4 (class IIa HDAC), and HDAC6 (class IIb HDAC) overexpression, suggesting that they were due to inhibition of HDAC activity. CONCLUSIONS: Our study demonstrated that HDAC inhibitor treatment in OA-FLS significantly increased miR-146a expression and mediated markedly negative regulation to inhibit IL-1ß-induced signaling and cytokine secretion. Our results indicate the potential rationale of anti-inflammatory effects for HDAC inhibitors.

miR-146a regulates mechanotransduction and pressure-induced inflammation in small airway epithelium.

Mechanical ventilation generates biophysical forces, including high transmural pressures, which exacerbate lung inflammation. This study sought to determine whether microRNAs (miRNAs) respond to this mechanical force and play a role in regulating mechanically induced inflammation. Primary human small airway epithelial cells (HSAEpCs) were exposed to 12 h of oscillatory pressure and/or the proinflammatory cytokine TNF-α. Experiments were also conducted after manipulating miRNA expression and silencing the transcription factor NF-κB or toll-like receptor proteins IRAK1 and TRAF6. NF-κB activation, IL-6/IL-8/IL-1ß cytokine secretion, miRNA expression, and IRAK1/TRAF6 protein levels were monitored. A total of 12 h of oscillatory pressure and TNF-α resulted in a 5- to 7-fold increase in IL-6/IL-8 cytokine secretion, and oscillatory pressure also resulted in a time-dependent increase in IL-6/IL-8/IL-1ß cytokine secretion. Pressure and TNF-α also resulted in distinct patterns of miRNA expression, with miR-146a being the most deregulated miRNA. Manipulating miR-146a expression altered pressure-induced cytokine secretion. Silencing of IRAK1 or TRAF6, confirmed targets of miR-146a, resulted in a 3-fold decrease in pressure-induced cytokine secretion. Cotransfection experiments demonstrate that miR-146a's regulation of pressure-induced cytokine secretion depends on its targeting of both IRAK1 and TRAF6. MiR-146a is a mechanosensitive miRNA that is rapidly up-regulated by oscillatory pressure and plays an important role in regulating mechanically induced inflammation in lung epithelia.

Euscaphic acid isolated from roots of Rosa rugosa inhibits LPS-induced inflammatory responses via TLR4-mediated NF-κB inactivation in RAW 264.7 macrophages.

As an attempt to search for bioactive natural products exerting anti-inflammatory activity, we have evaluated the anti-inflammatory effects of euscaphic acid (19α-hydroxyursane-type triterpenoids, EA) isolated from roots of Rosa rugosa and its underlying molecular mechanisms in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. EA concentration-dependently reduced the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) induced by LPS in RAW 264.7 macgophages. Consistent with these data, expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and iNOS, COX-2, TNF-α, and IL-1ß mRNA were inhibited by EA in a concentration-dependent manner. In addition, EA attenuated LPS-induced DNA binding and transcriptional activity of nuclear factor-kappa B (NF-κB), which was accompanied by a parallel reduction of degradation and phosphorylation of inhibitory kappa Bα (IκBα) and consequently by decreased nuclear translocation of p65 subunit of NF-κB. Pretreatment with EA significantly inhibited the LPS-induced phosphorylation of IκB kinase ß (IKKß), p38, and JNK, whereas the phosphorylation of ERK1/2 was unaffected. Furthermore, EA interfered with the LPS-induced clustering of TNF receptor-associated factor 6 (TRAF6) with interleukin receptor associated kinase 1 (IRAK1) and transforming growth factor-ß-activated kinase 1 (TAK1). Taken together, these results suggest that EA inhibits LPS-induced inflammatory responses by interference with the clustering of TRAF6 with IRAK1 and TAK1, resulting in blocking the activation of IKK and MAPKs signal transduction to downregulate NF-κB activations.

TNF receptor-associated factor 6 suppression inhibits inflammatory response to Porphyromonas gingivialis in human periodontal ligament cells.

OBJECTIVE: Periodontitis is a group of inflammatory diseases caused by microorganisms. Porphyromonas gingivalis, a gram-negative bacteria, is strongly associated with the onset of periodontitis. Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) represents an important target in the regulation of many disease processes, including immunity, inflammation, and osteoporosis. The aim of this study was to investigate the role of TRAF6 for inflammatory response in P gingivalis-infected human periodontal ligament cells (HPDLCs). METHOD AND MATERIALS: HPDLCs were stimulated with 1 x 108 CFU/mL P gingivalis, or 10 ug/mL P gingivalis lipopolysaccharide (LPS), separately in the absence or presence of small interfering RNA (siRNA) for TRAF6. The expression of TRAF6 was examined by real-time polymerase chain reaction and Western blot analysis. Concentrations of IL-1B, IL-6, and IL-8 in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In this study, we found that both P gingivalis and its LPS treatment increased the expression of TRAF6 and proinflammatory cytokine production in HPDLCs. In addition, we used siRNA for TRAF6, and the inhibition of TRAF6 expression reduced the production of proinflammatory cytokines in HPDLCs stimulated with P gingivalis and its LPS. CONCLUSION: The results suggested that TRAF6 may be a key molecule to control proinflammatory cytokine production induced by P gingivalis and its LPS. TRAF6 suppression may inhibit inflammatory responses in HPDLCs infected by P gingivalis and its LPS.

Leptospira santorosai Serovar Shermani detergent extract induces an increase in fibronectin production through a Toll-like receptor 2-mediated pathway.

Leptospirosis can activate inflammatory responses through Toll-like receptors (TLRs) and may cause renal tubulointerstitial fibrosis characterized by the accumulation of extracellular matrix (ECM). We have previously demonstrated that Leptospira santorosai serovar Shermani detergent extract stimulates ECM accumulation in vitro. The aim of this study was to examine the mechanistic basis of these previous observations and, in particular, to examine the potential involvement of TLRs. The addition of serovar Shermani detergent extract led to an increase in fibronectin gene expression and production. Inhibition of TLR2 but not TLR4 expression abrogated serovar Shermani detergent extract-mediated increases in fibronectin production. This response was also blocked by the knockdown of the gene expression of the TLR2 downstream transducers myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). Serovar Shermani detergent extract also activated nuclear factor-κB, and its inhibition by curcumin-attenuated serovar Shermani detergent extract induced increases in fibronectin production. These effects were also mimicked by the specific TLR2 agonist, Pam(3)CsK(4), a response that was also abrogated by the knockdown of MyD88 and TRAF6. Similarly, the administration of live leptospires to cells also induced fibronectin production that was blocked by inhibition of TLR2 and MyD88 expression. In conclusion, serovar Shermani detergent extract can induce fibronectin production through the TLR2-associated cascade, providing evidence of an association between TLRs and leptospirosis-mediated ECM deposition.

[Effect of Dureping injection on TIR signal pathway on Ana-1 cells].

OBJECTIVE: To investigate the influence of Dureping injection to the murinal celiac macrophage Ana-1 on TIR signal pathway. METHOD: Ana-1 cell line was infected by influenza virus FM1 strain and treated with the Dureping injection in different concentrations (10.1 mg x L(-1) group) for 12 h and 24 h. Then we collected the cells, extracted mRNA and measured the expressions of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 respectively by RT-PCR. RESULT: Dureping injection down-regulated the expression of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 mRNA in Ana-1 cell line infected by influenza virus, in a dose-dependent manner significantly. CONCLUSION: Dureping injection has an obvious effect against influenza virus FM1 strain by regulating the TIR signal pathway.