Diagnostic and Predictive Levels of Calcium-binding Protein A8 and Tumor Necrosis Factor Receptor-associated Factor 6 in Sepsis-associated Encephalopathy： A Prospective Observational Study

Background： Despite its high prevalence, morbidity, and mortality, sepsis-associated encephalopathy （SAE） is still poorly understood. The aim of this prospective and observational study was to investigate the clinical significance of calcium-binding protein A8 （S 100AS） in serum and tumor necrosis factor receptor-associated factor 6 （TRAF6） in peripheral blood mononuclear cells （PBMCs） in diagnosing SAE and predicting its prognosis. Methods： Data of septic patients were collected within 24 h after Intensive Care Unit admission fi-om July 2014 to March 2015. Healthy medical personnel served as the control group. SAE was defined as cerebral dysfhnction in the presence of sepsis that fulfilled the exclusion criteria. The biochemical indicators, Glasgow Coma Scale, Acute Physiology and Chronic Health Evaluation score II, TRAF6 in PBMC, serum S 100A8, S 10013, and neuron-specific enolase were evaluated in SAE patients afresh. TRAF6 and S 100A8 were also measured in the control group. Results： Of the 57 enrolled patients, 29 were diagnosed with SAE. The S 100A8 and TRAF6 concentrations in SAE patients were both significantly higher than that in no-encephalopathy （NE） patients, and higher in NE than that in controls （3.74 ± 3.13 vs. 1.08 ± 0.75 vs. 0.37 ± 0.14 ng/ml, P 〈 0.01 ; 3.18 ± 1.55 vs. 1.02 ± 0.63 vs. 0.47 ± 0.10, P 〈 0.01）. S 100A8 levels of 1.93 ng/ml were diagnostic of SAE with 92.90% specificity and 69.00% sensitivity in the receiver operating characteristic （ROC） curve, and the area under the curve was 0.86 （95% confidence interval [CI]： 0.76-0.95）. TRAF6-relative levels of 1.44 were diagnostic of SAE with 85.70% specificity and 86.20% sensitivity, and the area under the curve was 0.94 （95% CI： 0.88-0.99）. In addition, S 100A8 levels of 2.41 ng/ml predicted 28-day mortality of SAE with 90.00% specificity and 73.70% sensitivity in the ROC curve, and the area under the curve was 0.88. TRAF6 relative levels of 2.94 predicted 28-day mortality of SAE with 80.00% specificity and 68.40% sensitivity, and the area under the curve was 0.77. Compared with TRAF6, the specificity of serum S 100A8 in diagnosing SAE and predicting mortality was higher, although the sensitivity was low. In contrast, the TRAF6 had higher sensitivity for diagnosis. Conclusions： Peripheral blood levels of S 100A8 and TRAF6 in SAE patients were elevated and might be related to the severity of SAE and predict the outcome of SAE. The efficacy and specificity of S 100A8 for SAE diagnosis were superior, despite its weak sensitivity. S100A8 might be a better biomarker for diagnosis of SAE and predicting prognosis.

Inhibitory Effects of Parthenolide on the Activity of NF-κB in Multiple Myeloma via Targeting TRAF6

This study examined the mechanism of the inhibitory effect of parthenolide（PTL） on the activity of NF-κB in multiple myeloma（MM）. Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6（TRAF6） and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.

TRAF6 polymorphisms not associated with the susceptibility to and severity of sepsis ina Chinese population

BACKGROUND： The tumor necrosis factor recepter associated factor （TRAF） 6 is an important intracellular adapter protein that plays a pivotal role in activating multiple inflammatory and immune related processes induced by cytokines. TRAF6 represents a strong candidate susceptibility factor for sepsis. We investigated whether polymorphisms at the TRAF6 gene are associated with the susceptibility to and severity of sepsis.METHODS： A hospital-based case-control study was conducted with 255 patients with sepsis and 260 controls who were recruited from Zhengzhou, China. Haplotype tagging single nucleotide polymorphisms （htSNPs） were selected from the HapMap database and genotyped using the SNPstream genotyping platform. The associations with the susceptibility and disease severity of sepsis were estimated by logistic regression, and adjusted for age, sex, smoking, drinking, chronic diseases status, APACHEII score and critical illness status.RESULTS： A total of 13 TRAF6 SNPs were tagged by 7 htSNPs. Five htSNPs （rs5030490, rs5030411, rs5030416, rs5030445 and rs3740961） were genotyped in the case control study. Genotype frequencies of the htSNPs were conformed to the Hardy-Weinberg equilibrium in both patients and controls. No significant association was found between the 5 htSNPs and the susceptibility to and severity of sepsis. Compared with the main haplotype -11120A/-10688T/-9423A/805G/12967G, no certain haplotype was associated with the signi？ cantly susceptibility to or severity of sepsis.CONCLUSION： TRAF6 gene polymorphisms might not play a major role in mediating the susceptibility to and severity of sepsis in the Chinese population. A larger population-based case-control study is warranted.

Influence of Silencing TRAF6 with shRNA on LPS/TLR4 Signaling in vitro

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide(LPS)/toll-like receptor(TLR)-4 signaling pathway in vitro.Four plasmids(pGCsi-TRAF6-shRNA1,2,3,4) containing different shRNA sequences were designed and synthesized.The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay.Inflammatory cellular models were established by LPS stimulation.Levels of TNF-α,IL-1β and TGF-β1 in the supernatants,mRNA expressions of TRAF6,IL-6 and COX-2,protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA,real-time quantitative PCR and Western blotting,respectively.The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h.The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1,2 groups than in the TRAF6-shRNA3,4 groups.Therefore,pGCsi-TRAF6-shRNA1,2 were selected for the subsequent experiments.Our results still showed that pGCsi-TRAF6-shRNA1,2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-α,IL-1β,IL-6 and COX-2,and inhibit NF-κB nuclear translocation.Moreover,pGCsi-TRAF6-shRNA1,2 could suppress the release of TGF-β1 at the protein level.It was concluded that the recombinant plasmid pTRAF6-shRNA can,to some extent,inhibit inflammatory response stimulated by LPS at the initial phase.TRAF6 may become the potential therapeutic target of many inflammation-related diseases.

Regulatory Effects of AT1R-TRAF6-MAPKs Signaling on Proliferation of Intermittent Hypoxia-induced Human Umbilical Vein Endothelial Cells

Summary： Endothelial dysfunction induced by intermittent hypoxia （IH） participates in obstructive sleep apnea syndrome （OSAS）-associated cardiovascular disorders. Myeloid differentiation primary response 88 （MyD88） and tumor necrosis factor receptor-associated factor 6 （TRAF6） regulate nu- merous downstream adaptors like mitogen-activated protein kinases （MAPKs） and the subsequent oxidative stress and inflammatory responses. This study aimed to characterize the role of MyD88/TRAF6 in IH-treated cell function and its associated signaling. Human umbilical vein endo- thelial cells （HUVECs） were randomly exposed to IH or normoxia for 0, 2, 4 and 6 h. Western blot- ting was used to detect the expression pattern of target gene proteins [angiotensin 1 receptor （AT1R）, p-ERK1/2, p-p38MAPK, MyD88 and TRAF6], and the relationships among these target genes down-regulated by the corresponding inhibitors were studied. Finally, the influence of these target genes on proliferation of HUVECs was also assessed by EdU analysis. Protein levels of AT1R, TRAF6 and p-ERK1/2 were increased after IH exposure, with a slight rise in MyD88 and a dynamic change in p-p38MAPK. The down-regulation of TRAF6 by siRNA reduced ERK1/2 phosphorylation during IH without any effects on ATIR. Blockade of AT1R with valsartan decreased TRAF6 and p-ERK1/2 protein expression after IH exposure. ERK1/2 inhibition with PD98059 suppressed only AT1R expression. IH promoted HUVECs proliferation, which was significantly suppressed by the in- hibition of TRAF6, AT1R and ERK1/2. The findings demonstrate that TRAF6 regulates the prolifera- tion of HUVECs exposed to short-term IH by modulating cell signaling involving ERK1/2 down- stream of AT1R. Targeting the AT1R-TRAF6-p-ERK1/2 signaling pathway might be helpful in re- storing endothelial function.

Effect of TRAF6 gene silencing on hepatocellular carcinoma cell line SMCC7721 and its possible mechanism

Objective:To explore the effect of TRAF6 gene silencing on the function of hepatocellular carcinoma SMCC7721 and its possible mechanism.Method:Cell lines were constructed by cell transfection technology and verified by quantitative real-time PCR.Cell functional changes were observed by CCK8 method,Transwell test and Method of EdU.Western blotting was used to explore the possible mechanism of action.Result:TRAF6 RNA was abnormally up-regulated in HCC,and TRAF6 levels were detected in both HCC cell lines and L02 cells.SMCC7721 was selected as TRAF6 high expression cell.The results of CCK8 assay and EdU method showed that the decrease of TRAF6 expression significantly inhibited the proliferation of SMCC7721 cells.The results of CCK8 assay and EdU method showed that the decrease of TRAF6 expression significantly inhibited the proliferation of SMCC7721 cells.Overexpression of TRAF6 in TRAF6 knockdown cells can restore and enhance cell proliferation.Transwell assay confirmed that the invasiveness of SMCC 7721 cells treated with siRNA was significantly reduced.After treatment with LV-Rescue plasmid,the cell invasion was restored and enhanced.Western blotting showed that the protein levels of YB-1,Wnt,β-catenin,c-myc and Cyclin D1 were significantly down-regulated in siRNA group.On the contrary,the expression level of CYLD protein increased.Conclusion:As an important intracellular junctive protein in tumor cells,TRAF6 may improve the expression of pro-cancer factors C-myc and Cyclin D1 by modifying(ubiquitination)YB-1,thus improving the proliferation ability of cells.This process may be closely positively correlated with the Wnt/β-catenin pathway,and negatively correlated with the expression of CYLD protein.

An enriched environment reduces hippocampal inflammatory response and improves cognitive function in a mouse model of stroke

An enriched environment is used as a behavio ral intervention therapy that applies sensory,motor,and social stimulation,and has been used in basic and clinical research of va rious neurological diseases.In this study,we established mouse models of photothrombotic stroke and,24 hours later,raised them in a standard,enriched,or isolated environment for 4 weeks.Compared with the mice raised in a standard environment,the cognitive function of mice raised in an enriched environment was better and the pathological damage in the hippocampal CA1 region was remarkably alleviated.Furthermore,protein expression levels of tumor necrosis factor receptor-associated factor 6,nuclear factorκB p65,interleukin-6,and tumor necrosis factorα,and the mRNA expression level of tumor necrosis factor receptor-associated factor 6 were greatly lower,while the expression level of miR-146a-5p was higher.Compared with the mice raised in a standard environment,changes in these indices in mice raised in an isolated environment were opposite to mice raised in an enriched environment.These findings suggest that different living environments affect the hippocampal inflammatory response and cognitive function in a mouse model of stro ke.An enriched environment can improve cognitive function following stroke through up-regulation of miR-146a-5p expression and a reduction in the inflammatory response.

Flagellin of Pseudomonas aeruginosa induces transforming growth factor beta 1 expression in normal bronchial epithelial cells through mitogen activated protein kinase cascades

Background Acute lung infection due to Pseudomonas aeruginosa （P. Aeruginosa） is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P. Aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P.aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P. Aeruginosa in vitro. We used flagellin protein from P. Aeruginosa, a key initiator of acute P.aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P. Aeruginosa can cause fibrogenesis/remodeling.Methods We studied the effect of flagellin from P. Aeruginosa （flagellin for short） on the transforming growth factor beta 1 （TGF-β1） and interleukin-8 （IL-8） expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 （TRAF6）/mitogen activated protein kinase （MAPK） pathway. Flagellin was purified from the P. Aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-β1 and IL-8 were detected by enzyme-linked immunosorbant assay （ELISA）. Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH2-terminal kinase （JNK） and extracellular regulated kinase （ERK）.Results Expression of TGF-β1 in BEAS-2B cells was elevated by flagellin vs. Control groups （（104.3±20.8） vs.（44.6±4.4） pg/ml （P 〈0.01）） and was ablated by either p38 or JNK inhibitors compared with flagellin treatment （（45.1±18.8）vs. （104.3±20.8） pg/ml and （48.1±20.8） vs. （104.3±20.8） pg/ml, respectively （P 〈0.05））. Flagellin also elevated the expression of IL-8 in BEAS-2B cells vs. The control groups （（554.9±57.7） vs. （51.4±2.2.9） pg/ml （P 〈0.01））, and p38 MAPK inhibitors weaken the expression by flagellin （（301.1 ±155.1） vs. （554.9±57.7） pg/ml （P 〈0.05））. Western blotting revealed that all three MAPK proteins, p38, JNK and ERK were activated by flagellin challenge in an early phase, respectively in 15 minutes （P 〈0.01）, 30 minutes （P 〈0.01） and 15 minutes （P 〈0.01）. TRAF6 siRNA which decreased expression of TRAF6, altered the activation of JNK, p38, and ERK following flagellin treatment, but its influence on the expression of TGF-β1 and IL-8 has no statistical significance.Conclusions Flagellin from P. Aeruginosa PAO1 induces TGF-β1 expression in normal bronchial epithelial cells,BEAS-2B, through the MAPK signal cascade in vitro. It suggests that the fibrogenesis/remodeling process may be initiated from an early stage of acute lung infection due to P. Aeruginosa.

Inhibition Mechanism of Qingluo Tongbi Granule(清络通痹颗粒)on Osteoclast Differentiation Induced by Synovial Fibroblast and Monocytes Co-Culture in Adjuvant-Induced Arthritic Rats

Objective： To study the mechanism underlying the inhibitory effect of Qingluo Tongbi Granule （清络通痹颗粒, QTG） on osteoclast differentiation in rheumatoid arthritis in rats. Methods： Fibroblast and monocyte co-culture were used to induce osteoclast differentiation in adjuvant-induced arthritic （AIA） rats. Serum containing QTG was prepared and added to the osteoclasts, and activation of the tumor necrosis factor receptorassociated factor 6/mitogen-activated protein kinase/nuclear factor of activated T cells, cytoplasmic1 （TRAF6/ MAPK/NFATcl） pathways was examined. Results： The induced osteoclasts were multinucleated and stained positive for tartrate-resistant acid phosphatase （TRAP） staining. Serum containing QTG at 14.4, 7.2 or 3.6 g/kg inhibited the activation of TRAF6, extracellular regulated protein kinase （ERK）1/2, c-Jun N-terminal kinase （JNK） and p38 and decreased the percentage of cells with nuclear NFATcl in a dose-dependent manner, the high and middle doses exhibited clear inhibitory activity （P〈0.01 and P〈0.05, respectively）. After the addition of MAPK inhibitors, the NFATcl expression showed no significant difference compared with the control group （P〉0.05）. Conclusions： Serum containing QTG could generally inhibit the TRAF6/MAPK pathways and possibly inhibit the NFATcl pathway. In addition, QTG may regulate other signaling pathways that are related to osteoclast differentiation and maturation.