ABSTRACT
Objective To explore the potential relationship between ubiquitination of transforming growth factor kinase 1(TAK1)/nuclear factor-κB(NF-κB)signaling pathway mediated by ring finger protein 99(RNF99)and septic acute respiratory distress syndrome(ARDS).Methods Plasmid and siRNA transfection were conducted to overexpress or knock down RNF99 in MLE12,and expressions of p65 phosphate and p65 protein were analyzed.The protein interaction between RNF99 and TRAF6 or TAK1 was analyzed by immunoprecipitation assay.Forty mice were randomly divided into WT plus PBS,WT plus LPS,RNF99 specific expression(TG)plus PBS,and TG plus LPS groups,with 10 mice in each group.Sepsis was induced by intraperitoneal injection of 30 mg/kg LPS.Results As compared with vector group,protein expression levels of TRAF6 and TAK1 in MLE12 cells decreased significantly in RNF99 group(P<0.05).Ubiquitinated TRAF6 protein increased in MLE12 cells with RNF99 knockdown.As compared with LPS plus vector group,phosphorylation level of p65 in MLE12 cells was signifi-cantly lower in LPS plus RNF99 group(P<0.05).As compared with si-NC group,protein expression levels of RNF99 and IκBα in si-RNF99 group decreased significantly(P<0.05).As compared with LPS plus si-NC group,phosphorylation level of p65 in LPS plus si-RNF99 group increased significantly(P<0.05).The staining percentage of CD68 macrophages in lung tissues was significantly lower in TG plus LPS group than in WT plus LPS group(P<0.05).Phosphorylation level of p65 in lung tissues was significantly lower in TG plus LPS group than in WT plus LPS group(P<0.05).Conclusion RNF99 regulates NF-κB signaling pathway by interacting with the key regulator of NF-κB signaling pathway(TRAF6/TAK1),and improves lung injury after intraperitoneal injection of LPS in mice.
ABSTRACT
Objective: To study the effects and significances of lipopolysaccharide preconditioning on the activities of NF-?B and the expression of ICAM-1/LFA-1 in rats graft liver ischemia/reperfusion injury. Methods: Male Sprague-Dawley rats were divided into three groups: sham operation group(Sham group),orthotopic liver transplantation group (OLT group) and LPS preconditioning group (LPS group). Only dissecting hepatoduodenal ligament was perfomed in Sham group. Experiments of OLT were performed by two-cuff method in OLT group and LPS group. The activities of NF-?B and the expression of ICAM-1/LFA-1 in hepatic tissue ,the levels of ALT,AST in inferior caval vein blood were detected at 0,60,180 min after dissecting of hepatoduodenal ligament in Sham group and after portal vein reperfusion in OLT group and LPS group. Results: Compared with those in Sham group at the different time points respectively,the activities of NF-?B and the expression of ICAM-1/LFA-1 were higher in OLT group and LPS group(P0.05) at 0 min after reperfusion,they were evidently higher in OLT group than in LPS group (P
ABSTRACT
This study was carried out to examine the potency of the three surface components from Porphyromonas gingivalis to stimulate the murine macrophage cell line RAW264.7 to synthesize the pro-inflammatory cytokine tumor necrosis factor alpha(TNF-alpha) and nitric oxide (NO). Lipopolysaccharide(LPS), lipid A-associated proteins(LAP) and saline-extractable surface -associated material(SAM) were isolated from P. gingivalis 381. TNF-alpha release into culture supernatants was determined by two-site ELISA. NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Western blot analysis of iNOS and analysis of reverse transcription(RT)-PCR products were carried out. The surface components extracted from this bacterium were almost equally potent in stimulating release of TNF-alpha and NO by RAW264.7 cells. TNF-alpha that was being measured immunologically was due to activation of TNF-alpha gene transcription. The present study clearly shows that P. gingivalis surface components fully induced iNOS expression in RAW264.7 cells in the absence of other stimuli. The ability of P. gingivalis surface components to promote the production of TNF-alpha and NO may be important in the pathogenesis of inflammatory periodontal disease.