The Effect Of3-oxo-C12-HSL Mediated UPR On LPS-induced Inflammation And The Diagnosis Value Of NGAL, Hepcidin On Bacterial Translocation In A Rat Liver Cirrhosis Model

The effect of3-oxo-C12-HSL mediated UPR on LPS-induced inflammation Dr. Candidate:Zhang Jiangguo Supervisor:Prof. Song JianxinObjective:N-3-Oxododecanoyl homoserine lactone (3-oxo-C12-HSL), a quorum-sensing signal molecule produced by Pseudomonas aeruginosa, has a function not only in the expression of bacterial virulence factors but also in the modulation of host immune responses by directly disrupting nuclear factor-kappa B (NF-κB) signaling and inducing cell apoptosis. Unfolded protein response (UPR) triggered by endoplasmic reticulum stress can suppress inflammatory responses in the later phase by blocking the NF-κB activation. Latest advances have demonstrated that3-oxo-C12-HSL can induce UPR in human aortic endothelial cells. Therefore,3-oxo-C12-HSL may also possibly inhibit NF-κB activation and suppress inflammatory responses by activating UPR. However, little is known about this possible mechanism. In this study, we investigated the effects of3-oxo-C12-HSL on cellular viability, UPR activation, lipopolysaccharide (LPS)-induced NF-κB activation and inflammatory response in the mouse macrophage cell line RAW264.7cells.Materials and methods:RAW264.7cells were cultured under different concentration of3-oxo-C12-HSL for different hours. The viability and apoptosis of these cells was measured by WST-1cell cytotoxicity assay and flow cytometry to determine the proper concentration and incubation time of3-oxo-C12-HSL. After incubation with the proper concentration and time of3-oxo-C12-HSL, RAW264.7cells RNA was extracted and reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were performed to analyse the mRNA expression of molecular chaperones in UPR pathway. The protein expression mode of UPR pathway was also determined by Western blot analysis. The effects of3-oxo-C12-HSL on LPS-induced cytokines production were further examined by ELISA.Results:6.25μM3-oxo-C12-HSL did not obviously influence the cell viability of RAW264.7cells. Pretreating RAW264.7cells with6.25μM3-oxo-C12-HSL effectively triggered UPR and increased the expression of UPR target genes such as CCAAT/enhancer binding protein β (C/EBP β) and CCAAT/enhancer-binding protein-homologous protein (CHOP). The expression of C/EBP β and CHOP was inversely correlated with LPS-induced NF-κB activation.3-Oxo-C12-HSL pretreatment also inhibited LPS-stimulated proinflammatory cytokines production.Conclusion:3-oxo-C12-HSL may attenuate LPS-induced inflammation via UPR-mediated NF-κB inhibition without affecting cell viability. This possibly is another mechanism that P. Aeruginosa uses to evade the host immune system and maintain persistent infection. Objecive:Spontaneous bacterial peritonitis (SBP) is one of the most prevalent complications in patients with liver cirrhosis. Bacterial translocation (BT) is a critical pathogenesis mechanism of SBP. Studies of BT are limited in humans because of the requirement for surgery to remove mesenteric lymph nodes (MLNs). Neutrophil gelatinase associated lipocalin (NGAL) is released by human neutrophil granules. NGAL in peritoneal fluids can efficiently distinguish bacterial and nonbacterial ascites in patients with ascites. Hepcidin is produced by hepatocytes and macrophages and can be released into the circulation. Serum hepcidin is a useful marker of bacterial infection in the late-onset sepsis in very low birth weight infants. Therefore, combination use these two indicators may be a more powerful diagnosis tool for bacterial translocation. In present study, we investigated the level of NGAL and hepcidin and their relationship with bacterial translocation and the presence of bacterial DNA in MLNs in a liver cirrhosis rat model. We also explored the diagnosis value of NGAL and hepcidin on BT or the presence of bacterial DNA in MLNs. Whether NGAL and hepcidin in ascites could distinguish bacterial peritonitis from ascites without infection was furthermore examined. At last, the relationship between the level of NGAL, hepcidin in ascites and bacterial DNA presence was also investigated.Material and methods:Weekly doses of CC14were given to induce liver cirrhosis in Sprague-Dawley rats. Trypticase soy agar was used to culture bacteria. Bacterial DNA in MLNs and ascites was sequenced by ABIPRISM310automated sequencer. The level of NGAL and hepcidin in sera and ascites was determined by ELISA. Receiver operating characteristic curve was used to determine the value of NGAL and hepcidin in diagnosing BT and the existence of bacterial DNA in MLNs.Results:33rats were included in this study. The positive MLNs cultures (BT) were observed in6cirrhotic rats. The bacterial DNA was detected in MLNs from13cirrhotic animals,6being from culture-positive MLNs and the remaining7from culture-negative MLNs. The negative MLNs cultures with absence of bacterial DNA were identified in15cirrhotic rats. There was a100%match of bacterial sequence between MLNs and ascites cultures. The area under ROC curve for the diagnosis of BT was0.910for NGAL,0.858for hepcidin and0.940for their combination. Sensitivity and specificity for combination of NGAL>158ng/ml and hepcidin>15.65ng/ml was0.667,0.963. The area under ROC curve for the diagnosis of the presence of bacterial DNA in MLNs was0.906for NGAL,0.779for hepcidin and0.950for their combination. Sensitivity and specificity for combination of NGAL>97ng/ml and hepcidin>14.30ng/ml was1.000,0.900. The area under ROC curve for the diagnosis of BT from the only presence of bacterial DNA in MLNs was0.857for NGAL,0.857for hepcidin and0.857for their combination. Sensitivity and specificity for combination of NGAL>158ng/ml and hepcidin>17.20ng/ml was1.000,0.714. The area under ROC curve for the diagnosis of SBP was0.879for NGAL,0.883for hepcidin and0.977for their combination. Sensitivity and specificity for combination of NGAL>116.5ng/ml and hepcidin>19.30ng/ml was1.000,0.955. The area under ROC curve for the diagnosis of the presence of bacterial DNA in ascites was0.795for NGAL,0.804for hepcidin and0.956for their combination. Sensitivity and specificity for combination of NGAL>114.5ng/ml and hepcidin>16.85ng/ml was0.889,0.947. The area under ROC curve for the diagnosis of SBP from the only presence of bacterial DNA in ascites was1.000for NGAL,0.944for hepcidin and1.000for their combination. Sensitivity and specificity for combination of NGAL>138ng/ml and hepcidin>18.90ng/ml was1.000,1.000.Conclusion:BT and the only presence of bacterial DNA in MLNs were observed in a rat cirrhotic model. Serum NGAL and hepcidin can serve as sensitive and specific tests for diagnosis of BT or the presence of bacterial DNA in MLNs. Furthermore, NGAL and hepcidin in ascites can also serve as sensitive and specific tests for diagnosis of SBP or the presence of bacterial DNA in ascites. NGAL in combination with hepcidin can improve the accuracy of diagnosis.