The Role Of Twist-2 In The Development Mechanisms Of Endotoxin Tolerance In Kupffer Cells

Sepsis is a common complication in critical illness,such as severe trauma,sever infection, hypoxia, ischemia-reperfuion. Today, sepsis is still a nodus in clinical treatment, of which the mortality is more than 30%. Sepsis also has a high prevalence, high mortality, high treatment costs characteristics, and leads to be a serious threat to human health and economic development. Endotoxin is also named as lipopolysaccharide (LPS), which is the main component in the outer membrane of Gram-negative bacteria, and is also the most primary constituent of pathopoiesis. LPS has extensive biological activity can activate inflammatory cells, leading to the release of multiple pro-inflammatory mediators including TNF-α, IL-1 and so on. This results systemic inflammatory response syndrome (SIRS) and multiple organs dysfunct -ion.Endotoxin tolerance is defined that host or cells receiving low dose stimulation to bacterial endotoxin or LPS had a markedly reduced mortality or significant decreased in cytokine when rechallenged with a lethal exposure of LPS. Now, endotoxin tolerance is known as a negative feedback regulation and an adaptive response, which prevents host from continuous over-responding to LPS stimulation. The mechanism of endotoxin toleranc is not explained clearly so far.NF-κB is a multifunctional transcription factor that plays an important role in regulating pro-inflammatory genes. If activated NF-κB complex translocated into the nucleus and binds into the promoter regions of its targeted genes, that would lead to host produce and release a large number of inflammatory mediators and cause to media disease. In fact, activation of NF-κB by extracelluar stimuli is medicated by a distinctive signal pathway linked to degradation of inhibitors of NF-κB(IκB). Recent researches indicated that the existence of auto-regulatory loops for a net negative regulation of NF-κB function in body. Activation of NF-κB results not only in up-regulation of gene involving inflammation but also in synthesizing /re-synthesizing NF-κB dependment , negative regulators of NF-κB signaling. The negative regulatory loop of NF-κB ensures transient generation of intracellular signaling to prevent uncontrolled-NF-κB activation. It is very important to limit inflammatory injury by terminating and blocking pro-inflammatory cytokine-induced NF-κB activity.In recent times, numerous evidence suggest that twist-2 is a critial negative protein of transcription factor NF-κB signal pathway and is essential for down-regulation of inflammatory response, which may involve in the mechanisms of endotoxin tolerance. In this study, mouse or cell model of endotoxin tolerance were used to investigate the expression change of Twist-2 under endotoxin tolerance condition, and to observe the relationship between them. Furthermore, RNA interference technique was used to silence Twist-2 gene in Kupffer cells, through which we attempt to clarify the role of Twist-2 in the occurrence of endotoxin tolerane and may provide a new target for the clinical treatment of sepsis. PARTⅠTHE EXPRESSION OF TWIST-2 IN THE LIVER OF ENDOTOXIN TOLERANT MICEObjective To investigate the gene expression of Twist-2 in the liver of endotoxin tolerant mice before and after challenged with high dose LPS, and to explore the relationship between Twist-2 and endotoxin tolerance on organism level.Methods Mice were randomly divided into non-endotoxin tolerance group (NETT) and the tolerance group (ETT) by differerent treatment. The general situation was compared with two groups. The liver samples were collected for observation of histopathological and ultrastructural changes. The levels of plasma TNF-αwere measured by Enzyme linked immunosorbent assay (ELISA). The mRNA expression of TNF-αand Twist-2 in the liver was detected by Reverse transcription polymerase chain reaction (RT-PCR) analysis.Results (1) The mouse model of endotoxin tolerance was Successfully established. (2) The serum TNF-αlevels, the expression of TNF-αmRNA and the activities of NF-κB in liver tissuse of ETT group were lower than that of NETT group at different time (P<0.05). (3) The liver expression of Twist-2 mRNA in NETT group could not be detected until 24h after LPS stimulation. Howevre, there was slight expression of Twist-2 mRNA in the liver samples of ETT group before rechallenged LPS, and the expression was rapidly upregulated after LPS large dose treatment, peaking at 6h and being about two and half times higher than the level of NETT group at 24h.Conclusion LPS pretreatment can induce endotoxin tolerance of mice, which may be associated with Twist-2 mRNA expression in liver tissuse and the inhibition of Kupffer cell activation. PARTⅡTHE EXPRESSION OF TWIST-2 IN ENDOTOXIN TOLERANT KUPFFER CELLSObjective To study the expression and to explore its role of Twist-2 in endotoxin tolerance kupffer cells.Methods Kupffer cells were isolated from the mouse liver. All cells were divided randomly into NETT group and ETT group by differerent treatment. The mRNA expression of Twist-2 and TNF-αwas detected by RT-PCR, also as Twist-2 protein was detected by Western blotting analysis. The activation of NF-κB was examined by Trans AM NF-kB kit, and The TNF-αlevel in culture medium was measured by ELISA.Results (1) The Kupffer cells model of endotoxin tolerance was successfully established. (2) The TNF-αlevels in culture medium, the expression of TNF-αmRNA and the activities of NF-κB in Kupffer cells of ETT group were lower than that of NETT group at different time(P<0.05). (3) Western blotting analysis and RT-PCR showed that there was no Twist-2 mRNA and Twist-2 protein expression in NETT group until 24h after LPS stimulation. However, ETT group cells could expression this mRNA and protein before the large dose LPS stimulation, and this expression was upregulated and maintained at higher levels in a time-dependent manner.Conclusion Twist-2 gene transcription and translation levels were significantly increased, while the activation of the kupffer cells was inhibited, but not completely lost, which indicates that Twist-2 play an important role in the development of endotoxin tolerance, but it is not the unique mechanism, and there may be other mechanisms involved in endotoxin tolerance. PARTⅢTHE EFFECT OF TWIST-2 GENE SILENCING BY RNA INTERFERENCE ON ENDOTOXIN TOLERANCE OF KUPFFER CELLSObjective To silence Twist-2 gene in Kupffer cells by RNA interference and explore the role of Twist-2 in the mechanisms of endotoxin tolerance.Methods All cells were divided randomly into NETT group which was transfected with pTwist-2-shRNA and ETT group which was transfected with pGenesil-1. Two goups were stimulated by 100ng/ml LPS after with 10ng/ml LPS pretreatment. The mRNA expression of Twist-2 was detected by RT-PCR; also as Twist-2 protein was detected by Western blotting analysis. The activation of NF-κB was examined by Trans AM NF-kB kit, and The TNF-αlevel in culture medium was measured by ELISA.Results (1) The Twist-2 mRNA expressions in Kupffer cells was reduced by pTwist-2-shRNA transfection. PGenesil-1 transfection had no influence on Twist-2 expression. (2)After transfection with pTwist-2-sh RNA or pGenesil-1, The TNF-αlevels in culture medium, and the activities of NF-κB in Kupffer cells of pGenesil-1 group were lower than that of pTwist-2-shRNA group at different time(P<0.05); While the expression of Twist-2 mRNA and the protein of Twist-2 of pGenesil-1 group were higher than that of pTwist-2-shRNA group.Conclusion The endotoxin tolerance of Kupffer cells was significantly attenuated, which indicates that Twist-2 indeedly play an important role in the endotoxin tolerance...