| Bacterial infection is still a major threat to humans. Invasion of Gram-negative bacteria elicits immune responses, a major mediator of which is lipopolysaccharide(LPS), a component of the cell wall of Gram-negative bacteria. Liver is the first target organ to be attacked by the gut-derived lipopolysaccharide. LPS appears to provoke liver inflammation with focal hepatocyte injury and increases the sensitivity to CCU, ethanol liver injury. Recent study has shown that LPS can cause liver injury in normal subjects and exaggarate the liver injury in alcoholic liver disease. The prevailing hypothesis for LPS-induced signaling activation is that LPS/Toll-like receptor 4 (TLR4) signaling pathway is activated, proinflammatory factors are released and results in secondary tissue injury. However, the precise mechanism for LPS-induced liver injury remains unclear. It is conceivable that specific signaling and effector molecules are involved in the initiation and development of the processes.In this study, signaling molecules involved in LPS/TLR4 signaling pathway and some effector molecules were detected bymolecular biological and immunohistochemistry technology at indicated timepoints in LPS-induced liver injury model. Simultaneously, the functional molecules involved in the pathogenesis of LPS-induced liver injury were detected by use of proteomics methods.First, LPS-induced endotoxemic mouse were sacrificed at 3 hours, 6 hours, 12 hours, 24 hours and 30 hours respectively. The liver tissues were stained with hematoxylin and eosin for histopathologic analyses. Hepatocyte apoptosis and hepatic TNF-a was detected by immunohistochemical method. Simultaneously, the expression of TLR4mRNA and MyD88mRNA was detected by use of semi-quantitative RT-PCR. A large number of inflammatory cells infiltrated portal areas with additional focal hepatocellular necrosis 24 hours after i.p.LPS. Hepatocyte apoptosis and hepatic TNF- a expression increased significantly 3-6 hours after i.p LPS, while recruited to normal level at 12 hours. Hepatic TLR4mRNA expression was significantly depressed 6 through 12 hours after i.p.LPS, but recruited at 24 hours. Hepatic MyD88 mRNA expression did not change among different timepoints.Second, we identified several possible target proteins in order to provide some information regarding the mechanism of LPS-induced liver injury. The total liver protein was separated by two-dimensional gel electrophoresis at indicated different timepoints. After in-gel trypticdigestion, the peptide mass footprints were drawn up and the protein database was searched to identify the proteins. Then, analyzed how the indicated proteins participated in the pathogenesis of endotoxin-induced liver injury. After i.p. LPS: at earlier stage (6 hours), peak expression of TNFAIP1 was accompanied. TRAIL receptor 2 was downregulated. Mitochondrial related proteins (Metaxin2, TIMM8A) were upregulated. The proteasome related proteins were inhibited; Later stage (24 hours, even 3 mouse died at the 30 hour), significant histopathological changes took place with recruitment of proteasome related proteins and peak expression of metaxin2, TIMM8A . At the same time, p34 cdc2 kinase was also upregulated.It was concluded that LPS-induced liver injury was time dependent. At earlier stage: TNF- a related proteins reached peak expression and mitochondrial apoptotic pathway related proteins began upregulating. TNF-a-dependent hepatocyte apoptosis played predominant role at this stage. The expression of liver TLR4mRNA was depressed significantly. At latter stage: Mitochondrial apoptotic pathway related proteins reached peak expression and the repair program was initiated at the appropriate timepoint. Hepatocyte necrosis made a major role at this stage. The expression of liver TLR4mRNA was recruited from the initial depressed states. It indicated that the signal molecule TLR4mRNA took an important role in LPS-inducedliver injury and may participated in LPS tolerance. MyD88mRNA expression di...
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