In order to construct an expression vector carrying small hairpin （sh） RNA （shRNA） for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein （EGFP） and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide （LPS） stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mecha- nism, the reporter gene plasmid pEGFP-CI （4.7 kb） and psiRNA-hHlneo （2979 bp） were used. The HI promotor and double Bbs Ⅰ restrict endoenzyme site were cloned from plasmid psiRNA-hHlneo and reconstructed them into plasmid pEGFP-CI in the Mlu Ⅰ restrict endoenzymic site, forming plasmid pEGFP-HI/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H 1/siRNA forming plasnlid pEGFP-H 1/TLR4-siRNA. After transfection of pEGFP-H I/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha （TNF-α） release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-HI/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was （50.37±8.23） % and after transfection of the plasmid pEGFP-HI/ TLR4-siRNA the level of TNF-α released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-α release by RAW264.7 cells evoked by LPS.
Kupffer cells, expressing toll-like receptor 4 (TLR4), play a central role in hepatic ischemia/reperfusion (I/R) injury. Hyaluronic acid (HA) fragments, degradative products of high-molecular-weight HA (HMW-HA), acquire the ability to activate immune cells under inflammatory conditions. Here we inves- tigated whether HA fragments could activate Kupffer cells and analyzed the underlying mechanism. Kupffer cells were isolated from wild-type mice (WT, C3H/HeN) and TLR4 mutant mice (C3H/HeJ) and HA fragments were produced by the methods of enzyme digestion and chromatography. Then Kupffer cells were stimulated by HA fragments or other control stimuli. The activation of Kupffer cells was estimated as the release of pro-inflammatory cytokines. The activation of p38 MAPK pathway of Kupffer cells was checked and blocking experiments were done as well. The results indicated that HA fragments acquired the ability to activate Kupffer cells in vitro, which was TLR4 dependent and not due to contamination of lipopolysaccharide. Experiments of p38 MAPK kinase inhibition by SB-203580 verified p38 MAPK was required in HA fragments induced Kupffer cells activation. This suggests that HA fragments, degradative products of one of the major glycosaminoglycans of the extracellular matrix, play critical roles in Kupffer cell activation mediated by TLR4 signaling pathway, which is, at least partially, de- pendent on p38 MAPK activation.
ZHANG JinXiang1, WANG Hui2, XIAO Qing3, LIANG HuiFang4, LI ZhuoYa4, JIANG ChunFang1, WU HeShui5 & ZHENG QiChang5 1 Department of Emergency Surgery, Union Hospital Affiliated to Huazhong University of Science and Technology, Wuhan 430022, China 2 Department of Medical Genetics, Tongji Medical College Affiliated to Huazhong University of Science and Technology, Wuhan 430030, China 3 Department of Ophthalmology, Union Hospital Affiliated to Huazhong University of Science and Technology, Wuhan 430022, China 4 Department of Medical Immunology, Tongji Medical College Affiliated to Huazhong University of Science and Technology, Wuhan 430030, China 5 Department of General Surgery, Union Hospital Affiliated to Huazhong University of Science and Technology, Wuhan 430022, China
Background Restoration of blood flow to the ischemic liver lobes may paradoxically exacerbate tissue injury, which is called hepatic ischemia/reperfusion injury （IRI）. Toll-like receptor 4 （TLR4）, expressed on several liver cell types, and the nuclear factor-kappa B （NF-KB） signaling pathway are crucial to mediating hepatic inflammatory response. Because IRI is essentially a kind of profound acute inflammatory reaction evoked by many kinds of danger signals, we investigated TLR4/NF-KB signaling pathway activation in a murine model of partial hepatic IRI. Methods Wild-type mice （WT, C3H/HeN） or TLR4 mutant mice （C3H/HeJ） were subjected to 45 minutes of partial hepatic ischemia followed by 1 hour, 3 hours of reperfusion. Sham group accepted the same procedure without the obstruction of blood supply. At the end of reperfusion, the compromise of liver function and the histological change of liver sections were measured as the severity of liver injury. The level of endotoxin in the portal vein was measured by limulus assay. NF-KB activation was determined by electrophoretic mobility shift assay （EMSA）. The levels of tumor necrosis factor-a （TNF-a） and intedeukin-1β （IL-1β） in systemic blood after hepatic IRI were assessed by enzyme-linked immunosorbent assay （ELISA）. Results The compromise of liver function and the morphological injuries in mutant mice were relieved more markedly than those in WT mice after partial hepatic IRI. NF-KB activation in WT mice was stronger than that in TLR4 mutant mice, and both were stronger than those in the sham operated mice （P〈0.01）. Endotoxin in each group was undetectable. The levels of TNF-α and IL-1β in systemic blood were elevated in both strains, but lower in the sham operated group. These mediators were significantly decreased in TLR4 mutant mice compared with those in WT mice （P〈0.01）. Conclusions The TLR4/NF-KB signaling pathway may mediate hepatic IRI triggered by endogenous danger signals. Inhibition of the TLR4/NF-KB path
WANG HuiLI Zhuo-yaWU He-shuiWANG YangJIANG Chun-fangZHENG Qi-changZHANG Jin-xiang
