[The influence with block the endotoxin signal transduction for ischemia/reperfusion injury of graft liver in rats].

OBJECTIVE: To explore the feasibility of interleukin 1 receptor associated kinase-4 (IRAK-4) as gene therapy target for liver ischemia/reperfusion injury (I/RI) and effective approach in vivo for short hairpin RNA (shRNA) interference used to gene therapy in liver graft hqappened. METHODS: Sprague-Dawley rats were randomly divided into three groups: the control group, the in vivo transfection group (IVT) and the cold ischemia transfection group (CIT). Experiments of orthotopic liver transplantation were performed by two-cuff method. CIT were perfused with IRAK-4-shRNA plasmid (pSIIRAK-4) during cold ischemia phase, IVT received the equivalent volumes (2 mL) of pSIIRAK-4 after portal vein inosculated, and the control group leaved without any treatment. At 0 min, 60 min and 180 min after reperfusion, the expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The serum TNF-alpha level was detected by ELISA. Liver histopathological changes and cell apoptosis were observed by electron microscope and TUNEL. RESULTS: After reperfusion, the expression of IRAK-4 were largely depressed in CIT than that of IVT and the control group (P < 0.01), and furthermore, the serum TNF-alpha level, proportion of hepatocyte apoptosis and severity of hepatocyte injury were also lower than the latter. CONCLUSION: These results indicate that depression IRAK-4 expression with IRAK-4-shRNA through portal vein perfusion during cold ischemia phase could effectively blunt graft hepatic I/RI.

[Expression of CD14 and Toll-like receptor 4 on Kupffer cells and its role in ischemia-reperfusion injury on rat liver graft].

OBJECTIVE: To study the expression of lipopolysaccharide (LPS) receptor CD14 and Toll-like receptor 4 (TLR4) on Kupffer cells and its role in ischemia-reperfusion injury (IRI) on rat liver graft. METHODS: The Kupffer cells following IRI were isolated and divided into control, ischemia-reperfusion (IR), and anti-CD14 antibody group. The mRNA and protein expression of CD14 and TLR4, nuclear factor kappa B (NF-kappaB) activity and TNF-alpha level in supernatant were measured. RESULTS: The mRNA and protein expression of CD14 and TLR4 in IR group were significantly higher than those in control group (P < 0.01). The NF-kappaB activity and TNF-alpha level in IR group were significantly higher than those in control group (P < 0.01), and they greatly decreased after anti CD14 antibody treatment (compared with IR group, P < 0.05), but were still significantly higher than those in control group (P < 0.01). CONCLUSIONS: LPS following IRI could up-regulate CD14 and TLR4 gene and protein expression on Kupffer cells, and subsequently activate NF-kappaB to produce cytokines, but other signal transduction pathways might also participate in the IRI.

[The effect of glycine on CD14 and NF-kappa B in Kupffer cells from rat liver grafts after ischemia-reperfusion injury].

OBJECTIVE: To investigate the effect of glycine on CD14 and NF-kappa B in Kupffer cells from rat liver grafts after ischemia-reperfusion injury (IRI). METHODS: The rats were randomly divided into an IRI group, saline solution preconditioning group, and glycine preconditioning group. Their survival rates, graft functions, and hepatic histopathologic examinations were observed after IRI. Kupffer cells (KCs) following IRI were isolated and cultured to detect CD14 mRNA, NF-kappa B binding activity, and the TNF alpha and IL-1 level in the supernatant of the media. RESULTS: (1) Glycine preconditioning greatly enhanced the one-week survival rate (chi2 = 6.67 and 8.57 respectively), improved graft function, and ameliorated the histopathologic signs of injury. (2) The CD14 mRNA expression level (F = 7.64), NF-kappa B binding activity (F = 11.47), TNF alpha and IL-1 production (F = 14.08 and 9.56 respectively) in the glycine group were significantly lower than those in the other two groups. CONCLUSION: Glycine could efficiently protect rat liver grafts from ischemia-reperfusion injury by repressing the expression of CD14 and NF-kappa B binding activity in Kupffer cells and inhibiting the productions of TNF alpha and IL-1.

Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft.

AIM: To investigate the expression of toll-like receptor 4 (TLR4) and MD-2 gene and protein in Kupffer cells (KCs) and their role in ischemia-reperfusion (IR) injury of rat liver graft. METHODS: KCs were isolated at 0 (control group), 2, 12, 24 h (IR group) following IR in rat liver graft. mRNA expression of TLR4 and MD-2 was detected by RT-PCR analysis, protein expression of TLR4/MD-2 was detected by flow cytometric (FCM) analysis, and tumor necrosis factor-alpha (TNF-alpha) level in supernatant was measured by ELISA. Then isolated KCs were incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group), and TNF-alpha level was measured again. RESULTS: The mRNA and protein expression of TLR4/MD-2 and the level of TNF-alpha in IR group increased significantly at 2 h following IR, and reached the maximum at 12 h, and slightly decreased at 24 h, but were still significantly higher than those in the control group (P<0.01). The expression of these factors was markedly decreased after anti-TLR4 antibody treatment as compared with the IR group (P<0.01). CONCLUSION: Lipopolysaccharide (LPS) following IR can up-regulate TLR4/MD-2 gene and protein expression in KCs, and synthesize cytokine TNF-alpha. Anti TLR4 antibody can inhibit the production of TNF-alpha induced by LPS. TLR4 and its partner molecule MD-2 may play an important role in Kupffer cell activation and IR injury.

Protective effect of nitric oxide induced by ischemic preconditioning on reperfusion injury of rat liver graft.

AIM: Ischemic preconditioning (IP) is a brief ischemic episode, which confers a state of protection against the subsequent long-term ischemia-reperfusion injuries. However, little is known regarding the use of IP before the sustained cold storage and liver transplantation. The present study was designed to evaluate the protective effect of IP on the long-term preservation of liver graft and the prolonged anhepatic-phase injury. METHODS: Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation. All livers underwent 10 min of ischemia followed by 10 min of reperfusion before harvest. Rat liver transplantation was performed with the portal vein clamped for 25 min. Tolerance of transplanted liver to the reperfusion injury and liver damage were investigated. The changes in adenosine concentration in hepatic tissue and those of nitric oxide (NO) and tumor necrosis factor (TNF) in serum were also assessed. RESULTS: Recipients with IP significantly improved their one-week survival rate and liver function, they had increased levels of circulating NO and hepatic adenosine, and a reduced level of serum TNF, as compared to controls. Histological changes indicating hepatic injuries appeared improved in the IP group compared with those in control group. The protective effect of IP was also obtained by administration of adenosine, while blockage of the NO pathway using Nomega-nitro-L-arginine methyl ester abolished the protective effect of IP. CONCLUSION: IP appears to have a protective effect on the long-term preservation of liver graft and the prolonged anhepatic-phase injuries. NO may be involved in this process.