Cannulation Selection of Portal Venous and Splenic Venous Catheterization in Venovenous Bypass of Swine Orthotopic Liver Transplantation.

BACKGROUND The aim of this study was to compare the hemodynamic changes in 2 different cannulations in portal system (portal venous catheterization and splenic venous catheterization) during venovenous bypass (VVB) of swine orthotopic liver transplantation (OLT) MATERIAL AND METHODS Thirty pairs (a total of 60) of healthy Duroc pigs were selected for OLT. According to the difference of cannulation in portal venous system during VVB, these pigs were divided into 2 groups: the PVC group (pigs with portal venous catheterization, n=15) and the SVC group (pigs with splenic venous catheterization, n=15). Intraoperative hemodynamic parameters were monitored continuously. RESULTS Two recipients in the PVC group died: 1 died of unsmooth bypass during the operation and 1 died of disseminated intravascular coagulation (DIC). There was only 1 death in the SVC group, due to hemorrhagic shock. The duration of anhepatic phase (AP) in the SVC group was significantly shorter than in the PVC group (P<0.05). Moreover, hemodynamic parameters in phase III (5 min after start of portal vein suturing) and phase IV (5 min after graft reperfusion) were remarkably different between the SVC group and the PVC group (P<0.05). CONCLUSIONS Our results show that VVB via splenic venous catheterization in swine OLT: 1) shortens the AP time; 2) keeps the hemodynamics stable; and 3) reduces the occurrence of postoperative complications. Thus, SVC appears to be superior to PVC.

[Role of glucocorticoid-induced tumor necrosis factor-related protein ligand (GITRL) on lipopolysaccharide indu-ced Kupffer cells apoptosis].

AIM: To study the role of glucocorticoid-induced tumor necrosis factor-related protein ligand (GITRL) on apoptosis of mouse Kupffer cells (KCs) induced by lipopolysaccharide (LPS). METHODS: The KCs were isolated from BALB/c mice and transfected with Control siRNA or GITRL siRNA for 24 h. The KCs were randomly divided into four groups including control group: cultured in media alone, dexamethasone (Dex) group: media with Dex 10 µmol/L, LPS group: media with LPS 1 mg/L, and LPS+Dex group: media with LPS 1 mg/L and Dex 10 µmol/L. At 24 h after treatment, the expression of GITRL was detected by immunocytochemistry. The apoptosis of KCs was measured by Annexin V-FITC/PI double staining and FCM. RESULTS: The GITRL expression of KCs was increased by LPS challenge (P < 0.05), whereas Dex treatment attenuated the increase. LPS challenge induced KCs apoptosis, but the LPS induced apoptosis was inhibited by GITRL siRNA transfection or Dex treatment (P < 0.05, respectively). CONCLUSION: LPS could induce mouse KCs apoptosis, which may be depend on GITRL signal transduction.

Diagnosis and management of choledochoduodenal fistula.

Recent advances in hepatobiliary imaging techniques have led to the increased detection of choledochoduodenal fistula. However, the diagnosis and treatment of choledochoduodenal fistula is still a challenge. In this study, we summarize how patients were diagnosed and treated for choledochoduodenal fistula at our institution. Sixty-six patients with choledochoduodenal fistula were diagnosed and treated in our department from January 2000 to June 2009. Sixty-one patients were treated operatively, whereas five patients were treated with medicine. Patients with choledochoduodenal fistula were confirmed by endoscopic retrograde cholangiography. Of the 61 patients needing surgical intervention, clinical outcomes were excellent in 57 patients, and five patients underwent successful laparoscopic surgery for repairing the choledochoduodenal fistula. Follow-up of these patients for 6 months to 10 years showed they did not suffer from further cholangitis. A patients' past history of biliary disease, upper abdominal pain, fever, and jaundice may lead to choledochoduodenal fistula. Operative therapy, including laparoscopic surgery, was the primary treatment for most patients, regardless of the preoperative diagnosis.

Glycine attenuates endotoxin-induced liver injury by downregulating TLR4 signaling in Kupffer cells.

BACKGROUND: Several experimental studies have observed better outcomes after glycine treatment in patients with endotoxin-induced liver injuries, but its molecular mechanism is not yet fully understood. The purpose of this study was to evaluate the hypothesis that glycine attenuates endotoxin-induced liver injury by affecting endotoxin signal transduction in liver macrophages. METHODS: An animal model of endotoxin-induced liver injury was established by intraperitoneally injecting mice with 10 mg/kg body weight endotoxin fed a pretreatment diet with or without 5% (w/w) glycine. Blood and liver samples were obtained for analysis of liver morphology and to determine concentrations of alanine aminotransferase, endotoxin receptor Toll-like receptor 4 (TLR4), tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-10 at various time points after injection. To investigate the effect of glycine on liver macrophages, Kupffer cells (KCs) were isolated and challenged by LPS (100 ng/mL), with or without glycine (4 mmol/l) pretreatment, and the expressions of TLR4, IL-10, and TNF-alpha were assayed at mRNA and protein levels. DNA-binding activity of nuclear factor-kappa B (NF-kappaB) was also analyzed using enzyme-linked immunosorbent assay. RESULTS: Dietary glycine significantly improved the survival rate of endotoxemic mice (P < .05), whereas serum alanine aminotransferase and TNF-alpha levels were significantly decreased at different time points (P < .05); IL-10 levels were increased (P < .05). Concurrently, LPS-induced hepatic tissue injury was attenuated as indicated by morphologic analysis; secretion of IL-10 in liver tissue (P < .05) was enhanced; and expression of TLR4 and TNF-alpha in liver tissue was downregulated (P < .05). Consistent with these in vivo experiments, enhanced secretion of IL-10 and inhibited expression of TLR4 and TNF-alpha caused by glycine pretreatment were also observed in LPS-stimulated KCs. NF-kappaB DNA-binding activity was also significantly inhibited by glycine (P < .05, respectively). CONCLUSIONS: Dietary glycine improved survival rates and liver function in endotoxemic mice by regulating the production of proinflammatory or anti-inflammatory cytokines in liver. It attenuated liver injury by deactivating KCs through inhibiting TNF-alpha secretion and increasing IL-10 production. The downregulative effect of glycine on the endotoxin signaling pathway and TLR4/NF-kappaB/TNF-alpha may be a novel potential mechanism by which glycine inhibits KC activity.

Up-regulation of IRAK-M is essential for endotoxin tolerance induced by a low dose of lipopolysaccharide in Kupffer cells.

BACKGROUND: Endotoxin tolerance (ET) is an important mechanism to maintain the homeostasis of Kupffer cells (KCs), because KCs are continually exposed to various pathogen-associated molecular patterns including lipopolysaccharide (LPS). ET involves multiple changes in cell signal transduction pathways; however, not all signaling pathways are down-regulated and some proteins are up-regulated. The latter proteins may be counter regulatory, including interleukin-1 receptor-associated kinase M (IRAK-M) expression. The aim of this study is to clarify weather or not IRAK-M is involved in the mechanisms of ET in KCs through dampening nuclear factor-kappa B (NF-kappaB) mediated pathway. MATERIALS AND METHODS: KCs isolated from male C57BL/6J mice were seeded in 24-well plates at 1 x 10(6) cells/well and cultured overnight prior to transfection, were randomly divided into two groups: the pIRAK-M-short hairpin RNA (shRNA) group (transfected with IRAK-M shRNA) and the control group (transfected with control vector); 24 h after transfection, the two groups were further randomly divided into two subgroups: non-endotoxin pretreatment group (incubation in Dulbecco's modified Eagle's medium [Invitrogen, Carlsbad, CA] with 10% fetal bovine serum) and endotoxin pretreatment group (incubation in the same medium containing 10 ng/mL LPS), named pIRAK-M-EP, pIRAK-M-NEP, pCV-EP, and pCV-NEP, respectively. Each subgroup contained 6 wells; 24 h later, fresh media containing LPS (100 ng/mL) was added to each subgroup and incubated for an additional 3 h. The expression of IRAK-M gene and protein level were determined by reverse transcription-polymerase chain reaction and Western blot, the activities of NF-kappaB were estimated by electrophoretic mobility shift assay and enzyme-linked immunosorbent assay, and the supernatant tumor necrosis factor-alpha levels were analyzed by enzyme-linked immunosorbent assay. RESULTS: The recombinant plasmid of pIRAK-M-shRNA specifically inhibited IRAK-M expression after it was transfected into KCs. At 3 h after 100 ng/mL LPS was added to the medium, IRAK-M expression was significantly induced in pCV-EP than that in pCV-NEP; however, there was no difference between pIRAK-M-NEP and pIRAK-M-EP, accompanied with lowest level of NF-kappaB activation and tumor necrosis factor-alpha levels in pCV-EP, and a dramatic enhancement in the other three groups (P < 0.01). CONCLUSIONS: Although a primary low dose of LPS stimulation obviously attenuated KCs response to the second LPS stimulation, the inhibitive influences were partly refracted in pIRAK-M-EP than in pCV-EP, indicating that the absence of IRAK-M caused abnormal enhancement of inflammatory effects. IRAK-M negatively regulates toll-like receptors signaling and involves in the mechanisms of ET in KCs through dampening NF-kappaB mediated pathway; therefore it may be a key component of this important control system, and a new target for the clinical treatment of sepsis.

Glycine blunts transplantative liver ischemia-reperfusion injury by downregulating interleukin 1 receptor associated kinase-4.

AIM: To determine whether glycine could downregulate interleukin 1 receptor associated kinase-4 (IRAK-4) expression to interfere with lipopolysaccharides (LPS) signal transduction and blunt transplantative liver ischemia-reperfusion injury (I/RI). METHODS: SD rats were randomly divided into two groups: donor animals of the glycine group (n=40) were given glycine (1.5 mL; 300 mmol/L, iv) 1 h before harvest, and the control group were treated with 1.5 mL physiological saline (n= 40). Orthotopic liver transplantation was then performed according to the Kamada technique. Ten animals in each group were followed up for 7 d after surgery to assess survival. The remaining animals in each group were divided into 3 subgroups (n=10) at 1h, 2 h and 6 h after portal vein reperfusion. Levels of LPS, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin in portal circulation, as well as IRAK-4 and TNF-alpha expression, NF-kappaB transcriptional activity and morphological study of liver tissues were analyzed. RESULTS: Reperfusion resulted in a significant elevation of LPS concentrations in each group persisting to the end of our study. However, glycine, which led to improved survival rate and liver function, significantly alleviated liver parenchyma cell damage by downregulating IRAK-4, TNF-alpha expression and NF-kappaB transcriptional activity compared with the control group. CONCLUSION: Glycine can attenuate hepatic I/RI by downregulating IRAK-4 to interfere with LPS signal transduction.

[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.

[The protection mechanisms of glycine against liver injury induced by lipopolysaccharides].

OBJECTIVE: To explore the protective mechanisms of glycine (Gly) on lipopolysaccharides (LPS) induced liver injury. METHODS: BABL/c mice were randomly divided into a LPS group, in which the animals were intraperitoneally injected with 10 mg/kg LPS, and a Gly group, in which the mice were pretreated with a 5% Gly-containing diet for 3 days before receiving the same dose of LPS. The livers of the mice were examined for histopathological changes. The TNF alpha and interleukin-10 (IL-10) levels in the blood plasma were measured using ELISA analysis. The mRNA expression of TNF alpha, IL-10 and Toll-like receptor 4 (TLR4) in hepatic tissues were detected using RT-PCR analysis. Protein expression of TLR4 in livers was detected using immunohistochemistry. RESULTS: The Gly group mice had an improved survival rate and attenuated LPS-induced pathological changes in the liver tissues in comparison with those of the LPS group animals. The TNF alpha levels [(1,852.80+/-126.64) pg/ml vs (708.83+/-51.29) pg/ml, P<0.05] in plasma, as well as the expression of TNF alpha (A 1.59+/-0.14 vs. 0.91+/-0.11, P<0.05) and TLR4 (A 0.97+/-0.12 vs. 0.53+/-0.11, P<0.05) mRNA in liver tissues were decreased. However, the levels of plasma interleukin-10 [(344.09+/-31.70) pg/ml vs (418.64+/-38.86) pg/ml, P<0.05] were significantly increased and the peaking time left, shifted. CONCLUSIONS: Gly pretreatment could attenuate LPS -induced liver injury in mice, which may be associated with its role in down-regulating TLR4 expression and up-regulating IL-10 production.

[The mechanism and treatment phases chosen of glycine for inhibition lipopolysaccharide induced Kupffer cells activation].

OBJECTIVE: To explore the possible mechanism and optimal treatment phase of glycine for inhibition lipopolysaccharide (LPS) induced Kupffer cells (KCs) activation. METHODS: The KCs were isolated from 40 BALB/c mice and divided into four groups: the endotoxin group, the prevention group, the early treatment group and the later treatment group (n = 10). The endotoxin group was treated with 10 mg/L LPS, and in the other three groups, glycine (1 mmol/L) was given 24 h before, or at 0 h or 4 h respectively after LPS stimulation. At 0 h, 1 h, 2 h, 6 h and 12 h after LPS stimulation, the mRNA levels and protein expression of interleukin-1 receptor associated kinase-4 (IRAK-4) were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively, and nuclear factor-kappaB (NF-kappaB) activities as well as tumor necrosis factor alpha (TNF-alpha) levels were also detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: The climax values of IRAK-4, NF-kappaB and TNF-alpha were significantly higher in the endotoxin group and the later treatment group than that in the other two groups (t = 3.17, 4.33, 2.47, 126.73, P < 0.01). CONCLUSION: The results indicated that prophylactic or simultaneous treatment with glycine could effectively inhibit LPS-induced KCs activation by inhibiting IRAK-4 expression.

[Expression changes of interleukin-1 receptor associated kinase-4 during endotoxin tolerance development in kupffer cells].

OBJECTIVE: To explore the mechanism of endotoxin tolerance (ET) through observing the expression of interleukin 1 receptor associated kinase-4 (IRAK-4) during endotoxin tolerance development in Kupffer cells (KCs). METHODS: Isolated KCs of Balb/c mouse were divided into two groups: the non-endotoxin tolerance (NET) group and the endotoxin tolerance (ET) group, which were pretreated with 10 ng/ml lipopolysaccharide (LPS) for 24 h. Then, the two groups were treated with 100 ng/ml LPS. The expressions of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The activities of NF-kappaB of KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h after LPS stimulation. RESULTS: The ultimate level of IRAK-4, the activities of NF-kappaB and the TNFalpha level were evidently lower in the ET group than those in the NET group (t = 12.4, 17.4 and 138.9 respectively, P<0.01). CONCLUSIONS: Pretreatment with LPS on KCs could induce endotoxin tolerance of KCs and inhibition of IRAK-4 expression may be one of the reasons for its development.

[An experimental study of the inhibitory effects on the activation of endotoxin-induced Kupffer cells through short hairpin RNA targeting interleukin-1 receptor associated kinase-4 gene].

OBJECTIVE: To explore the inhibitory effects on the activation of endotoxin-induced Kupffer cells (KCs) through short hairpin RNA (shRNA) targeting interleukin-1 receptor associated kinase-4 (IRAK-4) gene. METHODS: Two effective transfection shRNA plasmid (pSIIRAK-4-A, pSIIRAK-4-B) and one invalidated plasmids (pSIIRAK-4-C) targeting IRAK-4 gene were constructed. The isolated mouse KCs were divided into three groups: the normal control group, the RNAi control group (pSIIRAK-4-C) and the RNAi effective group (pSIIRAK-4-A, pSIIRAK-4-B). Then KCs were stimulated with 0.1 microg/ml lipopolysaccharide (LPS) after 24 h transfection with the constructed plasmid. The expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot at 6 h after LPS stimulation, and the activities of NF-kappaB in KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h. RESULTS: The level of IRAK-4, the activities of NF-kappaB and the TNF-alpha level in the RNAi effective group were evidently lower than those in normal and RNAi control groups (P < 0.01) at 1 h, 3 h, and 6 h. Especially, the pSIIRAK-4-A group in which the changes of the above indices were of no difference (P > 0.05), had better inhibited effects than that of the pSIIRAK-4-B group (P < 0.01). CONCLUSION: The shRNA targeting IRAK-4 gene could effectively inhibit the activation of endotoxin-induced KCs.

[Expression of CD14 protein in U937 cells induced by vitamin D3].

AIM: To observe the 1, 25-dihydroxy vitamin D3 (Vit D3) induced CD14 expression in human U937 cell line and the reaction of the cells to LPS stimulation following the induction. METHODS: U937 cells were cocultured with 0.1 mumol/L Vit D3 for 24 hours to induce the expression of CD14 gene and the sensitiveness of U937 cells to stimulation of LPS at various concentrations and for different times were observed. RESULTS: Vit D3 stably induced U937 cells to express CD14 mRNA and CD14 protein. The sensitivity of U937 cells to LPS stimulation increased notably after Vit D3 induction. It was demonstrated that low concentration of LPS stimulated the activation of NF-kappaB in U937/CD14 cells, and enhanced the transcription and expression of TNF-alpha gene, and even release of TNF-alpha into the culture supernatant. CONCLUSION: Vit D3 can induce U937 cells to express CD14 gene and CD14 protein and enhance the reactivity of U937/CD14 cells to LPS stimulation.

The U937 cell line induced to express CD14 protein by 1,25-dihydroxyvitamin D3 and be sensitive to endotoxin stimulation.

BACKGROUND: CD14 was first described as a differentiation antigen on the surface of myeloid lineage cells. It acts as a glycosylphosphatidylinositol (GPI)-anchored receptor for the complex of lipopolysaccharide (LPS) and plays a key role in the activation of LPS-induced monocytes. The purpose of this study was to observe the expression of CD14 protein and its gene in the human U937 promonocytic cell line when these cells were exposed to 1,25-dihydroxyvitamin D3(VitD3) and investigate their sensitivity to endotoxin stimulation. METHODS: U937 cells were exposed to (0.1 micromol) VitD3 for 24 hours and were induced to express the CD14 mRNA gene and CD14 protein, then their responses were observed when they were stimulated with different concentrations of LPS for different time. RESULTS: The U937 cells induced by VitD3 were found to stably express CD14 mRNA and CD14 protein. And CD14 protein enhanced the sensitivity of U937/CD14 cells to lipopolysaccharide (LPS) stimulation. NF-kappaB in U937/CD14 cells can be activated with low concentration of LPS (1 ng/ml-10 ng/ml), the TNF-alpha mRNA gene was induced, and then TNF-alpha was produced and released into the supernatant of culture. CONCLUSION: VitD3 can induce U937 cell to express the CD14 gene and CD14 protein and enhance the response of this type of cells to LPS stimulation.