Integrative analysis reveals that SLC38A1 promotes hepatocellular carcinoma development via PI3K/AKT/mTOR signaling via glutamine mediated energy metabolism.

Although hepatocellular carcinoma (HCC) is rather frequent, little is known about the molecular pathways underlying its development, progression, and prognosis. In the current study, we comprehensively analyzed the deferentially expressed metabolism-related genes (MRGs) in HCC based on TCGA datasets attempting to discover the potentially prognostic genes in HCC. The up-regulated MRGs were further subjected to analyze their prognostic values and protein expressions. Twenty-seven genes were identified because their high expressions were significant in OS, PFS, DFS, DSS, and HCC tumor samples. They were then used for GO, KEGG, methylation, genetics changes, immune infiltration analyses. Moreover, we established a prognostic model in HCC using univariate assays and LASSO regression based on these MRGs. Additionally, we also found that SLC38A1, an amino acid metabolism closely related transporter, was a potential prognostic gene in HCC, and its function in HCC was further studied using experiments. We found that the knockdown of SLC38A1 notably suppressed the growth and migration of HCC cells. Further studies revealed that SLC38A1 modulated the development of HCC cells by regulating PI3K/AKT/mTOR signaling via glutamine mediated energy metabolism. In conclusion, this study identified the potentially prognostic MRGs in HCC and uncovered that SLC38A1 regulated HCC development and progression by regulating PI3K/AKT/mTOR signaling via glutamine mediated energy metabolism, which might provide a novel marker and potential therapeutic target in HCC.

LPS induces HMGB1 relocation and release by activating the NF-κB-CBP signal transduction pathway in the murine macrophage-like cell line RAW264.7.

BACKGROUND: High mobility group protein B1 (HMGB1) is an important late inflammatory mediator in sepsis. Understanding the mechanisms that regulate HMGB1 release from cells and their downstream signal transduction pathways may lead to the ability to develop anti-HMGB1 therapies to treat inflammation. MATERIALS AND METHODS: We stimulated murine macrophage-like RAW 264.7 cells with lipopolysaccharide (LPS) and LPS+ ethylpyruvate (EP) and examined the resulting HMGB1 expression and release. We also studied the expression of related signal transduction factors (NF-κB, p38 MAPK, and CBP). RESULTS AND CONCLUSION: Gene expression of HMGB1 mRNA in RAW264.7 cell showed no significant change at 0-18 h after stimulation with LPS, but increased significantly at 24, 36, and 48 h. HMGB1 mRNA expression in the LPS+EP group was significantly lower than in LPS alone. HMGB1 was distributed mainly in the nucleus; the cytoplasmic level was low before LPS stimulation. After stimulation with LPS, cytoplasmic HMGB1 increased gradually and plateaued at a high level at 12-48 h. Nuclear HMGB1 decreased gradually at 12-24 h, then increased, maintaining a comparatively high level at 36-48 h. EP prevented this pattern significantly. LPS induced p38 MAPK activation and NF-κB signal pathways first, followed by CBP activation. Activated CBP acetylated HMGB1 was stored in a crino-lysosome and secreted activated NF-κB resulted in increased transcription and synthesis of HMGB1, but the expression of up-regulated HMGB1 mRNA was delayed. Extracellular HMGB1 originated from early synthetic reserves present in the nucleus. New HMGB1 protein was synthesized in the nucleus and transferred into the cytoplasm, causing an increase in HMGB1 in the nucleus and cytoplasm. EP inhibits HMGB1 mRNA up-regulation and release from LPS- stimulated macrophages. The molecular function of EP is to attenuate the activation p38 MAPK, NF-κB, and CBP signaling pathways.

Comparative analysis of first-line treatment regimens for advanced EGFR-mutant non-small cell lung cancer patients with stable brain metastases.

BACKGROUND: To compare the survival outcomes of first-line treatment regimens for advanced epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) patients with stable brain metastases. METHODS: We conducted a systematic review of available data from randomized controlled trials (RCTs) of first-line treatment regimens of NSCLC patients with stable brain metastases. Progression free survival (PFS) and overall survival (OS) were extracted and analysed from the RCT subgroups. A network meta-analysis was constructed using the Bayesian statistical model to synthesize the survival outcomes of all the treatments. RESULTS: The analysis included 6 eligible RCT subgroups with 417 patients and 7 treatment regimens osimertinib, afatinib, first-generation EGFR-TKI (gefitinib or erlotinib), erlotinib + bevacizumab, gefitinib + pemetrexed + carboplatin, gemcitabine + cisplatin, and pemetrexed + cisplatin. Of these seven treatment regimens, gefitinib + pemetrexed + carboplatin had the highest potential for favorable PFS and OS, followed by osimertinib, in the treatment of advanced EGFR-mutant NSCLC patients with stable brain metastases. None of the results met the predetermined statistical significance of P<0.05. CONCLUSIONS: The regimens of "Gefitinib + pemetrexed + carboplatin" and "Osimertinib" were associated with the most favorable PFS and OS compared to the other therapies in advanced EGFR-mutant NSCLC patients with stable brain metastases, although the difference between these regimens and the others was not statistically significantly different.

Prevention of common bile duct injury during laparoscopic cholecystectomy.

BACKGROUND: Since the widespread adoption of laparoscopic cholecystectomy (LC) in the late 1980s, a rise in common bile duct (CBD) injury has been reported. We analyzed the factors contributing to a record of zero CBD injuries in 10 000 consecutive LCs. METHODS: The retrospective investigation included 10 000 patients who underwent LC from July 1992 to June 2007. LC was performed by 4 teams of surgeons. The chief main surgeon of each team has had over 10 years of experience in hepatobiliary surgery. Calot's triangle was carefully dissected, and the relationship of the cystic duct to the CBD and common hepatic duct was clearly identified. A clip was applied to the cystic duct at the neck of the gallbladder and the duct was incised with scissors proximal to the clip. The cystic artery was dissected by the same method. Then, the gallbladder was dissected from its liver bed. A drain was routinely left at the gallbladder bed for 1-2 days postoperatively. RESULTS: No CBD injuries occurred in 10 000 consecutive LCs, and there were 16 duct leaks (0.16%). Among these, there were 10 Luschka duct leaks (0.1%) and 6 cystic duct leaks (0.06%). Four hundred thirty cases were converted to open cholecystectomy (OC), giving a conversion rate of 4.3%. After a mean follow-up of 17.5 months (range 6-24 months), no postoperative death due to LC occurred, and good results were observed in 95% of the patients. CONCLUSIONS: In our 10 000 LCs with zero CBD injuries, the techniques used and practices at our department have been successful. Surgeon's expertise in biliary surgery, preoperative imaging, precise operative procedures, and conversion from LC to OC when needed are important measures to prevent CBD injuries.

Effect of Autologous Bone Marrow Stem Cell Therapy in Patients with Liver Cirrhosis: A Meta-analysis.

Background and Aims: Although autologous bone marrow stem cell (BMSC) transplantation is an effective treatment for liver cirrhosis, there are few reports describing the optimal delivery route and number of injected BMSCs. Methods: A literature search was conducted using PubMed, ISI Web of Science, Cochrane Central Register of Controlled Trials, and EBSCO. A meta-analysis was performed to assess the effect of BMSCs on liver and coagulation function indices. Subgroup analysis was performed based on number of injected BMSCs, delivery route, and length of follow-up. Results: A total of 15 studies were selected from among 1903 potential studies for analysis. Autologous BMSC transplantation significantly improved aspartate aminotransferase, total bilirubin, albumin, prothrombin time, prothrombin activity, prothrombin concentration, Child-Pugh score, and model for end-stage liver disease. In the subgroup analysis of cell numbers, all four of the indices were significantly improved when the number of BMSCs was >4 × 108. The subgroup analysis referring to the delivery route showed that arterial infusion increased the therapeutic effect over venous infusion. Finally, in the subgroup analysis of follow-up length, the results showed that BMSC therapy significantly improved liver function at 2 weeks after transplantation. In addition, this therapy improved coagulation 4 weeks after the transplant, with a maintenance of efficacy for up to 24 weeks. Conclusions: Autologous BMSC therapy is beneficial for liver improvement and coagulation in patients with liver cirrhosis. The therapeutic effect was generated at 2-4 weeks after transplantation. The effect lasted for 24 weeks but no more than 48 weeks. The greatest benefit to patients was observed with a 4 × 108 autologous BMSC transplant via the hepatic artery.

[Reversal of adriamycin resistance of hepatocellular carcinoma by targeting it with recombined adenovirus carrying antisense multidrug resistance gene 1 RNA].

OBJECTIVE: To investigate if an adenovirus vector carrying antisense multidrug resistance gene 1 (MDR1) could reverse multidrug resistance (MDR) of HepG2/ adriamycin (ADM) cells in tumors transplanted in athymic mice. METHODS: An adenovirus vector carrying AFP promoter and antisense MDR1 was constructed. HepG2 MDR cells (HepG2/ADM) were induced by graded resistance to ADM and were subcutaneously inoculated into athymic mice to construct the transplanted tumor. After adeno-asmdr1 was injected, the volume of the transplanted tumor and the apoptotic body in the xenograft tumor cells were observed and reverse transcriptase polymerase chain reaction was employed to investigate the expression of the mdr1-mRNA from the mouse transplanted tumor cells. RESULTS: Following injection with adeno-asmdr1, the tumor volumes in this mice group did not increase. However the tumor volume in the PBS plus ADM group did increase significantly (P less than 0.05). In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and found to be reduced at week 1, and at week 4 in the ADM+asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM+asmdr1 group compared to the ADM group at the 4th week. Evidence of apoptosis was observed in the tumor xenograft cells treated with adeno-asmdr1, but there was rarely any apoptosis in the group treated with ADM and PBS. CONCLUSION: Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.

Microrna Expression Profiling of Macrophage Line Raw264.7 Infected by Candida Albicans.

INTRODUCTION: The aim of this study was to clarify the microRNA (miRNA) expression profiles of RAW264.7 macrophages infected by Candida albicans to elucidate the roles of differentially expressed miRNAs and to further explore the mechanisms underlying the immune response to C. albicans infection. METHODS: High-throughput miRNA microarray analysis was performed to detect differentially expressed miRNAs in control and C. albicans-infected RAW264.7 cells. Quantitative real-time PCR analysis was used to verify the microarray results. Target genes of differentially expressed miRNAs were predicted with bioinformatics software. The cell biological processes and signaling pathways of these miRNA-targeted genes involved in C. albicans infection were predicted by gene ontology (GO) enrichment and pathway analyses. RESULTS: Significant upregulation of eight miRNAs (mmu-miR-140-5p, mmu-miR-96-5p, mmu-miR-8109, mmu-miR-466i-3p, mmu-miR-222-5p, mmu-miR-301b-3p, mmu-miR-466g, and mmu-miR-7235-5p) and downregulation of eight miRNAs (mmu-miR-3154, mmu-miR-223-3p, mmu-miR-494-3p, mmu-miR-6908-5p, mmu-miR-188-5p, mmu-miR-6769b-5p, mmu-miR-7002-5p, and mmu-miR-1224-5p) were observed, as compared with the control (fold change ≥2.0 and P < 0.05). GO analysis revealed that both mmu-miR-140-5p and mmu-miR-223-3p participated in immune responses, inflammatory reactions, and cell apoptosis in C. albicans infection. Also, the MAPK signaling pathway was found to play an important role in the immune response against C. albicans infection. CONCLUSIONS: This study revealed comprehensive expression and functional profiles of differentially expressed miRNAs in macrophage RAW264.7 cells infected by C. albicans. These findings should help to further elucidate the mechanisms underlying the immune response to C. albicans infection.

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.

[Expression of augmenter of liver regeneration in hepatic tumor cells and its clinical significance].

OBJECTIVE: To investigate the effects of augmenter of liver regeneration (ALR) on the proliferation of hepatocytes and hepatic tumor cells and the expression of ALR in herpatocellular carcinoma (HCC). METHODS: Primary rat hepatocytes, QGY and HepG2 cells were cultured separately with ALR from different species. Cell proliferation was detected by their 3H-TdR uptake. The expression of ALR was examined in 9 normal hepatic tissues and 21 HCC cases using immunohistochemistry method. RESULTS: Different ALRs could stimulate the proliferation of HepG2 and QGY cells in a dose-dependent way in vitro, but all ALR had no influence in the proliferation of primary rat hepatocytes. The expression of ALR was absent in normal hepatic tissues, but present in all HCC hepatic tissues. However, the expression of ALR had no relationship with the differentiation and size of the carcinomas. CONCLUSION: ALR might play an important role in the occurrence and development of HCC.

Inhibition effect of glycyrrhizin in lipopolysaccharide-induced high-mobility group box 1 releasing and expression from RAW264.7 cells.

INTRODUCTION: High-mobility group box 1 (HMGB1) is a therapeutic target for sepsis. Glycyrrhizin (GL) is the aglycone of glycyrrhizin derived from licorice. We clarified the anti-inflammatory effects of GL. We explored the anti-HMGB1 effect of GL and elucidated its molecular mechanism, which will be of benefit for sepsis treatment. METHODS: We stimulated murine macrophage-like RAW 264.7 cells with lipopolysaccharide (LPS) and LPS + GL, then measured the expression and release of HMGB1. The expression of related signal transduction factors was detected. RESULTS: High-mobility group box 1 was distributed mainly in the nucleus with lower cytoplasmic levels in RAW 264.7 cells before LPS stimulation. After stimulation, cytoplasmic HMGB1 levels increased gradually, whereas in nuclear fluctuation a trend of HMGB1 expression was observed. Significant upregulation of HMGB1 mRNA occurred 12 h after LPS stimulation. Glycyrrhizin prevented the transfer of HMGB1 from the nucleus to the cytoplasm and inhibited upregulation of HMGB1 mRNA induced by LPS. Phospho-p38 mitogen-activated protein kinase and activated activating protein 1 increased significantly 8 h after LPS stimulation. Tumor necrosis factor α and interleukin 6 increased 4 h after LPS stimulation and peaked at 48 h, and HMGB1 increased at 8 h. The Toll-like receptor 4/MD2/nuclear factor κB signaling pathway was activated 4 h after LPS stimulation. Glycyrrhizin inhibited this pathway. CONCLUSIONS: Glycyrrhizin inhibited the expression and release of HMGB1 through blocking the p38 mitogen-activated protein kinase/activating protein 1 signaling pathway then inhibited the massive release of tumor necrosis factor α and interleukin 6.

Lipopolysaccharide induced synthesis of CD14 proteins and its gene expression in hepatocytes during endotoxemia.

AIM: To observe synthesis of CD14 protein and expression of CD14 mRNA in hepatic tissue and hepatocytes of rats during endotoxemia. METHODS: The endotoxemia model of Wistar rat was established by injection of a dose of lipopolysaccharide (LPS) (5mg x kg(-1), Escherichia coli O111:B4) via the tail vein, then the rats were sacrificed after 3, 6, 12 and 24 h in batches respectively. Hepatocytes were isolated from normal and LPS-injected rats by in situ collagenase perfusion technique and were collected to measure the expression of CD14 mRNA and synthesis of CD14 protein by reverse transcript-polymerase chain reaction (RT-PCR) or Western blot analysis. The binding of fluorescein isothiocyanate (FITC)-CD14 polyclonal antibody to isolated hepatocytes was also assessed by flow cytometric analysis (FCM). RESULTS: In the rats with endotoxemia, the expressions of CD14 mRNA in hepatic tissue and isolated hepatocytes were stronger at 3, 6, and 12 h than that in control rats (3.48+/-0.15, 5.89+/-0.62, 4.33+/-0.18, vs 1.35+/-0.14 in hepatic tissue, P<0.01; 4.12+/-0.17, 6.24+/-0.64, 4.35+/-0.18, vs 1.87+/-0.15 in hepatocytoes, P<0.01). The synthesis of CD14 protein in hepatic tissue and isolated hepatocytes increases also obviously in 6 and 12 h when compared to that in control rats (13.27+/-1.27, 17.32+/-1.35, 11.42+/-1.20, vs 7.34+/-0.72 in hepatic tissue, P<0.01; 14.68+/-1.30, 17.95+/-1.34,11.65+/-1.19, vs 7.91+/-0.70 in hepatocytes, P<0.01). FCM showed that mean fluorescence intensity (MFI) and numbers of FITC-CD14 positive cells in the rats with endotoxemia increased obviously at 3,6,12 and 24h when compared with normal control group (43.4%, 70.2%, 91.4%, 32.6% vs 4.5%, P<0.01). CONCLUSION: LPS can markedly promote the synthesis of CD14 protein and up-regulate the expression of CD14 mRNA in isolated hepatocytes and hepatic tissue. Liver might be a main source for soluble CD14 production during endotoxemia.

Expression of CD14 protein and its gene in liver sinusoidal endothelial cells during endotoxemia.

AIM: To observe expression of CD14 protein and CD14 gene in rat liver sinusoidal endothelial cells (LSECs) during endotoxemia, and the role of CD14 protein in the activation of lipopolysaccharide (LPS)-induced LSECs. METHODS: Wistar rat endotoxemia model was established first by injection of a dose of LPS (5mg/kg, Escherichia coli O111:B4 ) via the tail vein, then sacrificed after 0 h,3h,6h, 12h, and 24h, respectively. LSECs were isolated from normal and LPS-injected rats by an in situ collagenase perfusion technique. The isolated LSECs were incubated with rabbit anti-rat CD14 polyclonal antibody, then stained with goat anti rabbit IgG conjugated fluorescein isothiocyanate(FITC) and flow cytometric analysis (FCM) was performed. The percentage and mean fluorescence intensity (MFI) of CD14-positive cells were taken as the indexes. LSECs were collected to measure the expression of CD14 mRNA by in situ hybridization analysis. The isolated LSECs from normal rats were incubated firstly with anti-CD14 antibody, then stimulated with different concentrations of LPS, and the supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)-a and Interleukin (IL)-6 with ELISA. RESULTS: In rats with endotoxemia, LSECs displayed a strong MFI distinct from that of control rats. CD14 positive cells in rats with endotoxemia were 54.32%, 65.83%, 85.64%, and 45.65% at 3h, 6h, 12h, and 24h respectively, there was significant difference when compared to normal group of animals (4.45%)(P<0.01). The expression of CD14 mRNA in isolated LSECs was stronger than that in control rats. In LPS group, the levels of TNF-alpha and IL-6 were 54+/-6 ng.L(-1), 85+/-9 ng.L(-1), 206+/-22 ng.L(-1), 350+/-41 ng.L(-1), 366+/-42 ng.L(-1) and 103+/-11 ng.L(-1), 187+/-20 ng.L(-1), 244+/-26 ng.L(-1), 290+/-31 ng.L(-1), and 299+/-34 ng.L(-1), respectively at different concentration points. In anti-CD14 group, the levels of TNF-alpha and IL-6 were 56+/-5 ng.L(-1), 67+/-8 ng.L(-1), 85+/-10 ng.L(-1), 113+/-12 ng.L(-1), 199+/-22 ng.L(-1) and 104+/-12 ng.L(-1), 125+/-12 ng.L(-1), 165+/-19 ng.L(-1), 185+/-21 ng.L(-1), and 222+/-23 ng.L(-1), respectively at different concentration points. There was significant difference between the two groups (P<0.01). CONCLUSION: LSECs can synthesize CD14 protein and express CD14 gene during endotoxemia. CD14 protein plays an important role in the activation of LPS-induced LSECs. This finding has important implications for the understanding of the mechanisms by which LPS may injure liver sinusoidal endothelial cells during sepsis.

Intestinal damage mediated by Kupffer cells in rats with endotoxemia.

AIM: To determine the in vivo effects of phagocytic blockade of Kupffer cell (KC) on the release of proinflammatory cytokines in small intestinal lesion and on the integrity of intestinal tract by using gadolinium chloride (GdCl(3)) during early endotoxemia. METHODS: Wistar rats were divided into three groups: Group A, rats were injected with endotoxin (E. coli O111:B(4), a dose of 12 mg x kg(-1)) only; Group B, rats were pretreated intravenously with 25 mg of GdCl(3) per kg 24 h are given endotoxin; and Group C, sham operation only. All animals were sacrificed 4 h after endotoxin injection. In portion of the rats of three groups, bile duct was cannulated, which the bile was collected externally. Morphological changes of ileum were observed under light microscopy and electronic microscopy. The KC were isolated from rats by collagenase perfusion and in KC, expression of TNF-alpha and IL-6 mRNA were determined by RT-PCR analysis. Plasma and bile TNF-alpha and IL-6 Levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In group A, there were neutrophil infiltration and superficial epithelial necrosis of the ileal villi, sloughing of mucosal epithelium, and disappearance of some villi. In group B, the ileal mucosal damage was much reduced. which in group C, no significant morphological changes were seen. GdCl(3) pretreatment decreased significantly the expression of TNF-alpha and IL-6 mRNA in group B (4.32+/-0.47 and 4.05+/-0.43) when compared to group A (9.46+/-1.21 and 9.04+/-1.09) (P<0.05). There was no significant expression of TNF-alpha and IL-6 mRNA in group C (1.03+/-0.14 and 10.4+/-0.13). In rats of group A, the levels of TNF-alpha and IL-6 in bile and plasma were 207+/-29 ng x L(-1), 1032+/-107 ng x L(-1), 213+/-33 ng x L(-1), and 1185+/-127 ng x L(-1), respectively. In group B, they were 113+/-18 ng x L(-1), 521+/-76 ng x L(-1), 147+/-22 ng x L(-1), and 572+/-54 ng x L(-1), respectively. In group C, they were 67+/-10 ng x L(-1), 72+/-13 ng x L(-1), 109+/-18 ng x L(-1), and 118+/-22 ng x L(-1) respectively. There were significant difference between the three group (P<0.05). CONCLUSION: KC release cytokines TNF-alpha and IL-6 causing damage to the integrity of intestinal epithelium and play a crucial role in the initiation and progression of intestinal mucosal damage during early endotoxemia.

Liver sinusoidal endothelial cell injury by neutrophils in rats with acute obstructive cholangitis.

AIM: The objective of this study is to elucidate the potential role of poly-morphonuclear neutrophils (PMN) in the development of such a sinusoidal endothelial cell (SEC) injury during early acute obstructive cholangitis (AOC) in rats. METHODS: Twenty one Wistar rats were divided into three groups: the AOC group, the bile duct ligated group (BDL group), and the sham operation group (SO group). The common bile duct (CBD) of rats in AOC group was dually ligated and 0.2 ml of the E. Coli O(111) B(4) (5 X 10(9)cfu/ml) suspension was injected into the upper segment, in BDL group, only the CBD was ligated and in SO group, neither injection of E. Coli suspension nor CBD ligation was done, but the same operative procedure. Such group consisted of seven rats, all animals were killed 6h after the operation. Morphological changes of the liver were observed under light and electron microscope. Expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in hepatic tissue was determined with reverse transcription polymerase chain reaction (RT-PCR). The serum levels of alanine aminotransferase (ALT) were determined with an autoanalyger and cytokine-induced neutrophil chemoattractant (CINC) was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Neutrophils was accumulated in the hepatic sinusoids and sinusoidal endothelial cell injury existed in AOC group. In contrast, in rats of BDL group, all the features of SEC damage were greatly reduced. Expression of ICAM-1 mRNA in hepatic tissue in three groups were 7.54 +/- 0.82, 2.87 +/- 0.34, and 1.01 +/- 0.12, respectively. There were significant differences among three groups (P<0.05). The serum CINC levels in the three groups were 188 +/- 21 ng.L(-1), 94+/-11 ng.L(-1), and 57+/-8 ng.L(-1), respectively. There were also significant differences among the three groups (P<0.05). Activity of the serum ALT was 917 +/- 167 nkat.L(-1), 901 +/- 171 nkat.L(-1), and 908 +/- 164 nkat.L(-1), respectively, (P<0.05). CONCLUSION: Hepatic SEC injury occurs earlier than hepatic parenchymal cells during AOC. Recruitments of circulating neutrophils in the hepatic sinusoidal space might mediate the SEC injury, and ICAM-1 in the liver may modulate the PMN of accumulation.

Nuclear factor kB activity in patients with acute severe cholangitis.

AIM: To determine the NF-kB activity in peripheral blood mononuclear cells (PBMC) in patients with acute cholangitis of severe type (ACST) and correlate the degree of NF-kB activation with severity of biliary tract infection and clinical outcome. METHODS: Twenty patients with ACST were divided into survivor group (13 cases) and nonsurvivor group (7 cases). Other ten patients undergoing elective gastrectomy or inguinal hernia repair were selected as control group. Peripheral blood samples were taken 24 hours postoperatively. PBMC were separated by density gradient centrifugation, then nuclear proteins were isolated from PBMC, and Electrophoretic Mobility Shift Assay (EMSA) used determined. The results were quantified by scanning densitometer of a Bio-Image Analysis System and expressed as relative optical density (ROD). The levels of TNF-alpha, IL-6, and IL-10 in the plasma of patients with ACST and healthy control subjects were determined by using an enzyme-linked immunoassay (ELISA). RESULTS: The NF-kB activity was 5.02 +/- 1.03 in nonsurvivor group, 2.98 +/- 0.51 in survivor group and 1.06 +/- 0.34 in control group. There were statistical differences in three groups (P<0.05). The levels of TNF-alpha and IL-6 in plasma were (498 +/- 53)ng.L(-1)and (587 +/- 64)ng.L(-1)in nonsurvivor group, (284 +/- 32)ng.L(-1) and (318 +/- 49)ng.L(-1)in survivor group and (89 +/- 11)ng.L(-1) and (102 +/-13)ng.L(-1)in control group. All patients with ACST had increased levels of TNF-alpha and IL-6, which were many-fold greater than those of control group, and there was an evidence of significantly higher levels in those of nonsurvivor group than that in survivor group (P<0.05). The levels of IL-10 in plasma were (378+/-32)ng.L(-1), (384+/-37)ng.L(-1) and (68+/-11)ng.L(-1) in three groups, respectively. All patients had also increased levels of IL-10 when compared with control group (P<0.05), but the IL-10 levels were not significantly higher in nonsurvivors than in survivors (P>0.05). CONCLUSION: NF-kB activity in PBMC in patients with ACST increases markedly and the degree of NF-kB activation is correlated with severity of biliary tract infection and clinical outcome.

Role of NF-kB in multiple organ dysfunction during acute obstructive cholangitis.

AIM: To elucidate the role of NF-kB activation in the development of multiple organ dysfunction (MOD) during acute obstructive cholangitis (AOC) in rats. METHODS: Forty-two Wistar rats were divided into three groups: the AOC group, the group of bile duct ligation (BDL group), and the sham operation group (SO group). All the animals in the three groups were killed in the 6th and 48th hour after operation. Morphological changes of vital organs were observed under light and electron microscopy. NF-kB activation was determined with Electrophoretic Mobility Shift Assay (EMSA). Arterial blood gas analyses and the serum levels of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine were performed. The concentrations of TNF-alpha and IL-6 in plasma were also measured. RESULTS: The significant changes of histology and ultrastructure of vital organs were observed in AOC group. By contrast, in BDL group, all the features of organs damage were greatly reduced. Expression of NF-kB activation in various tissues increased in AOC group when compared to other two groups. At 6 h, the arterial pH in three groups was 7.52+/-0.01, 7.46+/-0.02, and 7.45+/-0.02, and the blood pCO(2) was 33.9+/-0.95 mmHg, 38.1+/-0.89 mmHg, 38.9+/-0.94 mmHg, there was difference in three groups (P<0.05). At 48 h, the blood pH values in three groups was 7.33+/-0.07, 7.67+/-0.04, and 7.46+/-0.03, and blood HCO(3)(-) was 20.1+/-1.29 mmol x L(-1), 26.7+/-1.45 mmol x L(-1) and 27.4+/-0.35 mmol x L(-1), there was also difference in three groups (P<0.05). In AOC group, Levels of LDH, ALT, BUN and creatinine were 1,6359.9+/-2,278.8 nkat x L(-1), 5,796.2+/-941.9 nkat.L(-1), 55.7+/-15.3 mg/dl, and 0.72+/-0.06 mg/dl, which were higher than in SO group (3,739.1+/- 570.1 nkat x L(-1), 288.4+/-71.7 nkat x L(-1), 12.5+/-2.14 mg/dl, and 0.47+/-0.03 mg/dl) (P<0.05). Levels of plasma TNF-alpha and IL-6 in AOC at 48 h were 429+/-56.62 ng x L(-1) and 562+/-57 ng x L(-1), which increased greatly when compared to BDL group (139+/-16 ng x L(-1), 227+/-43 ng x L(-1)) and SO group (74+/-10 ng x L(-1), 113+/-19 ng x L(-1)) (P<0.05). CONCLUSION: The pathological damages and the NF-kB activation of many vital organs excised during AOC. These findings have an important implication for the role of NF-kB activation in MOD during AOC.