Up-regulated SAMD9L modulated by TLR2 and HIF-1α as a promising biomarker in tuberculosis.

The aim of this study was to identify potential biomarkers of TB in blood and determine their function in Mtb-infected macrophages. First of all, WGCNA was used to analyse 9451 genes with significant changes in TB patients' whole blood. The 220 interferon-γ-related genes were identified, and then 30 key genes were screened using Cytoscape. Then, the AUC values of key genes were calculated to further narrow the gene range. Finally, we identified 9 genes from GSE19444. ROC analysis showed that SAMD9L, among 9 genes, had a high diagnostic value (AUC = 0.925) and a differential diagnostic value (AUC>0.865). To further narrow down the range of DEGs, the top 10 hub-connecting genes were screened from monocytes (GSE19443). Finally, we obtained 4 genes (SAMD9L, GBP1, GBP5 and STAT1) by intersections of genes from monocytes and whole blood. Among them, it was found that the function of SAMD9L was unknown after data review, so this paper studied this gene. Our results showed that SAMD9L is up-regulated and suppresses cell necrosis, and might be regulated by TLR2 and HIF-1α during Mtb infection. In addition, miR-181b-5p is significantly up-regulated in the peripheral blood plasma of tuberculosis patients, which has a high diagnostic value (AUC = 0.969).

Laser speckle contrast imaging and Oxygen to See for assessing microcirculatory liver blood flow changes following different volumes of hepatectomy.

OBJECTIVE: Portal hyperperfusion after extended hepatectomy or small-for-size liver transplantation may induce organ dysfunction and failure. This study was designed to monitor and characterize the hepatic microcirculatory perfusion following different volumes of hepatectomy in rats by using laser speckle contrast image (LSCI) and Oxygen to See (O2C), a spectrometric device. METHODS: The microcirculatory liver blood flow of the rats that underwent 68%, 85% and 90% hepatectomy (68PH, 85PH and 90PH) was monitored with LSCI and O2C before and following the hepatectomy. The portal venous flow (PVF) and hepatic arterial flow (HAF) were measured with an ultrasonic flowmeter. Liver regeneration, liver injury, histologic evaluation and gene expression were also assessed at 12h, 24h, 3d and 7d post hepatectomy. RESULTS: All the 68PH and 85PH rats survived, and 57% of the 90PH rats survived. After hepatectomy, both PVF and HAF decreased transiently, with the PVF of the 85PH and 90PH rats significantly lower than that of the 68PH rats. In contrast, the PVF and HAF per gram of liver weight were greatly increased after liver resection and were proportional to the volume of resected liver. Correspondingly, the microcirculatory liver blood flow of the 68PH, 85PH and 90PH rats, as assessed by both LSCI and O2C, were increased after hepatectomy, and the 90PH group was significantly higher than the 68PH and 85PH groups. The hyperperfusion continued for approximately 3days and returned to baseline following the completion of liver regeneration. The liver venous oxygen saturation of the three groups decreased immediately after hepatectomy and returned to baseline from 24h after hepatectomy. The 90PH rats also showed delayed liver regeneration and the most severe liver injury, as reflected by increased serum ALT, AST and TBIL levels, hepatocellular vacuolization, and inflammatory and endothelial constriction gene expressions (TNF-α, IL-1ß, MIP-1α, ET-1 and TM-1). CONCLUSION: Hepatic microcirculation hyperperfusion resulting from major and extended liver resection could be assessed by LSCI and O2C methods. The 90PH in rats led to extraordinary sinusoidal hyperperfusion, severe endothelial injury and liver failure. Monitoring the changes of hepatic microcirculation perfusion following extended hepatectomy or small-for-size liver transplantation may help to analyze the extent of hyperperfusion.

Hepatoma-derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells.

We aimed to elucidate the effects of hepatoma-derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real-time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP-2 and MMP-9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF-siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF-siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright © 2016 John Wiley & Sons, Ltd.

Portal vein clamping alone confers protection against hepatic ischemia-reperfusion injury via preserving hepatocyte function in cirrhotic rats.

BACKGROUND: Chronic liver diseases always increase the risk of liver failure after hepatectomy. We aimed to explore the protective effect of portal vein clamping without hepatic artery blood control (PVC) on a cirrhotic rat liver that underwent ischemia and reperfusion. METHODS: Carbon tetrachloride-induced cirrhotic rats were randomly assigned to four groups as follows: cirrhotic control, PVC, portal triad clamping (PTC), and intermittent portal triad clamping (IC). After 45 min of portal vascular clamping, hepatic injury and liver function were investigated by assessing the 7-d survival rate, liver blood loss, serum alanine aminotransferase, liver tissue malondialdehyde, liver tissue adenosine triphosphate, indocyanine green retention rate, and morphology changes of the rat liver. RESULTS: The 7-d survival rates in the PVC and IC groups were much higher than in the PTC group. The PVC group had more liver blood loss during the hepatectomy than the PTC group, but had much less than the cirrhotic control group (P < 0.01). In addition, there were no differences between the IC group and PVC group. The PVC rats had a significantly higher adenosine triphosphate level in the liver tissue and a markedly lower indocyanine green retention rate than the PTC and IC rats (P < 0.05). At 1, 6, and 24 h after reperfusion, the alanine aminotransferase and malondialdehyde levels in the PTC group were much higher than those in the PVC and IC groups (P < 0.05). Based on the histopathologic analysis, hepatic injury in the PVC and IC groups were similar but less prominent than in the PTC group. CONCLUSIONS: Although both PVC and IC can confer protection against hepatic ischemic-reperfusion injury in cirrhotic rats, the PVC method is more efficient in preserving the energy and function of hepatocytes than the IC method, suggesting better prognosis after hepatectomy.

mTOR-Dependent Suppression of Remnant Liver Regeneration in Liver Failure After Massive Liver Resection in Rats.

BACKGROUND AND AIMS: Massive hepatectomy often leads to fatal liver failure because of a small remnant liver volume. The aim of this study was to investigate the potential mechanisms leading to liver failure. METHODS: Sprague-Dawley rats had performed a sham operation, 85 % partial hepatectomy (PH) or 90 % PH, and all had free access to water with or without supplemented glucose. Liver function and survival were evaluated. Liver parenchymal injury was assessed by evaluating hepatic pathology, blood biochemistry, and apoptotic and necrotic alterations. The regeneration response was assessed by the weight gain of the remnant liver, hepatocyte proliferation markers, and regeneration-related molecules. RESULTS: The 90 % hepatectomy resulted in a significantly lower survival rate and impaired liver function; however, no significant more serious liver parenchymal injuries were detected. TNF-α, HGF, myc and IL-6 were either similarly expressed or overexpressed; however, the increase in remnant liver weight, mitotic index, and the presence of Ki-67 and PCNA were significantly lower in the 90 %-hepatectomized rats. mTOR, p70S6K and 4EBP1 were not activated in the remnant liver after a 90 % hepatectomy as obviously as those after an 85 % hepatectomy, which was concomitant with the higher expression of phospho-AMPK and a lower intrahepatic ATP level. Glucose treatment significantly improved the survival rate of 90 %-hepatectomized rats. CONCLUSIONS: Suppression of remnant liver regeneration was observed in the 90 % PH and contributed to fatal liver failure. This suppressed liver regenerative capacity was related to the inhibited activation of mTOR signaling.

Establishment of a stable mouse model of brain death by the method of the gradually increasing intracranial pressure.

OBJECTIVE: To establish a stable and modified mouse model of brain death (BD) and to share our experiences in BD induction and maintenance. METHODS: Totally 35 C57BL/6 male mice were randomized into BD group (n=25) or sham control group (n=10). BD was induced by inserting a 2F Fogarty catheter connected to a syringe pump after trepanation of the left frontoparietal area and injecting volume at the speed of 6 µl/min until spontaneous respiration ceased. BD was diagnosed by electroencephalogram, apnea testing,as well as testing of brain stem reflexes. Mechanical ventilation was performed by orotracheal intubation. Right carotid artery was intubated by a PE-10 cannula for the continuous monitoring of mean blood pressure (MAP) and heart rate (HR). The right external jugular vein was catheterized for volume resuscitation.The sham control group underwent the same procedure with catheter insertion but without balloon inflation.Livers were removed and fixed in paraffin to evaluate the histological alterations with the light microscopy. RESULTS: Mouse models of BD were successfully established about 20 minutes after balloon inflation, and the mean balloon volume at the time of BD was (105.77 ± 21.57)µl. The MAP and HR rapidly increased on occurrence of BD and the peak value was (128.28 ± 17.16) mmHg and (434.16 ± 55.75) beat/min, respectively, which were significant higher than those in the sham control group at the same time point (P=0.000). During the 4-hour follow-up time, MAP and HR in 72% (18/25) of BD animals remained haemodynamically stable. No animal died due to anesthesia and surgical operation.Hepatic tissues in BD mice showed mild focal ischemic damages (cellular edema, congestion, and inflammatory infiltration), which were slighter and fewer in sham control group. CONCLUSION: The mouse model of BD was successfully established with lower surgical difficulty and can be performed in a standardized, reproducible and successful way.

The monitoring of microvascular liver blood flow changes during ischemia and reperfusion using laser speckle contrast imaging.

OBJECTIVE: The recovery of microvascular liver blood flow (LBF) after ischemia is an important determinant of the degree of hepatocellular injury. Laser speckle contrast imaging (LSCI) was recently suggested to be a suitable instrument for monitoring the LBF. This study was designed to evaluate LSCI in monitoring the LBF changes during liver ischemia and reperfusion (IR). METHODS: A rat model with 120-min ischemia and 60-min reperfusion to 90% of the liver (entire liver except the caudate lobe, which was kept as portal blood bypass) was used. The LBF of the sham operation (SO) group and the IR group was measured with LSCI at the following time points: before ischemia (Baseline), 5 min after the start of ischemia (I-5 min), 5 min before the end of ischemia (I-115 min) and 5 and 60 min after the start of reperfusion (R-5 min and R-60 min). The reproducibility among different rats or repeated measurements, the liver histopathology, the liver biological zero (BZ) and the influence of liver movement on the LSCI measurements were investigated. RESULTS: The entire exposed liver surface after laparotomy was suitable for full-view LSCI imaging. Establishing many circular or oval regions of interest (ROIs) on the LSCI flux image was a simple and convenient method for calculating and comparing the LBF of different ROIs and different liver lobes. There was good-to-moderate intra-individual and inter-individual reproducibility for the LSCI measurements of the LBF in the rats of the SO group. In the IR group, the total blood inflow occlusion resulted in a notable drop of the LBF from the baseline (P<0.05) that remained for the 120 min of ischemia. The LBF decreased further after the reperfusion (P<0.05), reflecting the IR-induced liver microcirculation dysfunction. The histopathological examination revealed severe hepatic sinus congestion and damaged hepatocytes in the IR group. The no flow BZ and liver movement contributed to the LBF values. CONCLUSIONS: LSCI technology is a simple, convenient and accurate method for the real-time monitoring of microvascular LBF changes during ischemia and reperfusion, regardless of the contribution of biological zero and liver movement. This finding suggests the possible application of LSCI for monitoring the microvascular LBF changes intraoperatively.

The defects of cholangiocyte primary cilia in patients with graft cholangiopathies.

PURPOSE: To observe the morphologic changes in intrahepatic bile ducts and the defects of cholangiocyte primary cilia in patients with graft cholangiopathies. METHODS: Four patients who were diagnosed as graft cholangiopathies and underwent retransplantation were chosen as the study group; another four patients who underwent liver transplantation during the same period and recovered normally six months after the operation were the control group. The serum levels of biochemical indicators were measured, the morphologic changes in intrahepatic bile ducts and cholangiocyte primary cilia were observed, and the ciliary marker (α-tubulin) and membrane proteins (polycystin-1, TPPV4) were detected by immunofluorescence analysis and Western blot. RESULTS: In the study group, biliary structures were vague and some bile ducts disappeared in portal areas; some epithelial cells were lost; lots of collagen was deposited and many phlogocytes infiltrated; microliths were found in some ductal lumens; partial biliary epithelial cells were necrosed; primary cilia and microvilli disappeared. In the control group, the structures of intrahepatic bile ducts and biliary epithelial cells were integrated and the primary cilia were present. CONCLUSIONS: The morphologic changes in biliary epithelial cells and the defects of cholangiocyte primary cilia have a close correlation with graft cholangiopathies in liver transplantation.

Preserving hepatic artery flow during portal triad blood inflow occlusion reduces the outgrowth of hepatocarcinoma in mice after ischemia-reperfusion.

AIM: To investigate the effect of different hepatic vascular occlusion maneuvers on the growth of hepatocarcinoma after liver ischemia-reperfusion (I/R) injury. METHODS: A mice hepatocarcinoma model was established by portal vein injection of H22 hepatoma cells. After 3 days, the mice underwent sham operation, occlusion of portal triad (OPT), portal vein (OPV), or intermittent clamping (INT) operation. The hepatic I/R injury, pathological changes, hepatic replacement area, proliferative cell nuclear antigen expression, and extracellular signal-regulated kinase (ERK) 1/2 activation were assessed 5 days after reperfusion. RESULTS: Alanine aminotransferase and aspartate aminotransferase levels in the OPV group were significantly lower than those in the OPT and INT groups at 24 h after reperfusion. The hepatic injury of clamped liver lobes in the OPV group, represented by histopathological alterations and myeloperoxidase activity, was much slighter than that in the OPT and INT groups. The values of hepatic replacement area in the sham operation, OPT, OPV, and INT groups were 7.661 2.55%, 35.61 1 4.23%, 9.02 1 3.01%, and 19.95 1 4.10%, respectively. Proliferative cell nuclear antigen expression and ERK1/2 activation of tumor cells were the highest in the OPT group, and the lowest in the OPV and INT groups. CONCLUSION: Preserving hepatic artery flow during portal triad blood inflow occlusion substantially inhibits the outgrowth of hepatocarcinoma via attenuating hepatic I/R injury in a murine liver tumor model. These results suggest a better prevention of hepatic tumor outgrowth after hepatectomy by using the selective portal vein clamping method in liver cancer patients.

[Transient hepatic venous occlusion induced liver hemodynamic change and reperfusion injury in rats].

OBJECTIVE: To observe the hemodynamic change and reperfusion injury cause by transient hepatic venous occlusion and transient hepatic inflow occlusion in rats. METHODS: The rat liver was divided into 3 different areas: the ischemia reperfusion (IR) area: the inflow of the right superior lobe was clamped for half an hour; the non-isolated lobe congestive reperfusion (NIL-CR) area: the outflow of the right median lobe was clamped for half an hour; and the isolated lobe congestive reperfusion (IL-CR) area: the outflow of the left lobe was clamped for half an hour. The flux value and the oxygen saturation of microcirculation were monitored before at clamping for 30 minutes, and on 1 day, 3 days ,and 7 days after reperfusion. The hepatic damage and Suzuki's score were evaluated. RESULTS: After clamping for 30 minutes, the flux value in the IR area was significantly higher than in NIL-CR area (P<0.01) and IL-CR area (P<0.01), the oxygen saturation in the IR area was significantly higher than in NIL-CR area (P<0.01) and IL-CR area (P<0.05). Compared with IR area, both NIL-CR area and IL-CR area were found having more severe liver damage in terms of Suzuki's score in early postoperative period (at clamping for 30 minutes and on 1 day, P<0.01). However, there was no significant difference between NIL-CR area and IL-CR area in flux value, oxygen saturation, and Suzuki's score (P>0.05). CONCLUSIONS: Hepatic venous occlusion can more effectively decrease the blood perfrusion and oxygen saturation; thus, compared to the IR, CR can result in more severe liver damage. The presence of normal liver tissue around the congestion area can not influence liver damage in transient hepatic venous occlusion.

Preserving hepatic artery flow during portal triad blood inflow occlusion improves remnant liver regeneration in rats after partial hepatectomy.

BACKGROUND: In the present study, we compared preservation of the hepatic artery flow during liver blood inflow occlusion with total portal triad blood flow clamping (the Pringle maneuver) to examine their effects on liver regeneration in rats after partial hepatectomy. MATERIALS AND METHODS: Male Wistar rats were randomized to a control group (without hepatic inflow occlusion), an occlusion of the portal triad (OPT) group (OPT for 30 min under portal blood bypass), and an occlusion of the portal vein (OPV) group (OPV only for 30 min under portal blood bypass). All the rats underwent partial hepatectomy at the end of hepatic blood control. Liver regeneration was assessed on days 3 and 7 after hepatectomy. Liver damage, extracellular signal-regulated kinase-1/2 activation, and cytokine expression of the remnant liver in the first 24 h after hepatectomy were also assessed. RESULTS: Significantly greater liver regeneration, at a level similar to that of the control group, as indicated by the percentage of the initial liver weight, proliferating cell nuclear antigen and Ki-67 labeling indexes, and technetium-99m galactosyl human serum albumin liver uptake, was observed in the OPV group on day 3 after hepatectomy (P < 0.05 versus the OPT group). Liver damage, as represented by alanine aminotransferase and aspartate aminotransferase measurement and histopathologic examination, was substantially alleviated in the OPV group compared with the OPT group. In contrast to the control and OPV groups, the OPT group had markedly increased extracellular signal-regulated kinase-1/2 activation, heat shock protein 70, and interleukin-6 expression in response to ischemia and partial hepatectomy. CONCLUSIONS: Our results have indicated that compared with the Pringle maneuver, clamping the portal vein while preserving the hepatic artery flow during partial hepatectomy is better for remnant liver regeneration at an early posthepatectomy stage.

Increased susceptibility of ST2-deficient mice to polymicrobial sepsis is associated with an impaired bactericidal function.

ST2, a member of the Toll/IL-1R superfamily, negatively regulates both TLR2 and TLR4 signaling. In this study, we report that ST2-deficient mice were more susceptible to polymicrobial sepsis than their wild-type littermates, with increased production of proinflammatory cytokines. Bacterial clearance from the circulation and visceral organs following polymicrobial infection was markedly impaired in ST2-deficient mice. This was associated with substantially reduced uptake, phagocytosis, and intracellular killing of both Gram-positive and Gram-negative bacteria by ST2-deficient phagocytes. Consistent with a reduced antimicrobial response, phagocytes lacking ST2 displayed a defect in bactericidal activity in response to bacterial challenges with severely impaired phagosome maturation and NOX2 function. Thus, ST2-deficient mice exhibit an increased susceptibility to polymicrobial infection with impaired bacterial clearance, which is associated with defects in phagosome maturation and NOX2-derived production of reactive oxygen species characterized in ST2-deficient phagocytes.

Bile Acid Overload Induced by Bile Duct and Portal Vein Ligation Improves Survival after Staged Hepatectomy in Rats.

OBJECTIVE: Compared to portal vein ligation (PVL), simultaneous bile duct and portal vein ligation (BPL) can significantly enhance hypertrophy of the intact liver. This study aimed to investigate whether BPL could improve survival after extended hepatectomy independently of an increased remnant liver. METHODS: We adopted rat models of 90% BPL or 90% PVL. To investigate the role of bile acids (BAs) the BA pools in the PVL and BPL groups were altered by the diet. Staged resection preserving 10% of the estimated liver weight was performed 3 days after BPL; PVL; or sham operation. Histology, canalicular network (CN) continuity; and hepatocyte polarity were evaluated. RESULTS: At 3 days after BPL; PVL; or sham operation when the volumetric difference of the intended liver remained insignificant, the survival rates after extended hepatectomy were 86.7%, 47%, and 23.3%, respectively (P<0.01). BPL induced faster restoration of canalicular integrity along with an intensive but transient BA overload. Staged hepatectomy after BPL shortened the duration of the bile CN disturbance and limited BA retention. Decreasing the BA pools in the rats that underwent BPL could compromise these effects, whereas increasing the BA pools of rats that underwent PVL could induce similar effects. The changes in CN restoration were associated with activation of LKB1. CONCLUSION: In addition to increasing the future remnant liver, BPL shortened the duration of the spatial disturbance of the CN and could significantly improve the tolerance of the hypertrophied liver to staged resection. BPL may be a safe and efficient future option for patients with an insufficient remnant liver.

Bacterial lipoprotein-induced tolerance is reversed by overexpression of IRAK-1.

Tolerance to bacterial cell wall components including bacterial lipoprotein (BLP) represents an essential regulatory mechanism during bacterial infection. Reduced Toll-like receptor 2 (TLR2) and IL-1 receptor-associated kinase 1 (IRAK-1) expression is a characteristic of the downregulated TLR signaling pathway observed in BLP-tolerised cells. In this study, we attempted to clarify whether TLR2 and/or IRAK-1 are the key molecules responsible for BLP-induced tolerance. Transfection of HEK293 cells and THP-1 cells with the plasmid encoding TLR2 affected neither BLP tolerisation-induced NF-κB deactivation nor BLP tolerisation-attenuated pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) production, indicating that BLP tolerance develops despite overexpression of TLR2 in these cells. In contrast, overexpression of IRAK-1 reversed BLP-induced tolerance, as transfection of IRAK-1 expressing vector resulted in a dose-dependent NF-κB activation and TNF-α release in BLP-tolerised cells. Furthermore, BLP-tolerised cells exhibited markedly repressed NF-κB p65 phosphorylation and impaired binding of p65 to several pro-inflammatory cytokine gene promoters including TNF-α and interleukin-6 (IL-6). Overexpression of IRAK-1 restored the nuclear transactivation of p65 at both TNF-α and IL-6 promoters. These results indicate a crucial role for IRAK-1 in BLP-induced tolerance, and suggest IRAK-1 as a potential target for manipulation of the TLR-mediated inflammatory response during microbial sepsis.

Preserving hepatic artery flow during portal triad blood inflow occlusion reduces liver ischemia-reperfusion injury in rats.

BACKGROUND: Temporary portal triad clamping (Pringle maneuver) during liver resection can reduce intraoperative blood loss, but also correlates with liver ischemia and reperfusion (I/R) injury. The hepatic artery supplies 20%-30% blood but more than 50% O(2) to the liver. In this study, we explored if preservation of hepatic artery flow when performing portal triad blood inflow occlusion could reduce liver I/R injury while not increasing the blood loss. MATERIALS AND METHODS: Three groups of rats were created: group SO (sham operation), group OPT (occlusion of portal triad under portal blood bypass), and group OPV (occlusion of portal vein under portal blood bypass). Blood flow was occluded for 90, 100, 110, and 120 min before reperfusion. Liver I/R injury was assessed by measuring the survival of rats within 7 d after operation, liver blood loss, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver tissue malondialdehyde (MDA), and Na(+)-K(+)-ATPase, and liver histology. RESULTS: The 7-d survival of rats in group OPV was higher than in group OPT. The safe tolerance limit was 90 min for group OPT and 110 min for group OPV. Liver blood loss in group OPT and OPV were significantly less than in group SO. However, no significant difference was observed in the amount of blood loss between group OPT and group OPV. The group OPV had significantly lower ALT, AST, and MDA values on the first hour and first day post-reperfusion than in group OPT. The Na(+)-K(+)-ATPase activity in OPV group was significantly higher than in group OPT 1 h post-reperfusion. Hepatocyte injury was significantly less in group OPV than in group OPT on histopathology. CONCLUSIONS: These data indicate that continuously clamping the portal vein while preserving the hepatic artery did not increase blood loss significantly in a rat liver I/R model, however this maneuver induced less liver I/R injury. It is therefore suggested that preserving hepatic artery inflow during portal triad blood inflow occlusion might become an alternative maneuver in liver surgery due to its ability to extend the safe tolerant time limit in normothermic hepatic ischemia.

Rapid separation and identification of anthocyanins from flowers of Viola yedoensis and V. prionantha by high-performance liquid chromatography-photodiode array detection-electrospray ionisation mass spectrometry.

INTRODUCTION: Anthocyanins are important plant secondary metabolites. They show strong antioxidant activities and have potential as anti-cancer agents. Viola yedoensis and V. prionantha are traditional Chinese medicines and ornamental plants. However, the anthocyanin compositions of these two species are still unresolved. OBJECTIVE: To develop a rapid and reliable high-performance liquid chromatography (HPLC) method for the separation and identification of anthocyanins from V. yedoensis and V. prionantha. METHODOLOGY: Samples were extracted in methanol-water-formic acid-TFA (70:27:2:1, v/v). HPLC analysis was done on a C(18) column (TSK-GEL ODS-80Ts: 150 × 4.6 mm i.d.). Four solvent systems were tested to optimise the separation of anthocyanins using different gradient separation systems. HPLC-photodiode array detection (DAD) coupled to electrospray ionisation mass spectrometry (ESI-MS) was used to carry out the comprehensive characterisation of anthocyanins. RESULTS: Fourteen anthocyanins were characterised within 40 min with satisfactory peak resolution by a gradient composed of 10% aqueous formic acid and formic acid-acetonitrile-water (10:40:50, v/v). The calibration curve showed an excellent linear regression (r(2) = 0.9995) and low intra- and inter-day variations (RSD < 3.67%). The detected anthocyanins derived from Dp, Cy, Pt, Mv and Pn, could be divided into three groups: non-acylated glycosides, acetylglycosides and coumaroylglycosides. Anthocyanins distribution exhibited remarkable differences in aglycone levels and acylation patterns. CONCLUSION: The optimised method was successfully applied for the analysis of 14 anthocyanins from V. yedoensis and V. prionantha. The identification of anthocyanin constitutions is valuable for breeding and will open up new prospects for their medicinal application.

[Effects of different hepatic inflow occlusion methods on liver regeneration following partial hepatectomy in rats].

OBJECTIVE: To explore the effects of different hepatic inflow occlusion methods on liver regeneration in rats after partial hepatectomy (PH). METHODS: Male Wistar-Furth rats were randomly assigned to three groups: control group, underwent 68% hepatectomy alone; occlusion of portal triad (OPT) group, subjected to occlusion of portal triad under portal blood bypass; and occlusion of portal vein (OPV) group, subjected to occlusion of portal vein under portal blood bypass. Blood flow was occluded for 20, 30, and 40 minutes before 68% hepatectomy. According to the 7-day survival of each group, a same occlusion time T was set. Each group was divided into two subgroups (n = 8), in which animals were killed 3 and 7 days later. Liver regeneration was calculated as a percent of initial liver weight. Immunohistochemistry for proliferating cell nuclear antigen (PCNA) and Ki-67 was performed to quantify proliferating cells. In addition, functional liver volume represented by 99Tc(m)-GSA radioactivity was assessed. RESULTS: The safe tolerance limit time was 30 minutes for OPT group and 40 minutes for OPV group. At 3 days after PH, no significant difference was observed in the regeneration rate of each group (P > 0.05). However, liver radioactive activity, PCNA labeling index, and Ki-67 index of OPV group was significantly higher than those of OPT group (P < 0.05); the latter were similar to those of control group (P > 0.05). At 7 days after PH, no significant difference was observed in all indexes among three groups (P > 0.05). CONCLUSION: Compared with Pringle maneuver, preserving the hepatic artery flow during portal triad blood inflow occlusion can promote remnant liver regeneration early after PH.

Effects of partial portal vein arterialization on the hilar bile duct in a rat model.

BACKGROUND: Liver revascularization is frequently required during the enlarged radical operation for hilar cholangiocarcinoma involving the hepatic artery. Researchers have carried out a number of experiments applying partial portal vein arterialization (PVA) in clinical practice. In this study we aimed to establish a theoretical basis for clinical application of partial PVA and to investigate the effects of partial PVA on rat hilar bile duct and hepatic functions. METHODS: Thirty rats were randomly and equally assigned into 3 groups: control (group A), hepatic artery ligation+bile duct recanalization (group B), and partial PVA+bile duct recanalization (group C). Proliferation and apoptosis of rat hilar bile duct epithelial cells, arteriolar counts of the peribiliary plexus (PBP) of the bile duct wall, changes in serum biochemistry, and pathologic changes in the bile duct were assessed 1 month after operation. RESULTS: The proliferation of hilar bile duct epithelial cells in group B was greater than in groups A and C (P<0.01). No apoptotic hilar bile duct epithelial cells were detected in any of the groups. The PBP arteriolar counts of the hilar bile duct wall were similar in groups A and C (P>0.05), but the count was lower in group B than in group A (P<0.01). No statistically significant differences in alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and albumin were found in the 3 groups. The gamma-glutamyltransferase value was higher in group B than in groups A and C (P<0.01). The hepatic tissues of groups A and C showed no significant abnormality. Chronic inflammatory changes in the hilar bile duct walls were observed only in group B. CONCLUSION: Partial PVA can restore the arterial blood supply of the hilar bile duct and significantly extenuate the injury to hilar bile duct epithelial cells resulting from hepatic artery ligation.

[Study on the nuclear translocation mechanism in the inhibition of nuclear factor-ΚB activation in bacterial lipoprotein-tolerant THP-1 cells].

OBJECTIVE: To approach the nuclear factor-ΚB (NF-ΚB) nuclear translocation mechanism in bacterial lipoprotein (BLP) tolerance. METHODS: Human monocytic THP-1 cells were first pretreated with 10, 100, 1 000 ng/ml BLP for 20 hours to induce BLP tolerance. Then THP-1 cells without BLP pretreatment (control group) or with BLP pretreatment (tolerance group) were stimulated with 0, 10, 100, 1 000 ng/ml BLP again for 6 hours. The tumor necrosis factor-α (TNF-α) content in culture medium was measured by enzyme linked immunosorbent assay (ELISA) in order to determine the most suitable BLP pretreatment and stimulation concentration. Western blotting was used to detect the protein level, nuclear translocation and phosphorylation of NF-ΚB p50 and p65 in the cells of control and tolerance groups treated with respective conditions for 0, 0.5, 1, 2 and 6 hours. RESULTS: In control group BLP stimulation (10, 100, 1 000 ng/ml) could induce THP-1 activation and TNF-α production (pg/ml: 184.86±32.51, 3 215.88±167.09, 6 042.96±245.37) in a dose-dependent manner. In tolerance group, 100 ng/ml BLP pretreatment resulted in almost complete inhibition of TNF-α production as induced by 101 000 ng/ml BLP stimulation. Therefore, 100 ng/ml BLP pretreatment and 1 000 ng/ml stimulation were selected for following cell treatment. Western blotting analysis showed that there was an increase of p50 protein level in BLP-tolerant cells comparing with control group (0 hour: 542.9±15.6 vs. 272.8±13.2, 0.5 hour: 558.0±16.9 vs. 236.4±11.8, 1 hour: 524.7±17.5 vs. 211.6±9.8, 2 hours: 584.9±15.6 vs. 222.4±12.3, all P<0.01), whereas the p65 protein level was similar between the two groups. BLP stimulation also induced the nuclear translocation of p50 and p65 in control group (1-hour p50: 344.2±13.6 vs. 79.0±5.2, p65: 78.4±4.5 vs. 0, both P<0.05), but not in tolerance group. In addition, the phosphorylation of p65 at serine 536 was induced after BLP stimulation in control THP-1 cells (0.5 hour: 0.67±0.08 vs. 0.04±0.01, 1 hour: 0.71±0.11 vs. 0.04±0.01, both P<0.05), but this change was not detected in BLP-tolerant cells. CONCLUSION: It was found that in BLP-tolerant cells, the expression of inhibitory subunit p50 was increased and the nuclear translocation and phosphorylation of p65 with trans-activation ability was inhibited. These changes are likely responsible for the reduced gene expression of NF-ΚB dependent genes in BLP-tolerant cells.

[Experimental study of a battlefield shock resuscitation fluid with new formula].

OBJECTIVE: To propose a resuscitation fluid with a new formula for resuscitation of shock in battlefield on the basis of pathogenesis of hemorrhagic shock and clinical experiences, and to evaluate its safety and effectiveness in a rat hemorrhagic shock model. METHODS: After hemorrhagic shock was reproduced in rats, a mixture of lactate Ringer solution and hydroxyethyl starch solution at 2:1 proportion (referred to as LH) was used for resuscitation in animals of control group; LH supplemented with dexamethasone and furosemide (referred to as LHDF) was used as resuscitation fluid for experimental group. After 4 hours of infusion, blood and major organs were obtained for serum biochemical tests, lung water content measurement and histopathological observation. RESULTS: The mean arterial pressure of rats of both control and experimental groups recovered rapidly after resuscitation. There was no significant difference in the parameters of serum biochemistry between control group and experimental group. The wet/dry weight ratio of lung tissue in experimental group was significantly lower than control group (4.56 ± 0.14 vs. 4.88 ± 0.29, P <0.05). The blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in experimental group were also significantly lower than in control group [ALT: (73.02 ± 41.89) U/L vs. (193.85 ± 104.49) U/L; AST: (199.06 ± 108.7) U/L vs. (395.25 ± 137.08) U/L, both P <0.05). Diuretic effect was obviously observed in experimental group than control group [time of start urination: (76 ± 20) minutes vs. (153 ± 14) minutes; urine volume: (9.6 ± 5.2) ml vs. (1.5 ± 2.2) ml, P <0.01 and P <0.05] , and the amount of ascitic fluid in experimental group was significantly lower than in control group [(1.3 ± 0.6) ml vs. (5.0 ± 3.0) ml , P <0.05). Histopathological observation of the liver, lung and intestine also showed less pathological changes in experimental group than in control group. CONCLUSION: The designed battlefield anti shock fluid in this study has been shown to be effective in fluid resuscitation for hemorrhage shock in rats, with reduced tissue edema and less injury to the liver, lung and intestine.