Active hexose correlated compound inhibits the expression of proinflammatory biomarker iNOS in hepatocytes.

BACKGROUND/AIMS: Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated as proinflammatory biomarker in liver injury. The application of active hexose correlated compound (AHCC) as a functional food in complementary and alternative medicine has increased. The possibility that AHCC might inhibit iNOS induction was investigated as a potential liver-protective effect. METHODS: Hepatocytes were isolated from rats by collagenase perfusion and cultured. Primary cultured hepatocytes were treated with interleukin-1ß in the presence or absence of AHCC-sugar fraction (AHCC-SF). RESULTS AND CONCLUSION: AHCC-SF inhibited the production of NO and reduced expressions of iNOS mRNA and its protein. AHCC-SF had no effects on either IκB degradation or nuclear factor-κB (NF-κB) activation. In contrast, AHCC-SF inhibited the upregulation of type I interleukin-1 receptor (IL-1RI) through the inhibition of Akt phosphorylation. Transfection experiments with iNOS promoter-luciferase constructs revealed that AHCC-SF reduced the levels of iNOS mRNA at both promoter transactivation and mRNA stabilization steps. AHCC-SF inhibited the expression of iNOS gene antisense transcript, which is involved in iNOS mRNA stabilization. These findings demonstrate that AHCC-SF suppresses iNOS gene expression through a IκB/NF-κB-independent but Akt/IL-1RI-dependent pathway, resulting in the reduction of NO production. AHCC-SF may have therapeutic potential for various liver injuries.

Enhanced production of interleukin 6 in peripheral blood monocytes stimulated with mucins secreted into the bloodstream.

PURPOSE: It has been reported that tumor progression is correlated with the serum level of interleukin 6 (IL-6). The purpose of this study was to investigate by what mechanism, other than production from tumor cell, the serum level of IL-6 is elevated in the tumor-bearing state. EXPERIMENTAL DESIGN: Monocytes from healthy donors were cultured in the presence of sera from colon cancer patients, and the activity to elevate IL-6 production was estimated. This activity of serum was also examined after various biochemical treatments. RESULTS: When monocytes from healthy donors were cultured in the presence of sera from patients with colon cancer, secretion of IL-6 from the cells was markedly elevated. Serum proteins were fractionated on Sepharose 4B and the activity to elevate IL-6 production was found in the excluded fractions. Sialyl Tn antigen was detected in these same fractions. By excluding some mucins from the serum, the inducing activity was reduced to 40% of the original level. Furthermore, we purified mucins from the conditioned medium of colon cancer cells. Production of IL-6 was effectively elevated by a small amount of purified mucins in a dose-dependent manner. When the inducing activity was examined in the presence of binding or competitive inhibitors to the scavenger receptor, the effect was remarkably reduced. CONCLUSIONS: Mucins secreted from colon cancer cells into the bloodstream induce production of IL-6 in peripheral blood monocytes through the scavenger receptor, which may be responsible for the high level of serum IL-6 in colon cancer patients.

In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme.

A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes has been obtained through in vitro selection. They share a common motif with the '8-17' deoxyribozyme isolated under different conditions, including different design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA and DNA/RNA chimeric substrates. It can cleave any substrate containing rNG (where rN is any ribo-nucleotide base and G can be either ribo- or deoxy-ribo-G). The pH profile and reaction products of this deoxyribozyme are similar to those reported for hammerhead ribozyme. This deoxyribozyme has higher activity in the presence of transition metal ions compared to alkaline earth metal ions. At saturating concentrations of Zn(2+), the cleavage rate is 1.35 min(-1)at pH 6.0; based on pH profile this rate is estimated to be at least approximately 30 times faster at pH 7.5, where most assays of Mg(2+)-dependent DNA and RNA enzymes are carried out. This work represents a comprehensive characterization of a nucleic acid-based endonuclease that prefers transition metal ions to alkaline earth metal ions. The results demonstrate that nucleic acid enzymes are capable of binding transition metal ions such as Zn(2+)with high affinity, and the resulting enzymes are more efficient at RNA cleavage than most Mg(2+)-dependent nucleic acid enzymes under similar conditions.

Increased extra domain-A containing fibronectin and hepatic dysfunction during septic response: an in vivo and in vitro study.

A massive inflammatory reaction resulting from systemic cytokine release is the common pathway underlying sepsis or multiple organ dysfunction. The role of extra domain sequence A-containing fibronectin (EDA+FN) formation during the septic response is not known. The present study investigates the role of EDA+FN during the septic response under in vitro and in vivo conditions. The direct effects of interleukin-1, interleukin-6, and tumor necrosis factor-alpha on EDA+FN production were evaluated in primary cultured human hepatocytes and fibroblasts. Serial plasma EDA+FN levels were measured using an enzyme-linked immunosorbent assay in 24 patients who developed postoperative sepsis following general abdominal surgery of which there were 17 survivors and 7 non-survivors. EDA+FN secretion was significantly increased in cultured hepatocytes but not fibroblasts at 24 and 48 h following exposure to IL-1 compared to controls. In the clinical setting plasma EDA+FN levels in non-survivors were significantly higher than in survivors. Moreover, the EDA+FN levels were correlated closely with liver function tests. EDA+FN levels may represent a specific marker of vascular injury or systemic inflammatory response syndrome that is associated with an adverse clinical outcome.

Use of technetium 99m diethylenetriamine-pentaacetic acid-galactosyl-human serum albumin liver scintigraphy in the evaluation of preoperative and postoperative hepatic functional reserve for hepatectomy.

BACKGROUND: Technetium 99m diethylenetriaminepentaacetic acid-galactosyl-human serum albumin (99mTc-GSA) is a new liver scintigraphy agent that binds to the asialoglycoprotein receptors. We evaluated the clinical use of 99mTc-GSA for the perioperative assessment of hepatectomy. METHODS: Thirty-six patients with hepatocellular carcinoma were admitted for elective hepatectomy. 99mTc-GSA scintigraphy was obtained after the intravenous injection of 99mTc-GSA, and a modified receptor index (MRI) was calculated. 99mTc-GSA scintigraphy, conventional liver function, the plasma disappearance rate, and the 15-minute retention rate of indocyanine green (ICGR15) were carried out before operation and every 1 to 3 months after operation. The relationships between several systemic hemodynamic parameters, histologic activity index (HAI), plasma disappearance rate, and ICGR15 or MRI values were estimated. RESULTS: A significant correlation was obtained between the MRI and ICGR15 (r = 0.6231, p < 0.001). Plasma disappearance rates correlated well with systolic volume and left cardiac work, whereas MRI values did not correlate with these systemic hemodynamics. Preoperative discrepancies between the values of MRI and ICGR15 were seen in eight cases. In these cases the MRI values correlated well with HAI scores (p < 0.05) but there was no significant correlation between ICGR15 values and the HAI scores. CONCLUSIONS: These results suggested the use of 99mTc-GSA scintigraphy as a easy and reliable method for determining liver functional reserve.

Cold preservation of rat pancreatic islets just above the freezing point using University of Wisconsin solution.

AIMS: To confirm whether rat islets stored at a temperature just above the freezing point using University of Wisconsin (UW) solution would remain viable for the short term. METHODOLOGY: Rat islets were stored for 24 hours in UW solution, either at 4 degrees C or at -0.6 degrees C (just above the specific freezing point of the UW solution). After cold storage, the islets were assessed for in vitro viability by static incubation and for in vivo viability by a transplantation study. One thousand islets preserved under different conditions were injected intraportally into a streptozotocin-induced diabetic rat as an isograft. Four weeks after the transplantation, an intravenous glucose tolerance test was performed. RESULTS: Islets stored at -0.6 degrees C showed higher insulin secretion rates than those stored at 4 degrees C on a static challenge. The interval from transplantation to the achievement of normoglycemia was also shorter in the -0.6 degrees C group than in the 4 degrees C group. After islet transplantation, the daily nonfasting plasma glucose concentration was higher in the 4 degrees C group than in the -0.6 degrees C group. When compared with the 4 degrees C group, the -0.6 degrees C group showed lower blood glucose values during all investigational periods on an intravenous glucose tolerance test. CONCLUSION: Islet preservation at -0.6 degrees C using UW solution is more advantageous for short term.

Laparoscopic cholecystectomy and choledocholithotomy in patients with a previous gastrectomy.

BACKGROUND: An increased incidence of cholelithiasis has been widely reported after truncal vagotomy and after gastric resection. In the early phase of patient selection, previous gastrectomy has been considered a relative contraindication to laparoscopic cholecystectomy (LC). In this study, we examined the management of LC in patients with previous gastrectomy. STUDY DESIGN: LC was attempted on 1,260 consecutive patients. Of these patients, 29 had a previous gastrectomy. Surgical procedures that had been performed included Billroth I gastrectomies (15), Billroth II gastrectomies (10), and total gastrectomies (4). There were 23 cases of cholelithiasis, 4 chronic cholecystitis, 2 gallbladder polyps, I porcelain gallbladder, and I gallbladder cancer. Nine patients were diagnosed with stones in their common bile duct or common hepatic duct. RESULTS: Preoperatively, seven of nine patients with common bile duct stones were subjected to endoscopic sphincterotomy, and the stones were removed successfully from five of these patients. In the remaining two patients, common bile duct stones were removed by laparoscopic choledocholithotomy by choledochotomy. The LC was completed in 26 patients (90%) who had undergone previous gastrectomy. In 449 patients who had previous abdominal surgery without a gastrectomy, only 4 patients (0.9%) required open surgery. In contrast, three patients (10%) with previous gastrectomy required open surgery. No major complications were recorded in this study series, and no residual or retained stones were seen during a followup period of 3 months. CONCLUSIONS: Clear visualization of anatomic structures and landmarks, and scrupulous hemostasis are needed to perform a safe LC in these patients. We conclude that in our study patients, a previous gastrectomy is considered an indication for LC and laparoscopic choledochotomy.

FK506, but not cyclosporin A, prevents mitochondrial dysfunction during hypoxia in rat hepatocytes.

Hepatic ischemia/reperfusion injury occurs in the clinical situations including liver transplantation. FK506 and cyclosporin A (CsA) are reported to be hepatotrophic agents in addition to being a powerful immunosuppressive agent. Studies were performed to determine whether the drugs influence a mitochondrial dysfunction under the hypoxic conditions in primary culture model of rat hepatocytes. The Anaeropack system was used for cell culture to create a hypoxia. Cells were treated with FK506 or CsA under the normoxic and hypoxic conditions. Hypoxia markedly decreased intracellular adenosine 5'-triphosphate (ATP) contents and the ketone body ratio (KBR, acetoacetate/beta-hydroxybutyrate) in culture medium as compared with normoxia. FK506 prevented the decreases of ATP contents and the KBR. In contrast, CsA had no effect on either ATP contents or the KBR. FK506, but not CsA, increased the KBR under the normoxic conditions. Under the hypoxic conditions, heat shock protein 70 (Hsp70) was detected after reoxygenation. FK506 enhanced the induction of Hsp70, but CsA again had no effect on Hsp70 induction. These results indicate that FK506 protects the hypoxia injury in part by preventing the mitochondrial dysfunction in concert with the enhancement of heat shock response in hepatocytes.

Spiral computed tomography scanning after intravenous infusion cholangiography for biliary duct anomalies.

BACKGROUND: Iatrogenic injury of the bile duct during cholecystectomy represents a failure of surgical technique, especially for laparoscopic surgery. Knowledge of the patient's individual ductal anatomy and anomalies preoperatively would be helpful in avoiding such injuries. Therefore, we investigated the anatomy of the biliary duct and any anomalies using spiral computed tomography (SCT) scanning following intravenous infusion cholangiography (IVC-SCT). MATERIALS: Laparoscopic cholecystectomies (LC) were attempted on 437 patients at the Kansai Medical University. Preoperative IVC-SCT and laparoscopic cholangiography were attempted in all of the patients. RESULTS: An overall anomalous union of the cystic duct was seen in 71 (16.2%) out of the 437 patients subjected to IVC-SCT. The following anomalies were observed: right hepatic duct entry in 7 cases (1.6%), parallel low entry in 17 cases (3.9%), posterior spiral entry in 35 cases (8.0%), anterior spiral entry in 7 cases (1.6%), and accessory duct entry in 5 cases (1.1%). The success rate for the LC was 99.5% (435/437). Three patients were switched to open surgery owing to advanced gallbladder cancer and severe adhesions. The success rate for the laparoscopic cholangiography was 97.2% (423 of 435). Intraoperative right hepatic duct injury occurred in only 1 patient with a bile duct anomaly, and it was repaired with laparoscopic T-tube drainage. CONCLUSIONS: The preoperative examination of the biliary tract by IVC-SCT was technically simple, less invasive, and may helpful in avoiding damage to the bile duct, especially in patients with biliary duct anomalies.

Gallbladder cancer with a low junction of the cystic duct or an anomalous pancreaticobiliary junction.

OBJECTIVE: To evaluate conditions similar to those of carcinogenesis of the gallbladder between the gallbladder with a low junction of the cystic duct (LJCD) and an anomalous pancreaticobiliary junction (APBJ). DESIGN: Retrospective and clinicopathological analysis of patients with gallbladder carcinoma. SETTING: First Department of Surgery, Kansai Medical University. PATIENTS: Examination of 47 patients (7 men and 40 women; average age: 67.8 years) with gallbladder carcinoma revealed 7 patients (14.9%; 1 man and 6 women; average age: 67.8 years) with LJCD and 6 patients (12.8%; 6 women; average age: 60.3 years) with APBJ. METHODS: Clinical findings in both groups were compared with those of the 34 patients who remained after exclusion of the data of the above 7 patients with LJCD and 6 patients with APBJ. The data of the three groups were examined by the chi 2 test at the 5% level of significance. RESULTS: Most of the gallbladder cancer patients with LJCD or APBJ had gallstones. The biliary amylase levels determined in the gallbladder of patients with LJCD or APBJ were remarkably high. CONCLUSION: The results indicate that patients with LJCD or APBJ are more likely to develop carcinoma of the gallbladder. The factors responsible for carcinogenesis may be alteration of the bile content due to reflux of pancreatic enzymes through the LJCD or APBJ, and mechanical irritation due to gallstones. Therefore, these pathological conditions in patients with LJCD are similar to those experienced in patients with APBJ.

A simplified model of hypoxic injury in primary cultured rat hepatocytes.

The Anaeropack system for cell culture, which was originally designed for the growth of anaerobic bacteria, was used to produce a hypoxic atmosphere for cultured hepatocytes. We measured changes in the oxygen and carbon dioxide concentrations and the atmospheric temperature in an airtight jar. We also measured changes in the pH of the medium during hypoxia to assess the accuracy of this system. Moreover, we used three durations (2, 3, and 4 h) of hypoxia and 8 h of reoxygenation in cultured rat hepatocytes, and then measured the lactate dehydrogenase (LDH), ketone body concentration (acetoacetate + beta-hydroxybutyrate), and the ketone body ratio (KBR: acetoacetate/beta-hydroxybutyrate) in the medium in order to assess the suitability of this system as a model for reperfusion following liver ischemia. The oxygen concentration dropped to 1% or less within 1 h. The concentration of carbon dioxide rose to about 5% at 30 min after the induction of the hypoxic conditions, and was maintained at this level for 5 h. No effect of the reaction heat produced by the oxygen absorbent in the jar was recognized. The extent of cell injury produced by changing the hypoxic parameters was satisfactorily reflected by the KBR, the ketone body concentration, and the LDH activity released into the medium. Because this model can duplicate the conditions of the hepatocytes during revascularization following ischemic liver, and the Anaeropack system for cell culture is easy to manipulate, it seems suitable for the experimental study of hypoxic injury and revascularization in vitro.

Fibronectin protects endotoxin-induced liver injury after partial hepatectomy in rats.

Endotoxemia following extended hepatectomy may be a cause of postoperative death. Multiple organ failure related to septemia is a common cause of early mortality after liver transplantation. Fibronectins (Fns) are involved in cellular adhesion, motility, differentiation, apoptosis, hemostasis, wound healing, and ischemic injury. Studies were performed to determine whether Fn influences the survival rate of rats subjected to endotoxin-induced liver injury after partial hepatectomy. Male Sprague-Dawley rats were intravenously administered lipopolysaccharide (LPS) 48 hours after 70% hepatectomy. Prior to LPS administration, plasma Fn or bovine serum albumin was given intravenously. The survival rate of the Fn-treated group was higher than that of the controls. Fn prevented increases in serum enzyme activity and total bilirubin related to liver injury. The levels of inflammatory cytokines including tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 interferon-gamma were also significantly lower in the Fn-treated than the control group. Furthermore, the number of apoptotic cells and the degree of necrosis in the remnant liver were significantly decreased in the Fn-treated rats compared with controls. These results indicate that Fn prevents endotoxin-induced liver injury after partial hepatectomy, at least in part through inhibiting the production of inflammatory cytokines, and the necrosis apoptosis in the remaining liver.

Safety of hepatectomy for living donors as evaluated using asialoscintigraphy.

In the living donor operation, accurate estimation of hepatic functional reserve is essential. Technetium-99m-galactosyl-human serum albumin (GSA) is a liver scintigraphy agent that binds to asialoglycoprotein receptors. We evaluated the preoperative assessment of the safety of an elective hepatectomy using GSA liver scintigraphy in 152 patients. GSA scintigraphy was performed after intravenous injection of GSA. The maximal removal rate of GSA (GSA-Rmax) was calculated using a radiopharmacokinetic model. We determined the areas for resection preoperatively depending on the operative procedures and calculated the local GSA-Rmax in the predicted residual liver (GSA-RL). A significant correlation was obtained between the GSA-Rmax and the 15-minute retention rate of indocyanine green. With sub- and monosegmentectomy, 2 patients had postoperative hepatic failure; in those 2 patients, the GSA-RL was 0.127 and 0.133, respectively, but these patients recovered well. Among those having di- and tri-segmentectomy, 5 patients experienced postoperative hepatic failure, in all subjects the GSA-RL was <0.15. Two patients died of postoperative liver failure 1 to 2 months after the operation. We concluded that GSA-RL is useful to select the procedure for hepatectomy in living donors and that GSA-RL should be >0.15 (mg/min/50 kg body weight) to avoid postoperative hepatic failure.

Functional hepatic regeneration following hepatectomy using galactosyl-human serum albumin liver scintigraphy.

In extended hepatectomy and liver transplantation, accurate estimation of functional hepatic regeneration is more important than volumetric regeneration. We investigated the usefulness of measuring the functional hepatic volume by 99m-technetium galactosyl-human serum albumin scintigraphy (GSA). Extended hepatectomy was performed in 32 patients. These patients were divided into subgroups with or without chronic hepatitis or cirrhosis. Functional hepatic volume GSA scintigraphy (GSA-LV) and determination of hepatic volume by CT (CT-LV) measurements were performed preoperatively, at 2 and 4 weeks and at 3 and 6 months after surgery. The preoperative GSA-LV values were significantly correlated with the hepatocyte volume and the 15-minute retention rate of indocyanine green (ICGR15). Similarly, the hepatocyte volume correlated well with the CT-LV and ICGR15. However, the CT-LV correlated only with the ICGR15. Recovery of the GSA-LV was delayed, and about 90% of the volumetric and functional regeneration was observed within 6 months after the hepatectomy. In contrast, the CT-LV in patients with normal liver remnants returned to approximately 90% of the initial volume within 1 month after the hepatectomy, whereas patients with injured livers regeneration showed gradual recovery to approximately 80% of the preoperative value by 6 months after hepatectomy. We conclude that measurement of functional hepatic volume using the GSA-LV is useful to evaluate hepatic function based on hepatocyte volume.

Fibronectin suppresses apoptosis and protects mice from endotoxic shock.

Multiple-organ failure related to septicemia is a common cause of early mortality after liver transplantation. Endotoxemia following living donor hepatectomy may be a cause of postoperative death. Plasma fibronectin (Fn) exerts a broad range of biological effects on cellular adhesion, motility, differentiation, apoptosis, hemostasis, wound healing, reticuloendothelial system function, and ischemic injury. We studied the therapeutic effect of plasma Fn in mice after an intraperitoneal injection of lipopolysaccharide (LPS) and d-galactosamine (GalN). Female Balb/c mice received simultaneous intraperitoneal injection of LPS (50 microg/kg) and GalN (400 mg/kg). Thirty minutes prior to GalN/LPS administration, plasma Fn or bovine serum albumin was given intravenously. A single administration of plasma Fn (500 mg/kg) protected in dose-dependent fashion against lethal shock after GalN/LPS challenge. Plasma Fn significantly reduced the serum tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 levels and significantly increased the serum interleukin-10 levels after GalN/LPS administration. Furthermore, plasma Fn significantly inhibited liver necrosis at 9 hours after GalN/LPS injection. The fraction of apoptotic-positive cells in these plasma Fn-treated mice was significantly lower than in the control group. These results support the protective treatment of endotoxin-induced liver injury by plasma Fn.

Effects of pirfenidone on endotoxin-induced liver injury after partial hepatectomy in rats.

BACKGROUND: In living donor liver transplantation, restrictions on graft size are a serious obstacle to expand indications for adult recipients. The sequence of gram-negative infection, septicemia, and multiple-organ failure is a common cause of early mortality after liver transplantation. An effective therapy has not been established for endotoxemia following extended hepatectomy in donors or small-for-size grafts in recipients. Pirfenidone (PFD), a new experimental antifibrotic agent, was used to ameliorate on endotoxin-induced liver injury following partial hepatectomy. METHODS: Male Sprague-Dawley rats were intravenously administered lipopolysaccharide (LPS) 48 hours after 70% hepatectomy. Prior to LPS administration, PFD (300 mg/kg) or its vehicle (0.5% carboxymethylcellulose) was given orally twice. RESULTS: The survival rate of the PFD-treated group was markedly improved compared with that of the controls. PFD prevented the increases in the activities of serum enzymes (aspartate transaminase [AST], alanine transaminase [ALT], and lactate dehydrogenase [LDH]) and total bilirubin. The serum and liver tissue levels of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma, and interleukin-6, were significantly lower among the PFD than the control group. Furthermore, the degree of necrosis in the remnant liver was significantly decreased in the PFD-treated rats compared with controls. CONCLUSION: These results indicate that PFD alleviates endotoxin-induced liver injury after partial hepatectomy through the inhibition of production of inflammatory cytokines in the residual liver. PFD may be useful to prevent endotoxin-induced liver injury after hepatectomy.

Effect of hepatocyte growth factor on induction of cytokine-induced neutrophil chemoattractant in rat hepatocytes.

BACKGROUND: Restrictions on graft size are a serious obstacle to the expansion of indications for adult recipients in living donor liver transplantation. Hepatocyte growth factor (HGF) has a crucial role in regeneration following hepatic injury. Rat cytokine-induced neutrophil chemoattractant (CINC), a member of the interleukin-8 superfamily in humans, has been implicated in chronic liver diseases or development of liver ischemia-reperfusion injury. Studies were performed to examine whether HGF influences the induction of CINC in hepatocytes. METHODS: Primary cultures of rat hepatocytes were treated with or without recombinant human (rh) HGF. The release of CINC into the culture medium and levels of CINC mRNA were measured using an enzyme-linked immunosorbent assay and Northern blot analysis. Transcription of nuclear factor (NF)-kappa B was detected by electrophoretic mobility shift assays. RESULTS: rhHGF increased the release of CINC in the medium dose- and time-dependently, showing a maximal effect at 100 ng/mL. Genistein (100 mumol/L) blocked the release of CINC stimulated by rhHGF. Levels of CINC mRNA were also increased, reaching a maximum at 8 hours after addition of rhHGF. Electrophoretic mobility shift assays revealed rhHGF activated transcription factor, NF-kappa B. CONCLUSION: These results suggest that HGF stimulates the induction of CINC gene expression through activation of NF-kappa B. CINC may be involved in the function of HGF during liver regeneration.

Pirfenidone protects endotoxin-induced liver injury after hepatic ischemia in rats.

BACKGROUND: Pirfenidone (PFD), an experimental antifibrotic agent, was investigated for its effects on endotoxin-induced liver injury after hepatic ischemia-reperfusion. METHODS: Male Sprague-Dawley rats were subjected to 30 minutes of partial hepatic ischemia, followed by reperfusion for 24 hours. Lipopolysaccharide (LPS) was injected at 30 minutes of reperfusion. PFD (300 mg/kg) or its vehicle (0.5% carboxymethylcellulose) was given orally following LPS administration. RESULTS: PFD prevented the increase in activities of serum alanine transaminase, aspartate transaminase, and lactate dehydrogenase after reperfusion. PFD inhibited the increase of cytokine-induced neutrophil chemoattractant in serum and liver tissue. The number of neutrophils infiltrating the liver was significantly lower in the PFD-treated group than the control group. CONCLUSION: These results indicate that PFD prevents endotoxin-induced liver injury after hepatic ischemia-reperfusion, in part through the decrease of neutrophil infiltration to the liver.

Effect of pirfenidone on induction of chemokines in rat hepatocytes.

BACKGROUND: Hepatic ischemia-reperfusion results in a neutrophil-dependent liver injury. The process of neutrophil recruitment and activation in this injury is at least partially dependent on the induction of chemokines, such as cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) in rats. In the liver, parenchymal cells (hepatocytes), in addition to nonparenchymal cells such as Kupffer cells, have been reported to produce chemokines in the regulation of hepatic inflammation. Pirfenidone (PFD) is a new experimental drug used as an antifibrotic agent. Studies were performed to determine whether PFD influences the production of CINC and MIP-2 stimulated by interleukin (IL)-1beta in a primary culture model of rat hepatocytes. METHODS: Primary cultures of rat hepatocytes were treated with IL-1beta in the presence and absence of PFD. The protein and mRNA of CINC and MIP-2 were analyzed using enzyme-linked immunosorbent assays and Northern blots. RESULTS: IL-1beta increased the release of CINC and MIP-2 into culture media in a dose- and time-dependent manner. PFD inhibited both CINC and MIP-2 release in dose-dependent fashion. However, PFD had no effect on the levels of CINC mRNA induced by IL-1beta. CONCLUSION: These results suggest that PFD inhibits the production of CINC and MIP-2 by IL-1beta at a posttranscriptional step in hepatocytes.

Induction of inducible nitric oxide synthase in hepatocytes isolated from rats with ischemia-reperfusion injury.

BACKGROUND: Recent evidence indicates that nitric oxide (NO) has a crucial role in hepatic ischemia-reperfusion (I/R) injury. However, little is known about how I/R influences the gene expression of inducible nitric oxide synthase (iNOS) in hepatocytes. Under inflammatory conditions, we compared the induction of iNOS in hepatocytes isolated from normal and I/R-treated rats. METHODS: Hepatocytes were isolated using the collagenase perfusion method from rats treated with I/R (30-minute ischemia of middle and left lobes, followed by 3-hour reperfusion) or sham operation (control): Primary cultures of rat hepatocytes were incubated with an inflammatory cytokine, interleukin-1beta (IL-1beta), to compare the iNOS induction/NO production between the 2 groups. RESULTS: Both control and I/R groups had no production of nitrite (a stable metabolite of NO) in the absence of IL-1beta. In the control group, IL-1beta stimulated dose- and time-dependent production of NO. The I/R group showed more than 2-fold increased levels of NO production. Western and Northern blot analyses revealed that the I/R group also showed increased levels of iNOS protein and its messenger RNA. CONCLUSION: These results suggest that I/R directly affects the inducibility of the iNOS gene in hepatocytes by IL-1beta. Increased NO may be associated with protective or toxic effects in hepatic I/R injury.