[Changes in mast cells and hepatic expression of c-kit and stem cell factor in the rat model of chronic hepatitis].

OBJECTIVE: To study the potential role of mast cells and the related molecular mechanism in chronic hepatitis (CH) using a rat model system. METHODS: Thirty Wistar rats (15 males, 15 females; weight range: 230-290 g) were randomly divided into the normal contrast (NC) group and experimental CH group. The CH group received subcutaneous injection of CCl4 and a diet high in cholesterol and alcohol content and low in protein and choline content. Throughout the 4-week modeling period, aseptic blood samples were taken to test plasma tryptase (TS) and hyaluronic acid (HA) levels. The rats were euthanized to assess the changes in liver mast cells by histology and morphology analyses and the changes in liver expression of c-kit and stem cell factor (SCF) proteins by immunohistochemistry and mRNAs by RT-PCR. RESULTS: Compared to the NC group, the CH group had higher plasma and liver concentration of HA (78.09 +/- 38.55 vs. 145.14 +/- 52.54 ng/ml, 51.58 +/- 20.45 vs. 106.59 +/- 43.15 ng/100 mg; t = 2.457 and 2.825 respectively, both P less than 0.05) and TS (0.416 +/- 0.143 vs 0.753 +/- 0.210 mg/ml; t = 4.165, P less than 0.05). The CH group also showed fatty degeneration and fibrosis with many degranulating and degranulated mast cells filled with purple granula located around the liver blood vessels and in fiber-intervals. The CH livers also showed a significantly higher number of mast cells (2.167 +/- 0.924 vs. NC: 10.92 +/- 1.575; t = 7.633, P less than 0.05) and stronger intensity of c-kit staining (2.783 +/- 0.577 vs. 12.86 +/- 3.126; t = 9.511, P less than 0.05) and SCF staining (3.383 +/- 1.583 vs. 15.58 +/- 6.431; t = 9.625, P less than 0.05). The expressions of c-kit and SCF were positively correlated with HA level (r = 0.478 and 0.556 respectively, both P less than 0.05). The c-kit and SCF mRNA expression levels were also significantly higher in the CH liver tissues. CONCLUSION: Mast cell degranulation and histamine release is significantly increased under conditions of chronic hepatitis, and the related mechanism may involve up-regulation of the membrane receptor c-kit and its ligand SCF.

Effects of glycine on phagocytosis and secretion by Kupffer cells in vitro.

AIM: To investigate the effects and mechanisms of action of glycine on phagocytosis and tumor necrosis factor (TNF)-α secretion by Kupffer cells in vitro. METHODS: Kupffer cells were isolated from normal rats by collagenase digestion and Percoll density gradient differential centrifugation. After culture for 24 h, Kupffer cells were incubated in fresh Dulbecco's Modification of Eagle's Medium containing glycine (G1: 1 mmol/L, G2: 10 mmol/L, G3: 100 mmol/L and G4: 300 mmol/L) for 3 h, then used to measure phagocytosis by a bead test, TNF-α secretion after lipopolysaccharide stimulation by radioactive immunoassay, and microfilament and microtubule expression by staining with phalloidin-fluorescein isothiocyanate (FITC) or a monoclonal anti-α tubulin-FITC antibody, respectively, and evaluated under a ultraviolet fluorescence microscope. RESULTS: Glycine decreased the phagocytosis of Kupffer cells at both 30 min and 60 min (P < 0.01, P < 0.05). The numbers of beads phagocytosed by Kupffer cells in 30 min were 16.9 ± 4.0 (control), 9.6 ± 4.1 (G1), 12.1 ± 5.7 (G2), 8.1 ± 3.2 (G3) and 7.5 ± 2.0 (G4), and were 22.5 ± 7.9 (control), 20.1 ± 5.8 (G1), 19.3 ± 4.8 (G2), 13.5 ± 4.7 (G3) and 9.2 ± 3.1 (G4) after 60 min. TNF-α secretion by Kupffer cells in G1 (0.19 ± 0.03), G2 (0.16 ± 0.04), G3 (0.14 ± 0.03) and G4 (0.13 ± 0.05) was significantly less than that in controls (0.26 ± 0.03, P < 0.01), and the decrease in secretion was dose-dependent (P < 0.05). Microfilaments of Kupffer cells in G2, G3 and G4 groups were arranged in a disorderly manner. The fluorescence densities of microtubules in G1 (53.4 ± 10.5), G2 (54.1 ± 14.6), G3 (64.9 ± 12.1) and G4 (52.1 ± 14.2) were all lower than those in the controls (102.2 ± 23.7, P < 0.01), but the decrease in microtubule fluorescence density was not dose-dependant. CONCLUSION: Glycine can decrease the phagocytosis and secretion by Kupffer cells in vitro, which may be related to the changes in the expression of microfilaments and microtubules induced by Kupffer cells.

Dynamic changes and mechanism of intestinal endotoxemia in partially hepatectomized rats.

AIM: To explore the mechanism of intestinal endo-toxemia (IETM) formation and its changes in partially hepatectomized (PH) rats. METHODS: One-hundred and two adult male Wistar rats were randomly divided into three groups: normal control (NC) group, partially hepatectomized (PH) group and a sham-operated (SO) group. To study the dynamic changes, rats were sacrificed before and at different time points after partial hepatectomy or the sham-operation ( 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, 120 h and 168 h). NC group was used as 0 h time point in observation, namely 0 h group. For each time point indicated, six rats were used in parallel. Endotoxin (ET) and diamine oxidase (DAO) levels were determined in serum using Limulus Lysate test with chromogenic substrate and spectrophotometry. Intestinal mucosa barrier was observed under optical or electron microscope. The number and functional state of Kupffer cells (KCs) in the remnant regenerating liver were measured by immunohistochemical staining. RESULTS: Serum ET levels significantly increased during 6-72 h period after PH compared with NC and SO groups, and there were two peak values at 12 and 48 h while serum DAO level significantly increased at 12 and 24 h. There was positive correlation (r = 0.757, P < 0.05) between the levels of DAO and ET dynamic changes. The optical examination showed neutrophil margination and superficial necrosis of the villi in the intestinal mucosa during 6-24 h period after PH. The penetrated electron microscope examination showed that the gaps between intestinal mucosa cells were increased and the Lanthanum (La) particles were observed among the intestinal mucosa cells during 6-48 h period. The numbers of KCs in the remnant regenerating liver were significantly increased during 24-168 h period after PH. However, the activation of KCs was predominantly observed at 48 h after PH. CONCLUSION: The mechanism of IETM in PH rats might be the injury of intestinal mucosa barrier and the decrease of the absolute number of KCs as well as the depression of functional state of KCs. This observation is of potential value in patients undergoing liver resection.

Experimental study on the role of endotoxin in the development of hepatopulmonary syndrome.

AIM: To evaluate the role of intestinal endotoxemia in the genesis of hepatopulmonary syndrome. METHODS: A rat model of cirrhosis was prepared with the method of compound factors. At the end of the eighth week, rats with cirrhosis were treated with 300 microg LPS/100 g body weight, and 1 g/rat of glycine about four h prior to LPS. After three h of LPS treatment, blood and tissues were collected for various measurements. Kupffer cells were isolated from male Wistar rats and cultured, and divided into five groups. Supernatant was harvested at 3 h after treatment with LPS for measurement of tumor necrosis factor-alpha (TNF-alpha). RESULTS: Our results showed that in rats with cirrhosis, slowed and deepened breath with occasional pause was. PaO2, PaCO2 and standard bicarbonate (SB) in arterial blood were decreased. Arterial O2 and actual bicarbonate (AB) were markedly decreased. There was a close correlation between decreased O2 and endotoxin. Metabolic acidosis accompanying respiratory alkalosis was the primary type of acid-base imbalance. The alveolar-arterial oxygen gradient was sharply widened. Massive accumulation of giant macrophages in the alveolar spaces and its wall and widened alveolar wall architecture were observed. The number of bacterial translocations in mesenteric lymph nodes increased. The ratio of TC99M-MAA brain-over-lung radioactivity rose. Endotoxin, and TNF-alpha, endothelin-1 (ET-1), nitric oxide (NO) in plasma and ET-1, carbon monoxide (CO) in lung homogenates increased. After administration of a given dosage of LPS in rats with cirrhosis, various pathological parameters worsened. Plasma level of endotoxin was related to TNF-alpha, ET-1, NO in plasma and ET-1, NO, CO in lung homogenates. TNF-alpha level was related to ET-1 and NO in plasma and lung homogenates and CO in lung homogenate as well. The level of TNF-alpha increased after infusion of LPS into culture supernatant of Kupffer cells in vitro. However, TNF-alpha significantly decreased after pretreatment with glycine, PD98059 and SB212850. Glycine could antagonize the effect of LPS in vivo and in vitro. CONCLUSION: Intestinal endotoxemia accompanying by cirrhosis may be an important mechanism in the development of hepatopulmonary syndrome in rats. Overproduction of TNF-alpha due to endotoxin stimulation of Kupffer cells via mitogen-activated protein kinase (MAPK) signal transduction pathway may be a major mechanism mediating the pathologic alterations of hepatopulmonary syndrome.

Endotoxins enhance hepatocarcinogenesis induced by oral intake of thioacetamide in rats.

AIM:To clarify whether endotoxin is of pathogenic importance for hepatocarcinogenesis,or the increased cancer risk results solely from the cirrhotic process.METHODS:The rat model of hepatoma was treated by the intake of 0.03% thioacetamide in drinking water for six months. During induction of hepatoma, rats were additionally treated with splenectomy and/or lipopolysaccharide administration.The liver nuclear DNA index and proliferation index were quantitatively analyzed by flow cytometry. Hepatic histology was examined with light and electron microscopes. Plasmic endotoxin concentration and gamma-glutamyl transpeptidase activity were measured, and hepatoma incidence was recorded.RESULTS: Thioacetamide induced cirrhosis and hepatoma in Wistar rats with histology or regenerative nodule, fibrosis and neoplastic foci were quite similar to the pathogenic process of human cirrhosis leading to hepatoma. In comparison with TAA controls (DNA index: 1.15 plus minus 0.21), exo-endotoxin increased the DNA index by 7.8% (1.24 plus minus0.25, P < 0.02) and hepatoma rate by 16.7. Splenectomy-induced enteric endotoxemia increased the DNA index by 25% (1.44plus minus0.15, P < 0.01) and hepatoma rate by 33%. A summation of the effects of these two factors increased the DNA index by 36% (P < 0.01)and hepatoma incidence by 50%, moreover, the level of endotoxemia showed a close relation with DNA index (r = 0.96, P < 0.01), as well as with the occurrence rate of hepatoma (r = 0.00, P < 0.01). Histological findings further verified such alterations.CONCLUSION:Lipopolysaccharide administration and/or splenectomy-induced enterogenic endotoxemia may enhance rat hepatocarcinogenesis induced by oral intake of thioacetamide.

Effect of endotoxin on fibronectin synthesis of rat primary cultured hepatocytes.

AIM:To investigate the effect of endotoxin on liver fibrosis and further define the role of hepatocytes in production of fibronectin in primary liver cell culture by endotoxin.METHODS:After isolation and seeding of hepatocytes, the obtained cells were added to various doses (0, 5, 10, 15 and 20mg/L) of LPS treated culture media. The cells were collected and counted at various periods (0, 12, 24, 48, 72, 96, 120h).The concentrations of fibronectin were tested by electrophoresis. RESULTS:The fibronectin levels tended to increase with prolongation of culture time. There was a sharp increase after 72h in 10 or 15 LPS treated group. The peak level of fibronectin was above 20mg/L. However, cell proliferation was inhibited during the course.Cell number of untreated control group (4.6 ± 0.1 10(6)) was about three fold that of 20 LPS treated group (1.6 ± 0.2 10(6)) at 120h.CONCLUSION:Hepatocytes have a potent ability to produce fibronectin stimulated by endotoxin, suggesting that hepatocytes might participate in the process of liver fibrosis.