Characteristics of the isocitrate dehydrogenase gene and telomerase reverse transcriptase promoter mutations in gliomas in Chinese patients.

OBJECTIVES: To explore the characteristics of IDH and TERT promoter mutations in gliomas in Chinese patients. METHODS: A total of 124 Chinese patients with gliomas were enrolled to study the frequencies of mutations in isocitrate dehydrogenase (IDH) and telomerase reverse transcriptase promoter (TERTp). Among the 124 patients, 59 patients were enrolled to study the classification of gliomas based on mutations in IDH and TERTp. RESULTS: Isocitrate dehydrogenase mutations are positively correlated with a good prognosis but mutations in TERTp cannot predict prognoses independently. The combined analysis of the mutations of IDH and TERTp can predict the prognosis more accurately. Patients with IDH and TERTp glioma mutations have the best prognosis, followed by only IDH mutation patients and only TERTp mutation patients, which have the worst prognosis. IDH and TERTp mutations occur frequently in males, younger patients or lower-grade patients. In contrast, only TERTp mutations occur frequently in females, older patients or higher-grade patients. CONCLUSIONS: Patients with IDH and TERTp glioma mutations have the best prognosis, and only IDH mutation patients and only TERTp mutation patients have the worst prognosis. Moreover, the molecular classification of gliomas by mutations of IDH and TERTp is not suitable for pediatric patients.

The role of intestinal endotoxemia in a rat model of aluminum neurotoxicity.

The present study aimed to investigate the effects of intestinal endotoxemia (IETM) in a rat model of aluminum neurotoxicity established by D-galactose and aluminum trichloride (AlCl3). Adult Wistar rats were administered D­galactose and AlCl3 to create the aluminum neurotoxicity model. The learning and memory abilities of the rats were subsequently observed using a Morris water maze test and the serum levels of lipopolysaccharide (LPS), tumor necrosis factor (TNF)­α, interleukin (IL)­1, diamine oxidase (DAO), glutamine (Gln) and glutaminase were measured. The expression of S­100ß in the serum was detected using an enzyme­linked immunosorbent assay. The expression levels of the amyloid ß­protein (Aß) precursor (APP), presenilin 1 (PS1), ß­site APP­cleaving enzyme (BACE), zona occludens protein (ZO)­1 and Aß 1­40 in the brain of rats were detected via reverse­transcription polymerase chain reaction, western blotting and immunohistochemistry. The levels of LPS, TNF­α, IL­1, DAO, Gln and S­100ß in serum and the mRNA and protein expression levels of APP, PS1, BACE and Aß1­40 in the brain were markedly increased in the model rats compared with controls. The level of glutaminase in the serum and the expression of ZO­1 in the brain were decreased in the model rats compared with controls. IETM was present in the rat model of aluminum neurotoxicity established by D­galactose and AlCl3 and may be important in the development of this neurotoxicity.

Effect of artesunate supplementation on bacterial translocation and dysbiosis of gut microbiota in rats with liver cirrhosis.

AIM: To evaluate the effect of artesunate (AS) supplementation on bacterial translocation (BT) and gut microbiota in a rat model of liver cirrhosis. METHODS: Fifty-four male Sprague-Dawley rats were randomly divided into a normal control group (N), a liver cirrhosis group (M) and a liver cirrhosis group intervened with AS (MA). Each group was sampled at 4, 6 and 8 wk. Liver cirrhosis was induced by injection of carbon tetrachloride (CCl4), intragastric administration of 10% ethanol, and feeding a high fat diet. Rats in the MA group were intragastrically administered with AS (25 mg/kg body weight, once daily). Injuries of the liver and intestinal mucosa were assessed by hematoxylin-eosin or Masson's trichrome staining. Liver index was calculated as a ratio of the organ weight (g) to body weight (g). The gut microbiota was examined by automated ribosomal intergenic-spacer analysis of fecal DNA. BT was assessed by standard microbiological techniques in the blood, mesenteric lymph nodes (MLNs), liver, spleen, and kidney. RESULTS: Compared to group N, the body weight was reduced significantly in groups M and MA due to the development of liver cirrhosis over the period of 8 wk. The body weight was higher in group MA than in group M. The liver indices were significantly elevated at 4, 6 and 8 wk in groups M and MA compared to group N. AS supplementation partially decreased the liver indices in group MA. Marked histopathologic changes in the liver and small intestinal mucosa in group M were observed, which were alleviated in group MA. Levels of pro-inflammatory interleukin-6 and tumor necrosis factor-α were significantly elevated at 8 wk in ileal homogenates in group M compared to group N, which were decreased after AS supplementation in group MA. The dysbiosis of gut microbiota indicated by the mean diversity (Shannon index) and mean similarity (Sorenson index) was severe as the liver cirrhosis developed, and AS supplementation had an apparent intervention effect on the dysbiosis of gut microbiota at 4 wk. The occurrence of BT was increased in the liver of group M compared to that of group N. AS supplementation reduced BT in group MA at 8 wk. BT also occurred in the MLNs, spleen, and kidney, which was reduced by AS supplementation. BT was not detected in the blood in any group. CONCLUSION: Dysbiosis of gut microbiota, injury of intestinal mucosal barrier and BT occurred as liver cirrhosis progressed, which might enhance inflammation and aggravate liver injury. AS may have other non-antimalarial effects that modulate gut microbiota, inhibit BT and alleviate inflammation, resulting in a reduction in CCl4, alcohol and high fat-caused damages to the liver and intestine.

[Protective effect of tanshinol on the hepatopulmonary syndrome in rat].

OBJECTIVE: To explore the mechanism of tanshinol on alleviate the inflammatory injury of lung tissue in rat hepatopulmonary syndrome (HPS). METHODS: SD rats were randomly divided into normal control group (n = 8), hepatopulmonary syndrome (HPS) group (n = 11) and tanshinol intervention group (n = 9). HE staining was used to observe the histopathology changes of pulmonary and hepatic tissues, and to count the number of macrophages in lung tissues. The activity of alanine transferase (ALT) and concentrations of endotoxin, tumor necrosis factor-a (TNF-alpha) and homocystein (Hcy) in plasma were detected. The concentrations of TNF-alpha, nitric oxide (NO) and malondialdehyde (MDA) and the activity of inducible nitric oxide synthase (iNOS) in the lung tissues were measured, respectively. RESULTS: Thickened alveolar septum and increased macrophages were observed in lungs in HPS rat. After administered with tanshinol, the pulmonary pathological changes were alleviated and the number of macrophages in lung tissue was decreased compared with HPS group. The activity of ALT and the concentrations of endotoxin, TNF-alpha and Hcy in plasma ,and TNF-alpha, iNOS, NO and MDA in lung tissue in HPS group were higher than those of normal control group; meanwhile, those tanshinol group were less those that of HPS group. CONCLUSION: Tanshinol may play an important role in delaying the development of HPS through protecting liver or directly antagonizing the effect of intestinal endotoxemia so as to alleviate the inflammatory reaction in lung tissue.

[Effects of antihistamine treatment on immune function in rats with experimental hepatitis].

OBJECTIVE: To investigate the effects of antihistamine treatment on immune function in rats with experimental hepatitis. METHODS: Thirty Wistar rats were randomly allocated into three groups:experimental hepatitis group (EH group), antihistamine treatment group (AH group) and normal control group (NC group). Rats in the EH group received the subcutaneous injection of 40% carbon tetrachloride oil solution and were fed on diet with low-protein, low-choline, high-fat and high-alcohol,while rats in the AH group received antihistamine treatment(ketotifen + vitamin C) additionally.They were sacrificed after 4 weeks, and the levels of serum alanine aminotransferase(ALT), total bilirubin (TBil), histamine(HA), IFNgamma, IL-12, IL-4 and IL-10 were determined. The levels of IL-12 mRNA and IFN-gamma mRNA in liver tissue were determined via real-time reverse transcriptional polymerase chain reaction(RT-PCR). RESULTS: (1) Compared to the NC group, in the EH group, the levels of ALT, TBil, and circulating and intrahepatic HA were significantly increased(P less than 0.05); intrahepatic HA were significantly decreased(P less than 0.05) after antihistamine treatment. (2) Compared to the NC group, in the EH group, the levels of IL-4, IL-10 were significantly increased((0.504+/-0.202)ng/ml and (29.025+/-1.478) pg/ml vs (0.811+/-0.244)ng/ml and (33.72+/-4.293)pg/ml respectively, P less than 0.05), and the levels of IL-12 were decreased ((6.515+/-2.893)pg/ml vs (3.519+/-1.113)pg/ml, P less than 0.05); and after antihistamine treatment the levels of IL-4 and IL-10 were significantly decreased (were (0.423+/-0.168)ng/ml and (30.412+/-3.275)pg/ml, P less than 0.05), the levels of IL-12 were significantly increased (P less than 0.05), but the level of IFNgamma had no significance (P more than 0.05). The levels of intrahepatic IL-12 mRNA and IFNgamma mRNA had similar results. CONCLUSION: Antihistamine treatment may improve liver function and correct Th1/Th2 unbalance.

[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.

Multiple pathogenic factor-induced complications of cirrhosis in rats: a new model of hepatopulmonary syndrome with intestinal endotoxemia.

AIM: To develop and characterize a practical model of Hepatopulmonary syndrome (HPS) in rats. METHODS: The experimental animals were randomized into five feeding groups: (1) control (fed standard diet), (2) control plus intraperitoneal injection with lipopolysaccharide (LPS), (3) cirrhosis (fed a diet of maize flour, lard, cholesterol, and alcohol plus subcutaneously injection with carbon tetrachloride (CCl(4)) oil solution), (4) cirrhosis plus LPS, and (5) cirrhosis plus glycine and LPS. The blood, liver and lung tissues of rats were sampled for analysis and characterization. Technetium 99m-labeled macroaggregated albumin (Tc99m-MAA) was used to test the dilatation of pulmonary microvasculature. RESULTS: Typical cirrhosis and subsequent hepato-pulmonary syndrome was observed in the cirrhosis groups after an 8 wk feeding period. In rats with cirrhosis, there were a decreased PaO(2) and PaCO(2) in arterial blood, markedly decreased arterial O(2) content, a significantly increased alveolar to arterial oxygen gradient, an increased number of bacterial translocated within mesenteric lymph node, a significant higher level of LPS and tumor necrosis factor-alpha (TNF-alpha) in plasma, and a significant greater ratio of Tc99m-MAA brain-over-lung radioactivity. After LPS administration in rats with cirrhosis, various pathological parameters got worse and pulmonary edema formed. The predisposition of glycine antagonized the effects of LPS and significantly alleviated various pathological alterations. CONCLUSION: The results suggest that: (1) a characteristic rat model of HPS can be non-invasively induced by multiple pathogenic factors including high fat diet, alcohol, cholesterol and CCl(4); (2) this model can be used for study of hepatopulmonary syndrome and is clinically relevant; and (3) intestinal endotoxemia (IETM) and its accompanying cytokines, such as TNF-alpha, exert a crucial role in the pathogenesis of HPS in this model.

Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15.

AIM: To observe the inhibition of hepatitis B virus replication and expression by transfecting vector-based small interference RNA (siRNA) pGenesil-HBV X targeting HBV X gene region into HepG2.2.15 cells. METHODS: pGenesil-HBV X was constructed and transfected into HepG2.2.15 cells via lipofection. HBV antigen secretion was determined 24, 48, and 72 h after transfection by time-resolved immunofluorometric assays (TRFIA). HBV replication was examined by fluorescence quantitative PCR, and the expression of cytoplasmic viral proteins was determined by immunohistochemistry. RESULTS: The secretion of HBsAg and HBeAg into the supernatant was found to be inhibited by 28.5% and 32.2% (P < 0.01), and by 38.67% (P < 0.05) and 42.86% (P < 0.01) at 48 h and 72 h after pGenesil-HBV X transfection, respectively. Immunohistochemical staining for cytoplasmic HBsAg showed a similar decline in HepG2.2.15 cells 48 h after transfection. The number of HBV genomes within culture supernatants was also significantly decreased 48 h and 72 h post-transfection as quantified by fluorescence PCR (P < 0.05). CONCLUSION: In HepG2.2.15 cells, HBV replication and expression is inhibited by vector-based siRNA pGenesil-HBV X targeting the HBV X coding region.

[Effect of Endotoxin on the expression of peroxisome proliferator-activated receptor alpha in the development of nonalcoholic steatohepatitis in rats].

OBJECTIVE: To study the effect of endotoxin on the expression of peroxisome proliferator-activated receptor alpha (PPARa) in the development of nonalcoholic steatohepatitis in rats. METHODS: A model of nonalcoholic steatohepatitis (NASH) was developed with Wistar rats fed a chow containing 20% maize oil for 14 weeks. The endotoxin group rats were intraperitoneally injected with lipopolysaccharide (LPS, 1 g/L, 3.0 ml/kg) once 4 hours before the end of the experiment. The concentrations of lipids, endotoxin, tumor necrosis factor-a, malondialdehyde, free fatty acid in plasma and hepatic tissues were determined and the degree of hepatocytic steatosis was studied. The expression of PPARa mRNA in hepatic tissues was measured using reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The expression of PPARa mRNA in the hepatic tissue of the LPS group was downregulated markedly in comparison to that of the control group. The level of free fatty acid and endotoxin by secreting tumor necrosis factor-a increased and triglyceride accumulated in the liver caused malondialdehyde content to increase, then lipid peroxidation process enhanced and ALT activity increased. Thus, hepatic injury and inflammatory reaction could be accelerated. CONCLUSION: Endotoxemia can enhance hepatocellular steatosis and lead to NASH due to its downregulating the expression of PPARa mRNA.

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.

Intestinal endotoxemia as a pathogenetic mechanism in liver failure.

Liver injury induced by various pathogenic factors (such as hepatitis virus, ethanol, drugs and hepatotoxicants, etc.) through their respective special pathogenesis is referred to as primary liver injury (PLI). Liver injury resulted from endotoxin (lipopolysaccharide, LPS) and the activation of Kupffer cells by LPS while intestinal endotoxemia (IETM) occurred during the occurrence and development of hepatitis is named the secondary liver injury (SLI). The latter which has lost their own specificities of primary pathogenic factors is ascribed to IETM. The secondary liver injury is of important action and impact on development and prognosis of hepatitis. More severe IETM commonly results in excessive inflammatory responses, with serious hepatic necrosis, further severe hepatitis and even induces acute liver failure. The milder IETM successively precipitates a cascade, including repeated and persistent hepatocytic impairment accompanied by infiltration of inflammatory cells, hepatic fibrosis, cirrhosis and hepatocarcinoma. Generally, the milder IETM ends with chronic hepatic failure. If PLI caused by various pathogenic factors through their independent specific mechanismis regarded as the first hit on liver, then SLI mediated by different chemical mediators from KCs activated by IETM in the course of hepatitis is the second hit on liver. Thus, fusing and overlapping of the primary and scondary liver injuries determine and influeuce the complexity of the illness and outcome of the patient with hepatitis. For this reason, the viewpoint of SLI induced by the second hit on liver inflicted by IETM suggests that medical professionals should attach great importance to both PLI and SLI caused by IETM. That is, try to adjust the function of KS(s) and eliminate endotoxemia of the patient.

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.