OBJECTIVETo explore the influence of exogenous putrescine on the function of liver and apoptosis of liver cells in rats.

METHODSNinety healthy clean SD rats were divided into control group (C, n = 10, intraperitoneally injected with 2 mL normal saline), low dosage putrescine group (LP, n = 40), and high dosage putrescine group (HP, n = 40) according to the random number table. Rats in the latter two groups were intraperitoneally injected with approximately 2 mL putrescine (2.5 or 5.0 g/L) with the dosage of 25 or 50 µg/g. Ten rats from group C at post injection hour (PIH) 24 and 10 rats from each of the latter two groups at PIH 24, 48, 72, 96 were sacrificed. Heart blood was obtained for determination of serum contents of ALT and AST. Liver was harvested for gross observation and histomorphological observation with HE staining. Apoptosis was shown with in situ end labeling, and apoptosis index (AI) was calculated. Data among the three groups and those at different time points within one group were processed with one-way analysis of variance or Welch test; LSD or Dunnett's T3 test was used for paired comparison; factorial design analysis of variance of two factors was applied for data between group LP and group HP.

RESULTS(1) No obvious abnormality was observed at gross observation of liver of rats in each group. Liver tissue of rats in group C was normal. Light edema was observed occasionally in liver of rats in groups LP and HP, but necrotic cells were not seen. (2) Content of ALT at PIH 24, 48, 96 and content of AST at PIH 72 and 96 in group LP were respectively (38 ± 10), (45 ± 6), (34 ± 4), (207 ± 18), (196 ± 19) U/L, and content of ALT at PIH 72 and 96 and content of AST at PIH 24, 72, 96 in group HP were respectively (38 ± 6), (48 ± 5), (213 ± 43), (209 ± 40), (230 ± 29) U/L. They were significantly higher than those of rats in group C [(29 ± 5), (163 ± 42) U/L, with P values all below 0.01]. There were statistically significant differences between group LP and group HP in the content of ALT at PIH 48, 72, 96 and content of AST at PIH 96 (with P values all below 0.05). Compared with that at PIH 24 of each group, content of ALT of rats in group LP at PIH 48 and that of rats in group HP at PIH 96, as well as content of AST of rats in group LP at PIH 48, 72, 96 and that of rats in group HP at PIH 48 were significantly increased or decreased (with P values all below 0.05). Factorial analysis showed that the differences due to different concentration of putrescine on content of AST were statistically significant (F = 12.21, P = 0.001), but not on content of ALT (F = 0.01, P = 0.974) between group LP and group HP. (3) AI values of rats in group LP at PIH 24, 48, 72 were respectively (5.69 ± 0.38)%, (13.80 ± 1.66)%, (11.56 ± 1.74)%, and AI values of rats in group HP at PIH 72 and 96 were respectively (10.29 ± 1.43)%, (15.29 ± 1.41)%. They were all obviously higher than AI value of control group at PIH 24 [(3.50 ± 0.30)%, with P values all below 0.01]. There were statistically significant differences between group LP and group HP in AI value at PIH 24, 48, 96 (with P values all below 0.05). Compared with that at PIH 24 of each group, AI value of rats in groups LP and HP at PIH 48, 72, 96 were significantly increased or decreased (with P values all below 0.05). Factorial analysis showed that the differences in the influence of concentration of putrescine and stimulation time on AI value were statistically significant (with F values respectively 22.95 and 130.44, P values all below 0.01).

CONCLUSIONSIntraperitoneal injection of exogenous putrescine in the dosage of 25 or 50 µg/g could lead to certain degree of functional damage of liver and apoptosis of liver cells of rat. The higher the dosage and the longer the stimulation time, the more obvious the damage and apoptosis would be.

Alanine Transaminase ; blood ; Animals ; Apoptosis ; drug effects ; Hepatocytes ; cytology ; drug effects ; Liver ; cytology ; pathology ; Putrescine ; toxicity ; Rats ; Rats, Sprague-Dawley

To investigate the neutralizing effect of N-acetylcysteine (NAC) and selenium (Se) aganist doxorubicin (DOX) toxicity in rats, NAC (140 mg/kg, p.o.) and Se (0.5 mg/kg, p.o.) were administered for 2 days before DOX injection and then 3 times a week. Cell viability and the level of lipid peroxidation were examined in cultured-rat astrocytes. Severe morphologic changes in the kidney of DOX group; thickening of Bowmans capsule, presence of multifocal tubular casts were observed, but not in the other treated groups. Vacuoles in some hepatic cells and focal aggregation of stellate macrophages were also detected in DOX group, but not in the other treated groups. However, the severe inhibition of spermatogenesis was found in all treated groups. The cell viability of DOX (10 mg/ml) treated group and NAC (5 mM) or Se (0.001 mg/ml) combinedtreated group was 52.5+/-2.0 % , 85.3+/-4.5 % and 75.5+/-1.6 %, respectively. In MDA (malondialdehyde) assay, the level of lipid peroxidation on DOX (10 mg/ml), NAC (5 mM) and Se (0.001 mg/ml) was 0.77+/-0.06, 0.35+/-0.06 and 0.54+/-0.11 nmol/mg protein, respectively. Thus, it is known that NAC and Se have protective effects in kidney and liver but not in the testes. Morphological change was not detected in brain and heart in all groups for experiment period. From this in vitro study, it is known that NAC and Se protect well the astrocytes against DOX induced-cell damage.
Acetylcysteine/*pharmacology ; Animals ; Astrocytes/*cytology/drug effects/pathology ; Cell Survival/drug effects ; Cells, Cultured ; Doxorubicin/antagonists & inhibitors/*toxicity ; Liver/cytology/*drug effects/pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Spermatocytes/cytology/drug effects/pathology

Animals ; Female ; Lactobacillus ; Liver ; pathology ; Lymphocyte Activation ; drug effects ; Lymphocytes ; cytology ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Selenium ; pharmacology

OBJECTIVETo investigate the mechanism of hepatocytes transdifferentiation to bile duct epithelial cells (BECs) and intervention of Huangqi decoction (HQD) on hepatic fibrosis formation in rats with secondary cholestasis.

METHODSSeventy-five SD male rats were made into cholestatic hepatic fibrosis model animals by bile duct ligation, and randomized into the control group (n = 50) and the HQD group (n = 15). Starting from one week after modeling, they were administered orally with saline and HQD respectively for four weeks. Besides, a sham-operated group was set up with 10 rats operated by choledochus segregating only and administered after then with saline. Rats were killed in batches at different time points, i.e. each five from the control group and sham-operated group at the end of the 1st week, five from the control group for each time at the end of the 2nd, 3rd and 4th week, and all the remaining rats at the end of the 5th week. Their liver tissues were taken for histological change examination, content of hydroxyproline (Hyp) determination; protein expression of BECs marker cytokeratin 7 (CK7) and the hepatocyte specific antigen HepPar detection by Western blot, and CK7-Hep Par co-localization by laser confocal microscopy. Then IPP software was used to analyze Sirius red stained positive areas of CK7 and Hep Par, as well as the average IOD of CK7/Hep Par co-localization.

RESULTSHepatocytes in hepatic tissues (Hep Par positive cell) in the model rats decreased gradually along was time went by after modeling (Sham > M1w > M2w > M3w > M4w > M5w), which was in parallel with the increase of BECs (CK7 positive cells), degree of fibrosis, Hyp content and CK7 protein expression. Increasing of co-localized positive cells of CK7/Hep Par began at 1 week and reached the peak 3 weeks after modeling, then it decreased gradually. The Hep Par protein expression was negatively correlated with that of CK7; the Hep Par positive cell expression was negatively correlated with CK7 positive cell expression and collagen deposition; while the CK7 positive cell expression was positively correlated with the collagen deposition in the liver tissue. Compared with the model control group, the mortality, CK7/Hep Par co-localized positive cells, fibrosis degree, Hyp content and CK7 protein expression were lesser obviously (P < 0.01), while Hep Par positive cell and protein expressions were higher significantly in the HQD group.

CONCLUSIONSHepatocytes transdifferentiation to BECs might be a key pathological element for secondary cholestatic hepatic fibrosis formation; the restraining action of HQD is possibly a major action mechanism of HQD for effectively intervening and treating secondary cholestasis hepatic fibrosis.

Animals ; Astragalus Plant ; Bile Ducts ; cytology ; Cell Transdifferentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; Hepatocytes ; cytology ; drug effects ; Liver ; Liver Cirrhosis, Biliary ; drug therapy ; pathology ; Male ; Phytotherapy ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the damage effect of different fractions from Polygonum multiflorum on normal human liver and liver cancer cells, in order to seek for fractions that can obviously kill cancer cells but have less impact on normal liver cells, and make a preliminary study on different mechanism of the two kinds of cells.

METHODP. multiflorum water-eluted fraction (RW), 50% ethanol-eluted fraction (R50) and 95% ethanol-eluted fraction (R95) were successively obtained from 70% ethanol extracts of P. multiflorum, after being eluted by water, 50% ethanol and 95% ethanol and then absorbed by AB-8 macroporous resin. Normal human liver L02 cells and liver cancer HepG2 cells were incubated with cell supernatants from different fractions and cells. MTT method and inverted microscope were adopted to observe the impact of L02 on growth of HepG2 cells, screening fractions with damage effect and detect their doses and time effect. Giemsa stain showed changes in cell nucleus after administration and flow cytometry analysis was used to detect cycle and apoptosis of L02 cells.

RESULTMTT method and inverted microscope showed that R50 had significant growth inhibition effects on L02 and HepG2 cells. According to giemsa stain and flow cytometry analysis, R50 showed different effect on inducing the two cells: there are much more apoptotic HepG2 cells than apoptotic L02 cells in each time phase (the proportion of the apoptosis cells in HepG2 group were 83.62%, 60.52% and 74.49%, and ID2 31.02%, 20.57% and 25.32% after treated with R50 for 24, 48, 72 h. Both cells showed less than 5% of apoptotic cells in the negative control group in each time phase). However, there is no significant impact on cycle of both cells.

CONCLUSIONR50 from P. multiflorum extracts had different damage effects on human liver L02 cells and liver cancer HepG2 cells, which was caused by different degree of induction on apoptosis of the two cells in nature.

Antineoplastic Agents ; adverse effects ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; adverse effects ; pharmacology ; Hep G2 Cells ; Humans ; Liver ; cytology ; pathology ; Liver Neoplasms ; pathology ; Polygonum ; chemistry

OBJECTIVETo explore the changes and significance of the level of reactive oxygen species (ROS) and mitochondria membrane potential (Delta Psi) in HepG2 cells under the stress of cisplatin (CDDP).

METHODSHepG2 cells were incubated with CDDP. The changes in the level of ROS were determined by a probe (2,7-dichloro fluorescein-ciactate, DCFH-DA) and the changes of Delta Psi were reflected as changes of intensities of fluorescence seen under a laser scan microscope using a probe (rhodamine-123). All these changes in cells at 0 h, 24 h, 48 h, 72 h, 120 h, 168 h were dynamically observed.

RESULTSThe level of ROS was much higher after the CDDP treatment than the non-treated, and the increase lasted for 24 h and 48 h. Then it started to decrease at 72 h, gradually returning to normal level at 120 h. Under the selective pressure of CDDP, the fluorescence intensity of rhodamine-123 in HepG2 cells was decreasing at 24 h and 48 h, then gradually started to increase at 72 h. There were no such changes in the cells of the controls.

CONCLUSIONThe changes of ROS and Delta Psi in HepG2 cells under the pressure of CDDP suggest that the cells change themselves adapting to such pressures.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cisplatin ; pharmacology ; Hepatocytes ; cytology ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Membrane Potentials ; drug effects ; Mitochondria, Liver ; physiology ; Reactive Oxygen Species ; metabolism ; Tumor Cells, Cultured

To study the effect of endotoxin on liver apoptosis, L02 liver cells were cultured and passaged in vitro, and then stimulated by endotoxin at 10 mg/mL for 4, 8, 16 and 24 h respectively. Liver apoptosis was flow cytometrically and fluorescently detected. Immunohistochemistry was used to detect the delivery of smac and caspase9. The delivery of liver cell smac and the activity of caspase3 were measured by caspase3 assay kit. The hepatic failure models of rats were established by using D-galactosamine. The blood serum and liver tissues were collected for the detection of the liver function, the level of endotoxin and the activity of caspase3 by using chromogenic substrate limulus amebocyte lysate method (LAL) and caspase3 active assay kit. The expression of smac and caspase9 in liver cells was detected by Western blotting. With in vitro study, the L02 cells stimulated by LPS condensed into conglobation and formed apoptotic bodies. After those cells were stained by hoechst, the apoptotic cells displayed blue color under the fluorescent microscope. The apoptosis rate was increased over time and the apoptosis was mainly of advanced stage. Meanwhile, the rate of smac delivery and activity of caspase9 and caspase3 were increased on L02 cell membrane. In vivo, hepatic failure and obvious endotoxemia were induced by injection of more than 200 mg/kg D-GalN. Hepatic mitochondria smac was reduced with dosage of D-GalN and, on the contrary, the activity of caspase3 was increased. D-GalN at 200 mg/kg increased Caspase9 while D-GalN at 300 mg/kg decreased caspase9. Mitochondria signal channel plays an important role in the endotoxin-induced apoptosis of hepatic cells by promoting the release of smac from mitochondria to cytoplasm and activating caspase9 and caspase3 in its low-level channel.
Apoptosis/*drug effects ; Carrier Proteins/*metabolism ; Caspase 3/metabolism ; Caspase 9/metabolism ; Cell Line ; Cells, Cultured ; Endotoxins/*pharmacology ; Liver/cytology ; Liver/*metabolism ; Liver/pathology ; Liver Failure/chemically induced ; Liver Failure/pathology ; Mitochondrial Proteins/*metabolism ; Rats, Wistar

OBJECTIVETo study the acting mechanism of Cordyceps mycelia extract (CME) for antagonizing hepatic sinusoidal capillarization (HSC) in rats with dimethylnitrosamine (DMN) induced liver cirrhosis.

METHODSRat liver cirrhosis model was established by peritoneal injection of DMN 10 mg/kg 3 times a week for 4 weeks. To rats in the CME-prevented group CME were administrated at a dose of 10 mL/kg, once a day, for 4 weeks. The observation time points were scheduled on the 3rd day (d3), and at the end of the 2nd (W2) and 4th week (W4) after modeling, and the following items were observed: hepatic ultrastructure was observed under electron microscope; expressions of CD44, von Willebrand factor (vWF) and type IV collagen (Col lV) in the liver sinusoidal walls by immunohistochemistry; matrix metalloproteinase-2 and-9 (MMP-2, MMP-9) activity under zymogram method; and serum hyaluronic acid (HA) content by radioimmunoassay.

RESULTSObservation at d3 showed MMP-2 and MMP-9 activity significantly increased, Col IV deposition and CD44 positive staining decreased, vWF positive staining increased in the liver sinusoidal walls, the fenestrae in the sinusoidal endothelial cells (SECs) decreased, and serum HA content increased (P<0.05); at W4, SECs defenestration and sub-SECs basal membrane formation were shown. In the CME-prevented group MMP-2 and MMP-9 activity significantly decreased (P<0.05); defenestration and basal membrane formation alleviated in the early stage (d3, W4); and at W2 and W4 decreases of HA content and vWF positive staining were shown, with increase of CD44 positive staining (P<0.05), more SECs fenestrae, and alleviated basal membrane formation.

CONCLUSIONSThe elevation of MMP-2 and MMP-9 activity in the early stage, which degrades the Col IV normally distributed under the sinusoidal endothelium, is an important factor for HSC formation. CME could inhibit the initiation of HSC by decreasing MMP-2 and MMP-9 activity in the early stage, and prevent its formation by decreasing SECs injury and phenotypic changes.

Animals ; Capillaries ; pathology ; Cordyceps ; Dimethylnitrosamine ; adverse effects ; Hepatic Veins ; cytology ; drug effects ; pathology ; Liver ; blood supply ; Liver Cirrhosis, Experimental ; pathology ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mycelium ; Neovascularization, Pathologic ; prevention & control ; Rats ; Rats, Wistar

To investigate the effect of ursolic acid on the invasion and migration of hepatocellular carcinoma (HCC) cells co-cultured with macrophages, and to explore the underlying mechanisms.
 Methods: The migration and invasion ability of HCC cells in the co-culture system with or without ursolic acid intervention were evaluated by transwell assay. The levels of epithelial-mesenchymal transition (EMT) markers E-cadherin, N-cadherin, and vimentin in HCC cells co-cultured with macrophages were detected by Western blot.
 Results: The migration and invasion ability and EMT were significantly enhanced when co-cultured with macrophages, and the expression of E-cadherin was significantly increased while N-cadherin and vimentin levels were significantly decreased. However, after ursolic acid treatment, the migration and invasion ability were significantly reduced, and the expression of E-cadherin was increased while N-cadherin and vimentin levels were decreased.
 Conclusion: Ursolic acid exerts inhibitory effect on the ability of migration, invasion, and EMT for HCC, which are enhanced by co-culturing with macrophages.
Cadherins ; genetics ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Coculture Techniques ; Epithelial-Mesenchymal Transition ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Liver Neoplasms ; pathology ; Macrophages ; cytology ; Neoplasm Invasiveness ; pathology ; Triterpenes ; pharmacology

OBJECTIVETo evaluate the protective efficacy of gossypin (3,3',4',5,7,8-hexahydroxyflavone 8-glucoside) by administering it intraperitoneally, for dose, time, and vehicle dependent effects against sulphur mustard (SM), administered through percutaneous route in mice.

METHODSSM (diluted in PEG-300) was administered percutaneously. The protective efficacy of gossypin was evaluated by administering it intraperitoneally (50, 100, 200, and 400 mg/kg), in various vehicles (water, PEG-300 and DMSO), and time intervals (30 min prior, simultaneous and 2 h post). The time dependent protection of gossypin (200 mg/kg in PEG-300; i.p.) was also evaluated using selected biochemical variables (GSH, GSSG, MDA, total antioxidant status, Hb, WBC count, RBC count, glutathione peroxidase, glutathione reductase, and superoxide dismutase) and liver histology. The protection of gossypin by oral route was also evaluated against percutaneously administered SM.

RESULTSThe protection against systemic toxicity of SM (LD50 8.1 mg/kg) was better when gossypin was given with PEG-300 (8.0 folds) than DMSO (5.7 folds). No protection was observed when gossypin was administered with water. Good protection (8.0 folds) was observed when gossypin was administered (200 mg/kg in PEG-300; i.p.) at 30 min prior or simultaneous to SM exposure, but no protection was observed when gossypin was administered 2 h post to SM exposure. A significant weight loss was observed 7 days after SM administration (2 LD50), with a significant increase in RBC and Hb. A significant decrease in total antioxidant status of plasma, liver GSH and GSSG levels, and in the activities of glutathione peroxidase, glutathione reductase and superoxide dismutase was also observed 7 days after SM administration. SM treated mouse liver also showed necrosis. A significant protection was observed when gossypin (200 mg/kg in PEG-300; i.p.) was administered either as a pretreatment (30 min before) or simultaneous treatment, and not as a post treatment (2 h). The protective efficacy of gossypin was better through oral route when administered with DMSO (4.8 folds) than with PEG-300 (2.4 folds). No protection was observed when gossypin was administered orally with water.

CONCLUSIONPercutaneous administration of SM induces oxidative stress and gossypin can protect it as a prophylactic agent by intraperitoneal or oral routes.

Administration, Cutaneous ; Animals ; Blood Cell Count ; Body Weight ; drug effects ; Chemical Warfare Agents ; toxicity ; Female ; Flavonoids ; administration & dosage ; pharmacology ; Hemoglobins ; metabolism ; Liver ; cytology ; drug effects ; pathology ; Mice ; Mustard Gas ; administration & dosage ; toxicity ; Oxidative Stress ; drug effects ; Time Factors