OBJECTIVETo investigate the anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc and its possible molecular mechanisms in vitro and in vivo.

METHODSTransonic alcohol-chloroform extraction method was used to extract toosendanin from the bark of Melia toosendan Sieb. et Zucc, and the content of toosendanin in the crude extract was measured by high performance liquid chromatography (HPLC). Anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc were investigated in in vivo and in vitro studies. In the in vitro experiment, human hepatocellular carcinoma cell lines SMMC-7721 and Hep3B were co-incubated with toosendanin crude extract of different concentrations, respectively. In the in vivo experiment, BALB/c mice were subcutaneously inoculated with mouse hepatocellular carcinoma H22 cells and treated with crude extract.

RESULTSHPLC revealed the content of toosendanin was about 15%. Crude extract from Melia toosendan Sieb. et Zucc inhibited cancer cells growth in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50, 72 h) was 0.6 mg/L for SMMC-7721 cells and 0.8 mg/L for Hep3B cells. Both high-dose [0.69 mg/(kg d)] and low-dose [0.138 mg/(kg d)] crude extract could markedly suppress cancer growth, and the inhibition rate was greater than 50%. Hematoxylin and eosin staining showed necrotic area in cancers and transmission electron microscopy displayed necrotic and apoptotic cancer cells with apoptotic bodies. Immunohistochemistry showed that the expression of Bax and Fas increased and the expression of Bcl-2 reduced.

CONCLUSIONSToosendanin extract has potent anti-cancer effects via suppressing proliferation and inducing apoptosis of cancer cells in vivo and in vitro. The mechanism of apoptosis involves in mitochondrial pathway and death receptor pathway.

Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; ultrastructure ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; therapeutic use ; Female ; Immunohistochemistry ; Liver Neoplasms ; drug therapy ; pathology ; ultrastructure ; Male ; Melia ; chemistry ; Mice, Inbred BALB C ; Mitochondria ; drug effects ; metabolism ; Neoplasm Transplantation ; Plant Extracts ; therapeutic use ; Reference Standards ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; metabolism

OBJECTIVE: To observe the micromorphological changes of ultrastructure, apoptosis-related proteins expression and tumor cell apoptosis after ablation with the high-intensity focused ultrasound (HIFU), and to explore the mechanisms responsible for the thermal and non-thermal effect.
 METHODS: Forty rabbits with hepatic VX2 tumors were randomly divided into a thermal group (n=20) and a non-thermal group (n=20), and were subjected to HIFU ablation with thermal or non-thermal condition, respectively. Five animals in each group were sacrificed on the 1st, 3rd, 7th or 14th day after the ablation. The changes of ultrastructure, apoptosis-related proteins expression and tumor cell apoptosis were detected.
 RESULTS: The results of transmission electron microscope (TEM) revealed more severe injury on tissue and cells in the non-thermal group than that in the thermal group. The changes of apoptosis-related proteins expression and tumor cell apoptosis in transient zone were significantly different in comparison with that in the ablated area or peripheral area between the two groups. The expression of vascular endothelial growth factor (VEGF) was at low level on the 1st and 3rd day and elevated gradually on the 7th and 14th day, with no significant difference (all P>0.05). The expression of caspase-3 reached peak on the 3rd day and decreased on the 7th and 14th day. It was significantly higher in the non-thermal group than that in the thermal group on the 3rd and 7th day (all P<0.05). The expression of NF-κB was elevated from the 3rd day and reached peak on the 7th day while decreased on the 14th day. There was no significant difference at every time point between the 2 groups (all P>0.05). The apoptosis index in the non-thermal group and the thermal group on the 3rd and 7th day were (28.60±1.14)% vs (21.80±1.92)% and (21.00±1.58)% vs (14.80±1.48)%, respectively. It was higher in the non-thermal group than that in the thermal group (both P<0.01).
 CONCLUSION Both the thermal and the non-thermal effect of HIFU can induce apoptosis in transient zone, but the latter have a stronger effect.
Animals ; Apoptosis ; Caspase 3 ; metabolism ; High-Intensity Focused Ultrasound Ablation ; Liver Neoplasms ; pathology ; ultrastructure ; NF-kappa B ; metabolism ; Neoplasms, Experimental ; pathology ; ultrastructure ; Rabbits ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo determine the roles of mitochondrial apoptosis and energy metabolism in hepatocytes during the pathogenic process of acute renal failure (ALF) by assessing disease-related differential activities of several key mitochondrial enzymes, including citrate synthase (CS), carnitine palmitoyltransferase-1 (CPT-1) and cytochrome c oxidase (COX).

METHODSThirty-two male Sprague Dawley rats were given D-galactosamine followed by and lipopolysaccharide (LPS) to induce acute liver failure and sacrificed after 4 (4 h group), 8 (8 h group) 12 (12 h group) and 24 hours (24 h group) of treatment. Eight unmodeled rats served as controls. Effects related to apoptosis were evaluated by pathological analysis of hepatic tissues and TUNEL staining. Ultrastructural changes in mitochondria were assessed by electron microscopy. The activity and expression of CS, CPT-1 and COX were measured.

RESULTSHepatocyte apoptosis was present in the 4 h treatment group and was increased obviously in the 8 h treatment group. Hepatocyte necrosis was first observed in the 12 h treatment group and was significantly higher in the 24 h treatment group, with inflammatory cell invasion. Ultrastructural changes in mitochondria were present in the 4 h treatment group, and the 24 h treatment group showed mitochondria with completely destroyed outer membranes, which resulted in mitochondrial collapse. Activity and protein expression of CS, CPT-1 and COX were increased in the 4 h group (vs. controls), were at their peak in the 8 h group (CS:t =1.481, P less than 0.01; CPT-1:t =2.619, P less than 0.05; COX:t =1.014, P less than 0.01) and showed a decreasing trend in the 12 h group. In addition, the activities of CS, CPT-1 and COX were enhanced at the stage of hepatocyte apoptosis, suggesting that these enzymes were involved in the initiation and development of ALF.

CONCLUSIONEnergy metabolism plays an important role in hepatocyte apoptosis during ALF.

Animals ; Apoptosis ; Carnitine O-Palmitoyltransferase ; metabolism ; Citrate (si)-Synthase ; metabolism ; Disease Models, Animal ; Electron Transport Complex IV ; metabolism ; Hepatocytes ; cytology ; enzymology ; Liver Failure, Acute ; metabolism ; pathology ; Male ; Mitochondria ; ultrastructure ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of ancient Chinese medical formula Xiayuxue Decoction ([symbols; see text], XYXD) on activation of hepatic stellate cells (HSCs) and defenestration of sinusoidal endothelial cells (SECs) in CCl4-induced fibrotic liver of mice.

METHODSHigh performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCl4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 (CD31). Whole liver lysates were detected of α-smooth muscle actin (α-SMA) and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and cellomics arrayscan, respectively.

RESULTSAmygdalin and emodin in XYXD were identified. XYXD (993 mg/kg) inhibited Sirius red positive area up to 70.1% (P<0.01), as well as protein levels of α-SMA and type-I collagen by 42.0% and 18.5% (P<0.05) respectively. In vitro, XYXD (12.5 μg/mL, 50 μg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% (P<0.05). Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% (P<0.05) and 49.5% (P<0.01), evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 (P<0.05).

CONCLUSIONSXYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.

Actins ; metabolism ; Animals ; Carbon Tetrachloride Poisoning ; drug therapy ; Collagen Type I ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Endothelium ; drug effects ; pathology ; Hepatic Stellate Cells ; drug effects ; pathology ; ultrastructure ; Liver Cirrhosis ; chemically induced ; drug therapy ; pathology ; Male ; Mice, Inbred C57BL ; Microscopy, Electron, Scanning ; Myofibroblasts ; drug effects ; pathology ; ultrastructure ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Primary Cell Culture ; Rats, Sprague-Dawley

OBJECTIVETo compare morphological differences of three drug-resistant hepatocellular carcinoma (HCC) cell subclones (Huh-7/ADM, Huh-7/CBP, Huh-7/MMC) and their parental Huh-7 cell line, to analyze differential microRNA (miRNA) expression profiles in these cells and, finally to screen for the abnormal expressed miRNAs in drug-resistant HCC cells.

METHODSCellular morphology was observed by histology and transmission electron microscopy. MiRNA microarray was used to analyze the differential miRNA expression profiles in these cells (Huh-7, Huh-7/ADM, Huh-7/CBP, Huh-7/MMC) followed by real time quantitative PCR validation.

RESULTSThe drug-resistant cells had more intracytoplasmic organelles and were larger in size along with increased cytological pleomorphism than the parental Huh-7 cells. Compared with the parental Huh-7 cells, 32 simultaneously up-regulated and 22 down-regulated miRNAs were found in three drug-resistant cells. Up-regulation of miR-15a, miR-16, miR-27b, miR-30b, miR-146a, miR-146b-5p, miR-181a, miR-181d and miR-194 was verified by RT-qPCR.

CONCLUSIONDrug-resistant HCC cells have abnormal expressed miRNAs, which may be explored to further investigate the association of miRNA expressions with multidrugs resistance in HCC.

Antineoplastic Agents ; pharmacology ; Carboplatin ; pharmacology ; Carcinoma, Hepatocellular ; genetics ; pathology ; ultrastructure ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Gene Expression Profiling ; Humans ; Liver Neoplasms ; genetics ; pathology ; ultrastructure ; MicroRNAs ; genetics ; metabolism ; Mitomycin ; pharmacology ; Oligonucleotide Array Sequence Analysis

OBJECTIVETo study the immunologic function of dendritic cells (DCs) cultured in two kinds of hepatoma cell line's supernatant and the enhancing effects of carboxymethylpachymaran (CMP) on DCs.

METHODSDCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines (HepG2 and HepG2.2.15) were collected for condition medium (CM) according to a volume ratio of supernatant to incomplete RPMI-1640 medium, which was 3:1. CMP was dissolved in incomplete RPMI-1640 medium. Experimental groups were divided according to the culture medium, either CM or with CMP in it. DCs subsets CD83, CD86, CD1a, and d-related human leukocyte antigens (HLA-DR) were analyzed by flow cytometry. The proliferation ability of allogeneic T cells in mixed lymphocyte reaction (MLR) stimulated by DCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. IL-12p70, interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) were detected by enzyme-linked immunosorbent assay analysis.

RESULTSThe proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group (P <0.01). Compared with the normal group, groups treated with CMP showed a higher expression level of DCs subsets, lymphocyte reproductive activity, as well as IL-12 and IFN-γ secretion levels. Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γ secretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group (P <0.05). Compared with the normal group, the expression level of NF-κB in DCs nuclear was higher in CMP groups but lower in two CM groups (P <0.05). After CMP was added, the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added (P <0.05). However, there was no significant difference between the two CM groups (P >0.05).

CONCLUSIONSTwo kinds of hepatoma cell line's supernatant can inhibit the immunologic function of DCs. This suppressive effect may be related to the inhibition of NF-κB/Rel pathway. CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.

Carcinoma, Hepatocellular ; pathology ; ultrastructure ; Cell Line, Tumor ; Cell Shape ; Dendritic Cells ; drug effects ; immunology ; Glucans ; pharmacology ; Humans ; Immunophenotyping ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Liver Neoplasms ; pathology ; ultrastructure ; Lymphocyte Culture Test, Mixed ; Signal Transduction ; drug effects ; Subcellular Fractions ; drug effects ; Transcription Factor RelA ; metabolism

OBJECTIVETo investigate the relationship between mast cell and hepatic fibrosis by histopathological method and semi-quantitative measurement.

METHODSSeventy-two Wistary male rats, the control group and the normal group of each only 16, experimental group of 40 rat liver fibrosis was induced by injection of DMN and was sampled at eight different time points. HE, histochemistry, immunohistochemistry (ABC method) and immunofluorescence were performed. The size of fibrosis and the number of mast cells were counted. The expression of MMP-2 and TIMP-2 was documented and electron microscopic examination was performed.

RESULTSAfter injection of DMN, the fibrosis was the most severe in the 2 week (3.72%) and the first month (3.73%, P = 0.2626), and then gradually diminished, although residual fibrosis was still present at 12 months (1.42%, P = 0.0003). The appearance of mast cells began at 2 weeks (1.73 per 200 power field in average by light microscope) after the injection and reached the peak at 4 months (3.06, P = 0.008). Residual amount of mast cells were present at 12 months (1.04, P = 0.045). However, the degree of fibrosis was not proportional or overlapping with the number of mast cells in this experiment model. Mast cells expressed MMP-2 but not TIMP-2.

CONCLUSIONSIn the DMN-induced rat liver fibrosis model, mast cell may be an integral player in the pathogenesis of liver fibrosis and may contribute to the degradation of fibrosis by synthesizing and secreting MMP-2.

Actins ; metabolism ; Animals ; Cell Count ; Dimethylnitrosamine ; Liver Cirrhosis ; chemically induced ; metabolism ; pathology ; Male ; Mast Cells ; metabolism ; pathology ; ultrastructure ; Matrix Metalloproteinase 2 ; metabolism ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Tryptases ; metabolism

OBJECTIVETo study the protective effect of Shaoganduogan (SGDG) on serum transaminase, liver pathology and hepatocyte mitochondria in rat with subacute liver injury induced by carbon tetrachloride.

METHODSubacute liver injury of rats were induced by carbon tetrachloride, and cured by different doses of SGDG through intragastric administration. The activity of serum ALT, AST, liver pathology and ultrastructure, activity of ATPase, SOD and content of MDA of hepatocyte mitochondria were observed.

RESULTSGDG can remarkably reduce the transaminase, alleviate the degeneration and necrosis of liver cells ,enhance activity of Na+ -K+ ATPase, Ca2+ ATPase, SOD, reduce content of MDA of mitochondria, alleviate ultrastructure change of mitochondria, reduce section area, perimeter equivalent diameter and average optical density perimeter of liver cells.

CONCLUSIONSGDG has obvious effect of liver protection, the mechanisms are related with alleviating mitochondria injury.

Animals ; Carbon Tetrachloride ; adverse effects ; Chemical and Drug Induced Liver Injury ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Hepatocytes ; drug effects ; pathology ; Male ; Malondialdehyde ; metabolism ; Mitochondria ; drug effects ; enzymology ; metabolism ; ultrastructure ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the anti-apoptosis effects of Rubus alceaefolius total alkaloids in rats with hepatic injury.

METHODThe hepatic injury model of rat was induced by intraperitoneal injection with CCl4. Sixty SD rats were randomly divided into the normal group, the model group, the R. alceaefolius total alkaloids intervened group, and the bifendate intervened group. The expressions of the levels of liver cell apoptosis were determined by TUNEL. Ultrastructures of the liver cells were observed with transmission electron microscope.

RESULTCompared with the model group, the degree of hepatic injury and the positive expressions of apoptosis in liver tissues in the R. alceaefolius total alkaloids intervened groups and the bifendate intervened group were significantly lower.

CONCLUSIONR. alceaefolius total alkaloids could reduce the pathological changes and degree of hepatic injury in rats, which may be partially through inhibiting the expressions of apoptosis in liver tissue.

Alkaloids ; pharmacology ; therapeutic use ; Animals ; Apoptosis ; drug effects ; Disease Models, Animal ; Female ; Hepatitis, Animal ; drug therapy ; metabolism ; pathology ; In Situ Nick-End Labeling ; Liver ; drug effects ; metabolism ; pathology ; ultrastructure ; Male ; Rats ; Rats, Sprague-Dawley ; Rosales ; chemistry

BACKGROUND AND OBJECTIVEIn recent years, incidence and mortality of lymphoma are markedly increasing worldwide. However, the pathogenesis and mechanism of invasion and metastasis for lymphoma are not yet fully clarified. It is mainly due to the lack of ideal animal models, which can precisely simulate the invasion and metastasis of lymphoma in the human body. So, it is very necessary to establish a highly metastatic nude mouse model of human lymphoma. This study developed a liver-metastatic model of primary gastric lymphoma in nude mice by using orthotopic surgical implantation of histologically intact patient specimens into the corresponding organs of the recipient small animals.

METHODSA histologically intact fragment of liver metastasis derived from a surgical specimen of a patient with primary gastric lymphoma was implanted into the submucosa of the stomach in nude mice. Tumorigenicity, invasion, metastasis, morphologic characteristics (via light microscopy, electron microscopy, and immunohistochemistry), karyotype analysis, and DNA content of the orthotopically transplanted tumors were studied.

RESULTSAn orthotopic liver metastatic model of human primary gastric lymphoma in nude mice (termed HGBL-0304) was successfully established. The histopathology of the transplanted tumors showed primary gastric diffuse large B-cell lymphoma. CD19, CD20, CD22, and CD79alpha were positive, but CD3 and CD7 were negative. The serum level of lactate dehydrogenase (LDH) was elevated [(1010.56+/-200.85) U/L]. The number of chromosomes ranged from 75 to 89. The DNA index (DI) was 1.45+/-0.25 (that is, heteroploid). So far, the HGBL-0304 model has been passed on for 45 generations of nude mice. A total of 263 nude mice were used for the transplantation. Both the growth and resuscitation rates of liquid nitrogen cryopreservation of the transplanted tumors were 100%. The transplanted tumors autonomically invasively grew and damaged a whole layer in the stomach of nude mice. The metastasis rates of liver, spleen, lymph node, and peritoneal seeding were 100%, 94.3%, 62.6%, and 43.5%, respectively.

CONCLUSIONSThe study successfully establishes an orthotopic liver metastatic model of human primary gastric lymphoma in nude mice. The HGBL-0304 model can completely simulate the natural clinical process of primary gastric lymphoma and provides an ideal animal model for the research on the biology of metastasis and antimetastatic experimental therapies of primary gastric lymphoma.

Aged ; Aneuploidy ; Animals ; Antigens, CD ; metabolism ; CD79 Antigens ; metabolism ; Disease Models, Animal ; Humans ; L-Lactate Dehydrogenase ; blood ; Liver ; pathology ; Liver Neoplasms ; genetics ; metabolism ; secondary ; ultrastructure ; Lymphatic Metastasis ; Lymphoma, Large B-Cell, Diffuse ; genetics ; metabolism ; pathology ; ultrastructure ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Splenic Neoplasms ; secondary ; Stomach Neoplasms ; genetics ; metabolism ; pathology