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Cheung PY, Zhang Q, Zhang YO, Bai GR, Lin MCM, Chan B, Fong CC, Shi L, Shi YF, Chun J, Kung HF, Yang M. Effect of WeiJia on carbon tetrachloride induced chronic liver injury. World J Gastroenterol 2006; 12:1912-7. [PMID: 16609998 PMCID: PMC4087517 DOI: 10.3748/wjg.v12.i12.1912] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the effect of WeiJia on chronic liver injury using carbon tetrachloride (CCl4) induced liver injury animal model.
METHODS: Wistar rats weighing 180-220g were randomly divided into three groups: normal control group (Group A), CCl4 induced liver injury control group (Group B) and CCl4 induction with WeiJia treatment group (Group C). Each group consisted of 14 rats. Liver damage and fibrosis was induced by subcutaneous injection with 40% CCl4 in olive oil at 3 mL/kg body weight twice a week for eight weeks for Groups B and C rats whereas olive oil was used for Group A rats. Starting from the third week, Group C rats also received daily intraperitoneal injection of WeiJia at a dose of 1.25 μg/kg body weight. Animals were sacrificed at the fifth week (4 male, 3 female), and eighth week (4 male, 3 female) respectively. Degree of fibrosis were measured and serological markers for liver fibrosis and function including hyaluronic acid (HA), type IV collagen (CIV), γ-glutamyl transferase (γ-GT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Alpha smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) immunohistochemistry were also performed.
RESULTS: CCl4 induction led to the damage of liver and development of fibrosis in Group B and Group C rats when compared to Group A rats. The treatment of WeiJia in Group C rats could reduce the fibrosis condition significantly compared to Group B rats. The effect could be observed after three weeks of treatment and was more obvious after eight weeks of treatment. Serum HA, CIV, ALT, AST and γ-GT levels after eight weeks of treatment for Group C rats were 58±22 µg/L (P<0.01), 57±21 µg/L (P<0.01), 47±10 U/L (P<0.01), 139±13 U/L (P<0.05) and 52±21 U/L (P>0.05) respectively, similar to normal control group (Group A), but significantly different from CCl4 induced liver injury control group (Group B). An increase in PCNA and decrease in α-SMA expression level was also observed.
CONCLUSION: WeiJia could improve liver function and reduce liver fibrosis which might be through the inhibition of stellate cell activity.
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Affiliation(s)
- Pik-Yuen Cheung
- Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, China
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2
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Mason WS, Jilbert AR, Summers J. Clonal expansion of hepatocytes during chronic woodchuck hepatitis virus infection. Proc Natl Acad Sci U S A 2005; 102:1139-44. [PMID: 15657132 PMCID: PMC544623 DOI: 10.1073/pnas.0409332102] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Chronic hepadnavirus infections cause liver damage with ongoing death and regeneration of hepatocytes. In the present study we set out to quantify the extent of liver turnover by measuring the clonal proliferation of hepatocytes by using integrated viral DNA as a genetic marker for individual hepatocyte lineages. Liver tissue from woodchucks with chronic woodchuck hepatitis virus (WHV) infection was assayed for randomly integrated viral DNA by using inverse PCR. Serial endpoint dilution of viral-cell junction fragments into 96-well plates, followed by nested PCR and DNA sequencing, was used to determine the copy number of specific viral cell junctions as a measure of the clonal distribution of infected cell subpopulations. The results indicated that the livers contained a minimum of 100,000 clones of >1,000 cells containing integrated DNA, representing at least 0.2% of the hepatocyte population of the liver. Because cells with integrated WHV DNA comprised only 1-2% of total liver cells, it is likely that the total number of clones far exceeds this estimate, with as much as one-half of the liver derived from high copy clones of >1,000 cells. It may be inferred that these clones have a strong selective growth or survival advantage. The results provide evidence for a large amount of hepatocyte proliferation and selection having occurred during the period of chronic WHV infection ( approximately 1.5 years) in these animals.
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Affiliation(s)
- William S Mason
- Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
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3
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Kawakami Y, Kitamoto M, Nakanishi T, Yasui W, Tahara E, Nakayama J, Ishikawa F, Tahara H, Ide T, Kajiyama G. Immuno-histochemical detection of human telomerase reverse transcriptase in human liver tissues. Oncogene 2000; 19:3888-93. [PMID: 10951582 DOI: 10.1038/sj.onc.1203733] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Although telomerase activity in hepatocellular carcinoma (HCC) increases in accordance with degree of histological undifferentiation, it is unknown whether the level of telomerase activity in HCC reflects of the degree of activity in individual cells or the frequency of telomerase-positive HCC cells. Non-cancerous liver tissues exhibit low but significant levels of telomerase activity, but the nature of telomerase-positive cells in these tissues is unclear. In this study, we performed immunohistochemical staining using specific antibody against telomerase reverse transcriptase (hTERT) protein in 15 HCC samples and 13 adjacent non-cancerous liver tissues. There were hTERT-positive hepatocytes, though very low frequency, in non-cancerous liver tissues. The frequencies in hTERT positive hepatocytes were very well correlated with clinicopathological parameters and telomerase activity levels: the average frequencies of chronic hepatitis was 0.2%, liver cirrhosis 0.2%, well-differentiated HCC 3.0%, moderately differentiated HCC 28%, and poorly differentiated HCC 95%. The intensity of staining varied among cells within a given specimen, and correlation with degree of histological undifferentiation was less obvious. Portions of migrating lymphocytes and biliary epithelial cells were also hTERT-positive. These findings indicate that the upregulation of telomerase activity with degree of undifferentiation of HCC is mainly due to the increase in frequency of hTERT positive HCC cells.
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Affiliation(s)
- Y Kawakami
- First Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima City, Japan
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Kuroki T, Seki S, Kawakita N, Nakatani K, Hisa T, Kitada T, Sakaguchi H. Expression of antigens related to apoptosis and cell proliferation in chronic nonsuppurative destructive cholangitis in primary biliary cirrhosis. Virchows Arch 1996; 429:119-29. [PMID: 8917713 DOI: 10.1007/bf00192434] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
The initial injury in primary biliary cirrhosis (PBC) is the destruction of portal bile ducts. Little information is available on apoptosis and cell proliferation in such bile ducts, so we used immunohistochemical techniques to locate molecules related to apoptosis [Fas antigen, Lewis Y antigen (BM1/JIMRO), and bcl-2 protein] and to cell proliferation (proliferating cell nuclear antigen, PCNA) in 21 patients with PBC. In addition, nick-end labelling was done to locate DNA fragmentation. The expression of these molecules in chronic nonsuppurative destructive cholangitis (CNSDC) was examined. Cell death and PCNA expression were both found in portal bile ducts affected by CNSDC in 7 of the 13 CNSDC patients examined. Fas antigen was found on the plasma membrane and rough endoplasmic reticulum of bile-duct cells with CNSDC in the frozen sections of all 6 patients with CNSDC out of the 9 patients inspected, and this antigen was found also in bile-duct cells without CNSDC in 2 of these 9 patients. It was not found in anatomically normal liver (from 2 patients with Gilbert's disease). The Lewis Y antigen was found in bile ducts with CNSDC and in proliferated ductules in all 16 patients examined. No bcl-2 protein was found in any bile-duct or ductule cells, but it was found in the cytoplasm of lymphocytes surrounding or invading CNSDC. DNA fragmentation was found in the nuclei of bile-duct cells with CNSDC by nick-end labelling. Our study indicated that Fas-mediated apoptosis might be involved in CNSDC, but that bcl-2 protein seems to participate less than Fas. Although the Lewis Y antigen was found in many bile ducts, the relationship between the antigen and apoptosis remains unknown because there was no evidence that this antigen mediates apoptosis.
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Affiliation(s)
- T Kuroki
- Third Department of Internal Medicine, Osaka City University Medical School, Japan
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5
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Abstract
The cellular mechanisms associated with the replicative response of hepatocytes to growth factor simulation is incompletely understood. Murine hepatocyte DNA synthesis is altered by cyclic AMP, suggesting that protein kinase A is involved in the cellular mechanisms associated with liver growth. The purpose of this study was to evaluate the role of protein kinase A in human hepatocyte DNA synthesis. human hepatocytes were isolated and maintained in primary culture on rat tail collagen. DNA synthesis was evaluated by determining [3H] thymidine incorporation. Human hepatocytes between 24 and 96 hr following harvest increased DNA synthesis in response to epidermal growth factor but not in response to glucagon, a stimulant of adenyl cyclase, or dibutyryl cyclic AMP. Mitogen-stimulated DNA synthesis was decreased by dibutyryl cyclic AMP. Cyclic AMP isomers that block or stimulate the effect of cyclic AMP on protein kinase A did not significantly alter resting or mitogens-stimulated human hepatocyte DNA synthesis. The results suggest that increased protein kinase A activity does not produce human hepatocyte replicative DNA synthesis.
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Affiliation(s)
- D L Kaminski
- Department of Surgery, St. Louis University Hospital, Missouri 63110-0250, USA
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6
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Kawakita N, Seki S, Sakaguchi H, Yanai A, Nakatani K, Yamada T, Kitada T, Sakai Y, Kuroki T, Kobayashi K. Immunohistochemical analysis of retinoblastoma gene product (pRB) expression in malignant and non-malignant liver diseases. LIVER 1994; 14:295-301. [PMID: 7877433 DOI: 10.1111/j.1600-0676.1994.tb00092.x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The retinoblastoma gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To analyze the expression of retinoblastoma gene product in the process of liver regeneration and the initiation of hepatocellular carcinoma, we studied immunohistochemically the expression of retinoblastoma gene product and DNA polymerase alpha (DPA) in 33 patients with various liver diseases. Only a few hepatocytes positive for retinoblastoma gene product were found in undamaged, nonregenerating liver tissues, whereas many hepatocytes positive for retinoblastoma gene product were detected in specimens of regenerating liver obtained from patients with acute or chronic liver diseases. Similarities were found between distribution patterns of hepatocytes positive for retinoblastoma gene product and those of hepatocytes positive for DPA, and a highly significant positive correlation was found between the number of hepatocyte nuclei stained for retinoblastoma gene product per 1000 nuclei examined (R-LI) and the number of hepatocyte nuclei stained for DPA per 1000 nuclei examined (D-LI) in tissues obtained from patients with nonmalignant liver disease. Hepatocellular carcinoma cells positive for DPA were detected in the 14 hepatocellular carcinoma specimens tested. In ten of these specimens, hepatocellular carcinoma cells positive for retinoblastoma gene product were found but not in the other four. For all hepatocellular carcinoma specimens, R-LI was proportional to D-LI. Thus in both nonmalignant and malignant liver, retinoblastoma gene product increased in proportion to proliferation of hepatocytes or hepatocellular carcinoma cells.
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Affiliation(s)
- N Kawakita
- Third Department of Internal Medicine, Osaka City University Medical School, Japan
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7
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Mancini R, Marucci L, Benedetti A, Jezequel AM, Orlandi F. Immunohistochemical analysis of S-phase cells in normal human and rat liver by PC10 monoclonal antibody. LIVER 1994; 14:57-64. [PMID: 7910934 DOI: 10.1111/j.1600-0676.1994.tb00048.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The expression of proliferating cell nuclear antigen (PCNA) was examined in normal human and rat liver fixed in either formaldehyde or methanol, and was compared with the incorporation of bromodeoxyuridine (BrdU) in S-phase cells. Codistribution of PCNA and BrdU was assessed in rat liver by double immunohistochemical staining using PC10 and anti-BrdU monoclonal antibodies to identify labelled nuclei of parenchymal and sinusoidal cells. In formaldehyde-fixed human biopsies (n = 13) PCNA-labelling index (PCNA LI) was 0.43 +/- 0.24% (mean +/- SEM) for hepatocytes and 0.09 +/- 0.03% for sinusoidal cells. A great interspecimen variability was observed and a preferential lobular distribution was not evident. In methanol-fixed human liver (n = 8) the immunostaining was strong. PCNA LI was 0.05 +/- 0.01% for hepatocytes and 0.14 +/- 0.01% for sinusoidal cells. 75% of labelled hepatocytes and 60% of labelled sinusoidal cells were found in acinar zone 1. In formaldehyde-fixed rat liver (n = 10) a weak nuclear staining and a great interspecimen variability were evident. LI was 0.13 +/- 0.07% for hepatocytes and 0.40 +/- 0.21% for sinusoidal cells without preferential acinar distribution. In methanol-fixed rat liver (n = 10), PCNA LI was 0.14 +/- 0.02% for hepatocytes and 0.40 +/- 0.04% for sinusoidal cells. 64% of labelled hepatocytes and 50% of labelled sinusoidal cells were found in zone 1. Only on methanol-fixed material did double immunohistochemistry show an almost complete overlap of BrdU and PCNA labelling. The PCNA LIs and the zonal distribution of labelled nuclei as obtained in methanol-fixed material are in keeping with previous reports using 3H-thymidine (3H-Thy) incorporation, suggesting that PCNA immunostaining represents a valid alternative to 3H-Thy.(ABSTRACT TRUNCATED AT 250 WORDS)
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Affiliation(s)
- R Mancini
- Department of Gastroenterology, University of Ancona, Italy
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8
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Shibata M, Watanabe M, Ueno Y, Sadamoto T, Sato G, Yasushi T, Yamagami T, Tuzimoto S, Enomoto M. Clinicopathological study of proliferating cell nuclear antigen (PCNA) of hepatocytes in primary biliary cirrhosis. J Gastroenterol 1994; 29:56-60. [PMID: 7911055 DOI: 10.1007/bf01229074] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The DNA synthesis activities of hepatocytes in primary biliary cirrhosis (PBC) and other chronic liver diseases and control subjects were examined by staining proliferating cell nuclear antigen (PCNA) with anti-PCNA monoclonal antibody. The number of PCNA-positive cells (PCNA value) was significantly higher in PBC (375 +/- 281 parts per thousand; ppt) than in other chronic liver diseases, i.e., chronic hepatitis (95 +/- 83 ppt), liver cirrhosis (72 +/- 71 ppt), and alcoholic liver disease (73 +/- 56 ppt), and in control subjects (11 +/- 14 ppt). The PCNA value of PBC in stages I-III of Scheuer's classification was remarkably high, while in stage IV it was low. Even in identical, Scheuer's stages, the PCNA value of PBC was higher in patients who were not given ursodeoxycholic acid (UDCA) than in those who received UDCA. In identical patients, the PCNA value was lowered significantly after UDCA treatment. It was concluded that the DNA synthesis activity of PBC in stages I-III was accelerated and that UDCA can alleviate the abnormality in DNA synthesis activity.
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Affiliation(s)
- M Shibata
- Department of Internal Medicine, Kawasaki Central Hospital, Kanagawa, Japan
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Seki S, Sakaguchi H, Kawakita N, Yanai A, Tamori A, Yabusako T, Nishiguchi S, Kuroki T, Kobayashi K, Monna T. Detection of the preneoplastic lesions of small hepatocellular carcinoma in cirrhotic livers. J Gastroenterol Hepatol 1993; 8:582-9. [PMID: 7904190 DOI: 10.1111/j.1440-1746.1993.tb01657.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
To identify the preneoplastic lesions of hepatocellular carcinoma and the fine structure of preneoplastic hepatocytes, we studied proliferative conditions in cirrhosis of the liver. In all, 46 foci of cellular alteration (FCA), three regions of adenomatous hyperplasia (ADH), and 21 small hepatocellular carcinomas (sHCC) were studied by published criteria for sHCC and by the proliferative activity of the lesions as examined with monoclonal antibodies against DNA polymerase alpha and proliferating cell nuclear antigen. The four patients with FCA composed of basophilic hepatocytes were classified by the criteria as having sHCC; cells had features similar to those of sHCC. Two of these four patients with FCA were found to have HCC several years later. The number of hepatocytes stained for proliferating cell nuclear antigen was 72 and 81 per 1000 hepatocyte nuclei in the two patients who developed HCC. In one of the three patients with ADH, a sHCC was found 1 year later, and dysplastic hepatocytes from the region of ADH in this patient had features similar to those of HCC cells by light and electron microscopy. In this patient, the number of hepatocytes stained for DNA polymerase alpha was 452 per 1000 nuclei. Therefore, FCA and ADH might be preneoplastic lesions of sHCC in cirrhosis of the liver. Preneoplastic hepatocytes seem to be small cells with basophilic cytoplasm, with a large nucleus to cytoplasm ratio, finely indented nuclei with a smaller amount of condensed chromatin than normal, and poorly to moderately developed organelles.
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Affiliation(s)
- S Seki
- Third Department of Internal Medicine, Osaka City University Medical School, Japan
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10
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Jonges GN, Vogels IM, Bosch KS, Dingemans KP, Van Noorden CJ. Experimentally induced colon cancer metastases in rat liver increase the proliferation rate and capacity for purine catabolism in liver cells. HISTOCHEMISTRY 1993; 100:41-51. [PMID: 8226108 DOI: 10.1007/bf00268877] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Metastases in rat liver were generated experimentally by intraportal injection of colon cancer cells to investigate the effects of cancerous growth on the metabolism of surrounding liver tissue. Maximum activities (capacity) of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, lactate dehydrogenase, succinate dehydrogenase, alkaline phosphatase, 5'-nucleotidase, xanthine oxidoreductase, purine nucleoside phosphorylase and adenosine triphosphatase have been determined. Two types of metastases were found, a small type surrounded by stroma and a larger type in direct contact with hepatocytes. Both types affected the adjacent tissue in a similar way suggesting that the interactions were not mediated by stroma. High capacity of the degradation pathway of extracellular purines released from dead cells of either tumours or host tissue was found in stroma and sinusoidal cells. Metastases induced both an increase in the number of Kupffer cells and proliferation of hepatocytes. The distribution pattern in the liver lobulus of most enzymes investigated did not change distinctly. However, activity of alkaline phosphatase, succinate dehydrogenase and phosphogluconate dehydrogenase was increased in hepatocytes directly surrounding metastases. These data imply that the overall metabolic zonation in liver lobuli is not dramatically disturbed by the presence of cancer cells despite the fact that various metabolic processes in liver cells are affected.
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Affiliation(s)
- G N Jonges
- Laboratory of Cell Biology and Histology, University of Amsterdam, The Netherlands
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11
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Seki S, Sakaguchi H, Kawakita N, Yanai A, Kuroki T, Kobayashi K. Analysis of proliferating biliary epithelial cells in human liver disease using a monoclonal antibody against DNA polymerase alpha. VIRCHOWS ARCHIV. A, PATHOLOGICAL ANATOMY AND HISTOPATHOLOGY 1993; 422:133-43. [PMID: 7682020 DOI: 10.1007/bf01607165] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The proliferative activity and ultrastructural characteristics of proliferating biliary epithelial cells were analysed immunohistocytochemically in 39 biopsied liver specimens from patients with acute viral hepatitis, chronic hepatitis and liver cirrhosis using a monoclonal antibody against DNA polymerase alpha (DNA-PA). In acute viral hepatitis with perivenular confluent necrosis, proliferation of typical bile ducts was found frequently in portal areas. In chronic aggressive hepatitis and cirrhosis, ductular proliferation of both typical and atypical forms was found in enlarged portal and periportal areas and in confluent necrotic areas. The number of proliferating biliary epithelial cells that stained positive for DNA-PA was small. There were very few positively stained cells in atypical bile ducts in confluent necrotic areas of cirrhosis. Atypical bile ducts seen in chronic aggressive hepatitis, cirrhosis and acute hepatitis with confluent necrosis were positively stained for both cytokeratins 8 and 19. In cirrhosis, the number of stained biliary epithelial cells in typical bile ducts was larger than the number of such cells in atypical bile ducts (P < 0.01). By electron microscopy, the cells positively stained for DNA-PA were mostly so-called clear cells with irregular nuclei containing coarse nucleoplasm, and a few small cells with scanty cytoplasm and few organelles.
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Affiliation(s)
- S Seki
- Third Department of Internal Medicine, Osaka City University Medical School, Japan
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