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Guedes PLR, Carvalho CPF, Carbonel AAF, Simões MJ, Icimoto MY, Aguiar JAK, Kouyoumdjian M, Gazarini ML, Nagaoka MR. Chondroitin Sulfate Protects the Liver in an Experimental Model of Extra-Hepatic Cholestasis Induced by Common Bile Duct Ligation. MOLECULES (BASEL, SWITZERLAND) 2022; 27:molecules27030654. [PMID: 35163920 PMCID: PMC8839946 DOI: 10.3390/molecules27030654] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Revised: 01/06/2022] [Accepted: 01/12/2022] [Indexed: 12/15/2022]
Abstract
During liver fibrogenesis, there is an imbalance between regeneration and wound healing. The current treatment is the withdrawal of the causing agent; thus, investigation of new and effective treatments is important. Studies have highlighted the action of chondroitin sulfate (CS) in different cells; thus, our aim was to analyze its effect on an experimental model of bile duct ligation (BDL). Adult Wistar rats were subjected to BDL and treated with CS for 7, 14, 21, or 28 days intraperitoneally. We performed histomorphometric analyses on Picrosirius-stained liver sections. Cell death was analyzed according to caspase-3 and cathepsin B activity and using a TUNEL assay. Regeneration was evaluated using PCNA immunohistochemistry. BDL led to increased collagen content with corresponding decreased liver parenchyma. CS treatment reduced total collagen and increased parenchyma content after 21 and 28 days. The treatment also promoted changes in the hepatic collagen type III/I ratio. Furthermore, it was observed that CS treatment reduced caspase-3 activity and the percentage of TUNEL-positive cells after 14 days and cathepsin B activity only after 28 days. The regeneration increased after 14, 21, and 28 days of CS treatment. In conclusion, our study showed a promising hepatoprotective action of CS in fibrogenesis induced by BDL.
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Affiliation(s)
- Pedro L. R. Guedes
- Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04023-062, Brazil;
| | - Carolina P. F. Carvalho
- Department of Biosciences, Instituto Saúde Sociedade, Universidade Federal de São Paulo, Santos 11015-020, Brazil; (C.P.F.C.); (M.L.G.)
| | - Adriana A. F. Carbonel
- Department of Gynecology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04039-001, Brazil;
| | - Manuel J. Simões
- Department of Morphology and Genetic, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04023-900, Brazil;
| | - Marcelo Y. Icimoto
- Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04039-032, Brazil;
| | - Jair A. K. Aguiar
- Department of Biochemistry, Universidade Federal de Juiz de Fora, Juiz de Fora 36036-900, Brazil;
| | - Maria Kouyoumdjian
- Department of Biochemistry, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04023-062, Brazil;
| | - Marcos L. Gazarini
- Department of Biosciences, Instituto Saúde Sociedade, Universidade Federal de São Paulo, Santos 11015-020, Brazil; (C.P.F.C.); (M.L.G.)
| | - Marcia R. Nagaoka
- Department of Biosciences, Instituto Saúde Sociedade, Universidade Federal de São Paulo, Santos 11015-020, Brazil; (C.P.F.C.); (M.L.G.)
- Correspondence:
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Potential Therapeutic Application of Estrogen in Gender Disparity of Nonalcoholic Fatty Liver Disease/Nonalcoholic Steatohepatitis. Cells 2019; 8:cells8101259. [PMID: 31619023 PMCID: PMC6835656 DOI: 10.3390/cells8101259] [Citation(s) in RCA: 67] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2019] [Revised: 10/10/2019] [Accepted: 10/12/2019] [Indexed: 02/07/2023] Open
Abstract
Nonalcoholic fatty liver disease (NAFLD) caused by fat accumulation in the liver is globally the most common cause of chronic liver disease. Simple steatosis can progress to nonalcoholic steatohepatitis (NASH), a more severe form of NAFLD. The most potent driver for NASH is hepatocyte death induced by lipotoxicity, which triggers inflammation and fibrosis, leading to cirrhosis and/or liver cancer. Despite the significant burden of NAFLD, there is no therapy for NAFLD/NASH. Accumulating evidence indicates gender-related NAFLD progression. A higher incidence of NAFLD is found in men and postmenopausal women than premenopausal women, and the experimental results, showing protective actions of estradiol in liver diseases, suggest that estrogen, as the main female hormone, is associated with the progression of NAFLD/NASH. However, the mechanism explaining the functions of estrogen in NAFLD remains unclear because of the lack of reliable animal models for NASH, the imbalance between the sexes in animal experiments, and subsequent insufficient results. Herein, we reviewed the pathogenesis of NAFLD/NASH focused on gender and proposed a feasible association of estradiol with NAFLD/NASH based on the findings reported thus far. This review would help to expand our knowledge of the gender differences in NAFLD and for developing gender-based treatment strategies for NAFLD/NASH.
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Wei Y, Zhang X, Wen S, Huang S, Huang Q, Lu S, Bai F, Nie J, Wei J, Lu Z, Lin X. Methyl helicterate inhibits hepatic stellate cell activation through downregulating the ERK1/2 signaling pathway. J Cell Biochem 2019; 120:14936-14945. [PMID: 31009108 DOI: 10.1002/jcb.28756] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2018] [Revised: 12/22/2018] [Accepted: 01/07/2019] [Indexed: 11/11/2022]
Abstract
The present study was to investigate the inhibitory effect of methyl helicterate (MH) on hepatic stellate cells (HSC-T6), primarily elucidating the underlying mechanism of MH against liver fibrosis. HSC-T6 cells were activated by platelet-derived growth factor (PDGF) stimulation, and then the effects of MH on cell viability, cytomembrane integrity, colony, migration, apoptosis, and cell cycle were detected. Moreover, the regulative mechanism of MH on HSCs was investigated by detecting the activation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. The results showed that MH significantly inhibited HSC-T6 cell viability and proliferation in a concentration-dependent manner. It notably promoted the release of lactate dehydrogenase, destroying cell membrane integrity. MH also markedly inhibited HSC-T6 cell clonogenicity and migration. Moreover, MH treatment significantly induced cell apoptosis and arrested cell cycle at the G2 phase. The further study showed that MH inhibited the expression of ERK1, ERK2, c-fos, c-myc, and Ets-1, blocking the ERK1/2 pathway. In conclusion, this study demonstrates that MH significantly inhibits HSC activation and promotes cell apoptosis via downregulation of the ERK1/2 signaling pathway.
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Affiliation(s)
- Yuanyuan Wei
- Life Sciences Institute and Pharmaceutical College, Guangxi Medical University, Nanning, China
| | - Xiaolin Zhang
- Life Sciences Institute and Pharmaceutical College, Guangxi Medical University, Nanning, China
| | - Shujuan Wen
- Life Sciences Institute and Pharmaceutical College, Guangxi Medical University, Nanning, China
| | - Shaode Huang
- Pharmaceutical College, Guangxi Agricultural Vocational College, Nanning, China
| | - Quanfang Huang
- Department of Pharmacy, The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning, China
| | - Shengjuan Lu
- Life Sciences Institute and Pharmaceutical College, Guangxi Medical University, Nanning, China
| | - Facheng Bai
- Life Sciences Institute and Pharmaceutical College, Guangxi Medical University, Nanning, China
| | - Jinlan Nie
- Life Sciences Institute and Pharmaceutical College, Guangxi Medical University, Nanning, China
| | - Jinbin Wei
- Life Sciences Institute and Pharmaceutical College, Guangxi Medical University, Nanning, China
| | - Zhongpeng Lu
- Department of Pharmacy, The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning, China
- Pharmaceutical College, University of Arkansas Medical School, Little Rock, Arkansas
| | - Xing Lin
- Life Sciences Institute and Pharmaceutical College, Guangxi Medical University, Nanning, China
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Wang J, Tang L, White J, Fang J. Inhibitory effect of gallic acid on CCl4-mediated liver fibrosis in mice. Cell Biochem Biophys 2014; 69:21-6. [PMID: 24096707 DOI: 10.1007/s12013-013-9761-y] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The aim of this study was to investigate the effect of gallic acid (GA) on liver fibrosis induced by carbon tetrachloride (CCl4). Male BALB/c mice were randomly divided into four groups: normal control group (group A), CCl4-induced liver injury control group (group B), and CCl4 induction with GA of low dose (5 mg/kg) and high dose (15 mg/kg) treatment group (group C and group D). GA was intra-gastric given for mice once a day after 2 weeks of CCl4 induction. Animals were killed at the eighth week. Degrees of fibrosis and collagen percentage were measured. Hyaluronic acid (HA), type IV collagen (cIV), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) were determined. Expression of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) mRNA levels were examined by RT-PCR. Western blotting was carried out to evaluate the changes of MMP-2 protein. HE and VG stainings showed GA in a dose-dependent manner improved significantly the fibrosis condition in CCl4-injured mice (P < 0.05 or P < 0.01). Also, the concentrations of HA, cIV, and MDA, as well as the serum levels of ALT, AST, and γ-GT were markedly reduced by GA (P < 0.05 or P < 0.01), and decreases in MMP-2, TIMP-1 mRNA, and MMP-2 protein were observed as well (P < 0.05 or P < 0.01). GA could exert protective effect on liver injury and reduce liver fibrosis induced by CCl4 in mice, which might be through the inhibition of hepatic stellate cell activity.
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Affiliation(s)
- Jing Wang
- School of Life Sciences, Jilin Agricultural University, Changchun, 130118, China
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Jung YR, Lee YJ, Lee NJ, Lin CM, Moon JH, Chai HY, Kang JK. Inhibitory Effect of 1-O-Hexyl-2,3,5-Trimethylhydroquinone on Dimethylnitrosamine-induced Liver Fibrosis in Male SD Rats. Toxicol Res 2013; 26:193-201. [PMID: 24278524 PMCID: PMC3834479 DOI: 10.5487/tr.2010.26.3.193] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2010] [Revised: 08/03/2010] [Accepted: 08/14/2010] [Indexed: 11/20/2022] Open
Abstract
Hepatic fibrosis represents the main complication of most chronic liver disorders and, regardless of its etiology, is characterized by excessive deposition of extracellular matrix components. In this study, we examined that 1-O-Hexyl-2,3,5-Trimethylhydroquinone (HTHQ) , a potent anti-oxidative agent, could prevent experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in male SD rats. Except for vehicle control group, other groups were induced hepatic fibrosis by intraperitoneal injection with DMN (10 mg/ml/kg) on 3 consecutive days weekly for 4 weeks. During the same 4 weeks, control and DMN groups were given vehicle and HTHQ 50, 100 and 200 groups were orally administered HTHQ (50, 100, 200 mg/kg respectively) . In HTHQ 100 and 200 groups, relative liver weight and serum chemistry level improved significantly. HTHQ reduced hydroxyproline (p < 0.05) and malondialdehyde (p < 0.05) level in the liver. Histopathological examination of H&E, Masson’s trichrome stain showed the reduced fibrotic septa in HTHQ 100 and 200 groups. HTHQ administration showed reduced mRNA level of PDGF (Plateletderived growth factor) , α-SMA (α-smooth muscle actin) and TGF-β (transforming growth factor-β) than DMN-induced hepetic fibrosis animals in the liver tissue. In this study, we showed that HTHQ improves against DMN-induced liver fibrosis in male SD rats.
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Affiliation(s)
- Yu-Ri Jung
- College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea
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Abstract
Hepatocytes, like other epithelia, are situated at the interface between the organism's exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes.
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Affiliation(s)
- Aleksandr Treyer
- Albert Einstein College of Medicine, Department of Developmental and Molecular Biology, Bronx, New York, USA
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The protective effect of resveratrol on dimethylnitrosamine-induced liver fibrosis in rats. Arch Pharm Res 2010; 33:601-9. [DOI: 10.1007/s12272-010-0415-y] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2009] [Revised: 01/19/2010] [Accepted: 01/24/2010] [Indexed: 01/08/2023]
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Kim KY, Cho SE, Yu BS. Intragastrically Applicated CCl 4-Thiopental Sodium Enhanced Lipid Peroxidation and Liver Fibrosis (Cirrhosis) in Rat: Malonedialdehyde as a Parameter of Lipid Peroxidation Correlated with Hydroxyproline as a Parameter of Collagen Synthesis (Deposition). Toxicol Res 2009; 25:71-78. [PMID: 32038822 PMCID: PMC7006345 DOI: 10.5487/tr.2009.25.2.071] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2009] [Revised: 04/30/2009] [Accepted: 05/15/2009] [Indexed: 11/20/2022] Open
Abstract
We investigated the pathogenesis of liver tissue damage during the lipid peroxidation and fibrogenesis with the observation of correlations between the parameters of collagen synthesis (and deposition) and lipid peroxidation in liver fibrosis (cirrhosis) rats. Rats were randomly divided into two groups, normal and CCl4-thiopental sod. intoxicated group. And the one group was treated intragastrically with the mixture of CCl4-thiopental sod. 3 times per week for 3 weeks. The liver tissue and sera were used for the measurement of hydroxyproline (HYP), malonedialdehyde (MDA) and superoxide dismutase (SOD). Biochemical parameters such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total-bilirubin and blood urea nitrogen (BUN) were measured. Additionally, the expression of collagen α1(III) and β-actin mRNA was observed by RT-PCR. The histological change in liver tissue was also observed by Masson's trichrome and H&E staining. Correlation analysis was carried by Spearman's rho method. All biochemical parameters except total-bilirubin were significantly higher in the CCl4-thiopental sod. treated group than that of the normal group (p < 0.01). In the CCl4-thiopental sod. treated group, Hyp as a parameter of collagen synthesis (deposition) and MDA as a metabolite of lipid peroxidation, were significantly elevated by 1.98 and 2.11 times higher than that of the normal group (p> 0.001) respectively. The activity of SOD in the CCl4-thiopental sod. treated group is decreased significantly by 44.8% (p> 0.001). And collagen α1(III) mRNA was more expressed in the CCl4-thiopental sod. treated group than that of the normal group. However, the expression of β-actin mRNA is showed similar in both of groups. A good correlation was observed between the content of hyp and MDA concentration (r = 0.70, n = 40) in the two groups. And the correlation between the levels of hyp and SOD (r = -0.71, n = 25) is also reliable. However, no correlation were observed between MDA concentration and SOD (r = -0.40, n = 25) in the two groups. Elevated levels of MDA in CCl4-thiopental sod. treated rats indicated enhancement of lipid peroxidation, which is accompanied by a decrease in SOD activity. Moreover, we could confirm that the parameters of collagen synthesis (and deposition) is in good correlation with the metabolite of lipid peroxidation (MDA) and the lipid peroxidation antagonizing enzyme (SOD). Hence, we propose that ➀ lipid peroxidation and collagen synthesis (and deposition) could be enhanced by intragastrically application of CCl4-thiopental sod. during a short terms. And ➁ the intoxication of CCl4-thiopental sod. could be used for monitoring of lipid peroxidation and collagen synthesis (and depositon) for test of antioxidant and antifibrotic agent.
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Affiliation(s)
- Ki-Young Kim
- Dept. of Pathology, Medical School and Professional Graduate School of Oriental Medicine, Korea
| | - Syung-Eun Cho
- 22Bionanochemistry Division, Natural Science College, Wonkwang University, 344-2 IKsan City, Jeonbook, Korea
| | - Byung-Soo Yu
- 22Bionanochemistry Division, Natural Science College, Wonkwang University, 344-2 IKsan City, Jeonbook, Korea
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Sezaki H, Suzuki F, Kawamura Y, Yatsuji H, Hosaka T, Akuta N, Kobayashi M, Suzuki Y, Saitoh S, Arase Y, Ikeda K, Miyakawa Y, Kumada H. Poor response to pegylated interferon and ribavirin in older women infected with hepatitis C virus of genotype 1b in high viral loads. Dig Dis Sci 2009; 54:1317-24. [PMID: 18958621 DOI: 10.1007/s10620-008-0500-y] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/30/2008] [Accepted: 08/22/2008] [Indexed: 12/14/2022]
Abstract
BACKGROUND Response to treatment in patients with chronic hepatitis C, with reference to age and gender, has not been examined fully. AIM The influence of gender and age on treatment with pegylated interferon (PEG-IFN) and ribavirin was evaluated in a retrospective study. METHODS PEG-IFN and ribavirin were given for 48 weeks to 179 men and 121 women infected with hepatitis C virus (HCV) of genotype 1b in high viral loads (>100 kIU/ml). RESULTS Sustained virological response at 24 weeks after treatment was poorer in women than men who were aged >or=50 years (22% vs 53%, P < 0.001). Among the patients aged >or=50 years who had received >or=80% of the doses of PEG-IFN, ribavirin, or both, women responded less often than men (26% vs 64%, P < 0.001; 33% vs 61%, P = 0.022; and 32% vs 63%, P = 0.016; respectively). In multivariate analysis, male gender, retention of indocyanine green, ribavirin dose and compliance with therapy increased sustained virological response. CONCLUSIONS Response to combined PEG-IFN and ribavirin is poorer in female than male patients with hepatitis C who are aged >or=50 years, irrespective of compliance with treatment. Low estrogen levels in older women could be responsible for their impaired response to PEG-IFN and ribavirin.
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Affiliation(s)
- Hitomi Sezaki
- Department of Hepatology, Toranomon Hospital, Tokyo, Japan.
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Ohara F, Nii A, Sakiyama Y, Tsuchiya M, Ogawa S. Pathophysiological characteristics of dimethylnitrosamine-induced liver fibrosis in acute and chronic injury models: a possible contribution of KLF5 to fibrogenic responses. Dig Dis Sci 2008; 53:2222-32. [PMID: 18095165 DOI: 10.1007/s10620-007-0112-y] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2007] [Accepted: 10/27/2007] [Indexed: 12/11/2022]
Abstract
Dimethylnitrosamine administration induces a rapid increase in collagen deposition with concomitant proliferation of hepatic stellate cells in rats. Here, we investigated the pathophysiological profiles of acute and chronic hepatic fibrosis states and attempted to determine the possible role of Kruppel-like factor-5 (KLF5) in this model. In acute study using a single drug injection, we observed a rapid transient increase of ALT and mRNA levels of KLF5 followed by increases in fibrosis-related genes. Repeated administration of dimethylnitrosamine once a week caused early damage with severe fibrosis and sustained hepatocyte injury, while intermittent injections at 2-week intervals induced only modest fibrosis from 3 weeks. Weekly administration also induced profound upregulation of collagen I, alpha-smooth muscle actin, and KLF5 mRNA. In contrast, such continued augmentation was not observed after intermittent injections; KLF5 increased only after 3 weeks. These results suggested that dimethylnitrosamine induced a rapid hepatic fibrogenic response with a possible participation of KLF5.
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Affiliation(s)
- Fumihiro Ohara
- St Louis Laboratories, Pfizer Global Research and Development, Chesterfield, MO 63017, USA.
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Lin YL, Hsu YC, Chiu YT, Huang YT. Antifibrotic effects of a herbal combination regimen on hepatic fibrotic rats. Phytother Res 2008; 22:69-76. [PMID: 17724770 DOI: 10.1002/ptr.2265] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Liver fibrosis has been characterized as chronic inflammatory processes involving multiple molecular pathogenetic pathways. This therapeutic study investigated whether a combination regimen of Salvia miltiorrhiza (S), Ligusticum chuanxiong (L) and Glycyrrhiza glabra (G) exerted in vivo antifibrotic effects on rats with hepatic fibrosis. Fibrosis was induced in rats by dimethylnitrosamine (DMN) administration for 4 weeks. Fibrotic rats were randomly assigned to one of the three groups: control, SLG (50 mg/kg) or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting 1 week after DMN injection. The results showed that fibrosis scores of livers from DMN-treated rats with SLG (1.13 +/- 0.13) were significantly reduced in comparison with DMN-treated rats receiving vehicle (1.63 +/- 0.18). Moreover, the hepatic collagen content of DMN rats was significantly reduced by either SLG or silymarin treatment. The double immunohistochemical staining results also showed that alpha-SMA positive cells with NF kappa B nuclear translocation were decreased in the fibrotic livers by SLG and silymarin treatments. The mRNA expression levels of TGF-beta1, alpha-SMA, collagen1 alpha 2, iNOS and ICAM-1 genes were attenuated by SLG and silymarin treatment. The results showed that SLG exerted antifibrotic effects in rats with DMN-induced hepatic fibrosis.
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Affiliation(s)
- Yun-Lian Lin
- National Research Institute of Chinese Medicine, Taipei, Taiwan
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Liu C, Sun M, Wang L, Wang G, Chen G, Liu C, Liu P. Effects of Yinchenhao Tang and related decoctions on DMN-induced cirrhosis/fibrosis in rats. Chin Med 2008; 3:1. [PMID: 18237412 PMCID: PMC2267793 DOI: 10.1186/1749-8546-3-1] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2007] [Accepted: 01/31/2008] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Chinese medicine decoctions such as Yinchenhao Tang (YCHT), Xiayuxue Tang (XYXT), Huangqi Tang (HQT), Yiguan Jian (YGJ) and Xiaochaihu Tang (XCHT)) were used to treat liver cirrhosis. The present study evaluates the effects of these decoctions on fibrosis in rats induced by dimethylnitrosamine (DMN). METHODS DMN solution (0.5%) was injected to rats for three consecutive days per week for four weeks. At the beginning of week 3, rats were randomly divided into 4-week DMN control group, YCHT, XYXT, HQT, YGJ, XCHT and vehicle groups. Each group was orally administered with specific decoctions daily for two weeks. Rats in the vehicle group were orally administered with only water. RESULTS Liver fibrosis and cirrhosis were observed in weeks 2 and 4 in DMN-intoxicated rats. Compared with normal rats, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) activities and level of total bilirubin acid (TBA) in serum and content of Hydroxyproline (Hyp) in liver tissue of model group rats rose significantly. However, the albumin (Alb) level in serum decreased significantly. Compared with the 4-week DMN group, the pathological conditions and functions of the liver in the YCHT group improved significantly, and the content of Hyp decreased remarkably: only one rat in this group developed liver cirrhosis and the ratio of cirrhosis was only 8.3%. On the other hand, the other decoctions did not show remarkable effects. YCHT inhibited alpha-SMA activation, including its gene expression into mRNA and protein. CONCLUSION Among the five Chinese medicine decoctions, YCHT exerted the most significant therapeutic effects on DMN-induced cirrhosis/fibrosis in rats.
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Affiliation(s)
- Cheng Liu
- Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Mingyu Sun
- Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Lei Wang
- Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Gaoqiang Wang
- Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Gaofeng Chen
- Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Chenghai Liu
- Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
- Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
- E-institutes of Shanghai Municipal Education Commission, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Ping Liu
- Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
- Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
- E-institutes of Shanghai Municipal Education Commission, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
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Hsu YC, Chiu YT, Lee CY, Wu CF, Huang YT. Anti-fibrotic effects of tetrandrine on bile-duct ligated rats. Can J Physiol Pharmacol 2007; 84:967-76. [PMID: 17218962 DOI: 10.1139/y06-050] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Tetrandrine (Tet) (C38H42O8N2; molecular weight, 622), an alkaloid isolated from the Chinese medicinal herb Stephania tetrandra, has been shown to elicit anti-inflammatory and anti-fibrotic effects in pulmonary diseases, but the mechanism of action has yet to be investigated. In this study, we tested whether Tet exerts anti-fibrotic effects on rat hepatic fibrosis through anti-NFkappaB pathways. After bile-duct ligation, rats were given Tet (1 or 5 mg/kg) or silymarin (50 mg/kg, as a positive control) by gavage twice daily for 3 weeks. Liver sections were taken for Sirius red quantitative scoring, immunofluorescence double staining of alpha-smooth muscle actin (alpha-SMA) and NFkappaB, and for quantitative determinations of the mRNA expression levels of TGF-beta1, alpha-SMA, collagen 1alpha2, inducible nitric oxide synthase (iNOS), intercellular adhesion molecule 1 (ICAM-1), metallothionein, vascular endothelial growth factor (VEGF), and VEGF type II receptor (VEGFR2) genes. The results showed that both Tet and silymarin treatment significantly reduced the fibrosis scores and hepatic collagen content of BDL rats, compared with no treatment. Both Tet and silymarin treatments decreased the number of alpha-SMA- and NFkappaB-positive cells in fibrotic livers. Moreover, Tet and silymarin treatments attenuated the mRNA expression levels of TGF-beta1,alpha-SMA, collagen 1alpha2, iNOS, ICAM-1, VEGF, and VEGFR2 genes, and induced the mRNA expression of the metallothionein gene. This study suggests that the anti-fibrotic effects of Tet were related to the reduction of fibrosis-related gene transcription, the attenuation of NFkappaB-activated pathways, and the induction of metallothionein gene transcription in the livers of BDL rats.
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Affiliation(s)
- Yi-Chao Hsu
- Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, and Department of Medical Research and Education, Taichung Veterans General Hospital, Taiwan
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14
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Hsu YC, Chiu YT, Cheng CC, Wu CF, Lin YL, Huang YT. Antifibrotic effects of tetrandrine on hepatic stellate cells and rats with liver fibrosis. J Gastroenterol Hepatol 2007; 22:99-111. [PMID: 17201889 DOI: 10.1111/j.1440-1746.2006.04361.x] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Anti-inflammation strategies are one of the proposed therapeutic approaches to hepatic fibrosis. Tetrandrine (C(38)H(42)O(8)N(2), molecular weight: 622; Tet), an alkaloid isolated from the Chinese medicinal herb Stephania tetrandra, has been shown to exert anti-inflammatory activity in pulmonary diseases. The purpose of the present study was to investigate the in vitro and in vivo effects of Tet on hepatic fibrosis. METHODS A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or tumor necrosis factor-alpha (TNF-alpha). The inhibitory effects of Tet on the nuclear factor kappaB (NFkappaB) signaling cascade and molecular markers including intercellular adhesion molecule-1 (ICAM-1) and alpha-smooth muscle actin (alpha-SMA) secretion were assessed. Fibrosis was induced by dimethylnitrosamine (DMN) administration in rats for 4 weeks. Fibrotic rats were randomly assigned to one of the four groups: vehicle (0.7% carboxyl methyl cellulose, CMC), Tet (1 mg/kg), Tet (5 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. At the end of the study, liver tissues were scored for fibrosis and analyzed for molecular markers of fibrosis. RESULTS Tetrandrine (0.5-5.0 micromol/L) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IkappaBalpha phosphorylation and mRNA expressions of ICAM-1 in HSC-T6 cells. In addition, Tet also inhibited TGF-beta1-induced alpha-SMA secretion and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats with high-dose Tet (1.3 +/- 0.3) were significantly reduced in comparison with DMN-treated rats receiving saline (2.0 +/- 0.2). Hepatic collagen content of DMN rats was significantly reduced by either Tet or silymarin treatment. Double-staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the fibrotic livers by Tet and silymarin treatment. In addition, mRNA expression of ICAM-1, alpha-SMA, and TGF-beta1 was attenuated by Tet treatment. Moreover, levels of plasma aspartate aminotransferase and alanine aminotransferase activities were reduced by Tet and silymarin treatment. CONCLUSION Tetrandrine exerts antifibrotic effects in both HSC-T6 cells and in rats with DMN-induced fibrosis.
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Affiliation(s)
- Yi-Chao Hsu
- Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
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15
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Chong LW, Hsu YC, Chiu YT, Yang KC, Huang YT. Anti-fibrotic effects of thalidomide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats. J Biomed Sci 2006; 13:403-18. [PMID: 16604421 DOI: 10.1007/s11373-006-9079-5] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2005] [Accepted: 11/25/2005] [Indexed: 02/08/2023] Open
Abstract
Tumor necrosis factor-alpha (TNF-alpha) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, alpha-N-phthalidoglutarimide, (C(13)H(10)N(2))(4), has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-alpha effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or TNF-alpha. The inhibitory effects of thalidomide on the NFkappaB signaling cascade and fibrosis markers including alpha-smooth muscle actin (alpha-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100-800 nM) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IKKalpha expression and IkappaBalpha phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 +/- 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 +/- 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition, real-time PCR analysis indicated that hepatic mRNA expressions of TGF-beta1, alpha-SMA, collagen 1alpha2, TNF-alpha and iNOS genes were attenuated by thalidomide treatment. In conclusion, our results showed that thalidomide inhibited activation of HSC-T6 cells by TNF-alpha and ameliorated liver fibrosis in DMN-intoxicated rats.
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Affiliation(s)
- Lee-Won Chong
- Graduate Institute of Clinical Medicine, School of Medicine, National Yang Ming University, Taipei, Taiwan
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16
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Hsu YC, Lin YL, Chiu YT, Shiao MS, Lee CY, Huang YT. Antifibrotic effects of Salvia miltiorrhiza on dimethylnitrosamine-intoxicated rats. J Biomed Sci 2005; 12:185-95. [PMID: 15864749 DOI: 10.1007/s11373-004-8167-7] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2004] [Accepted: 09/13/2004] [Indexed: 12/13/2022] Open
Abstract
Excessive oxidative stress is implicated in hepatic fibrogenesis. Extracts of Salvia miltiorrhiza (Sm) have been shown to protect cells against oxidative stress. In this study we investigated the in vitro and in vivo effects of Sm on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1). The inhibitory effects of Sm (50-400 microg/ml) on TGF-beta1-induced alpha-smooth muscle actin (alpha-SMA) secretion and the mRNA expressions of fibrosis-related genes, including alpha-SMA, connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed. Fibrosis was induced by dimethylnitrosamine (DMN) administration in rats. DMN-treated rats were randomly assigned to 1 of 4 groups: saline, Sm (20 mg/kg), Sm (100 mg/kg), or silymarin (100 mg/kg), each given by gavage twice daily for 5 weeks starting from the onset of DMN administration. Sm (200 and 400 microg/ml) significantly inhibited TGF-beta1-stimulated alpha-SMA secretion and the mRNA expressions of alpha-SMA, CTGF, and TIMP-1 in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats with either a low (1.8 +/- 0.2) or high (1.8 +/- 0.1) dose of Sm, or silymarin (1.4 +/- 0.2) were significantly reduced in comparison with DMN-treated rats receiving saline (3.1 +/- 0.1). Hepatic collagen contents were also significantly reduced by either Sm or silymarin treatment. The mRNA expression levels of alpha-SMA, TGF-beta1, and procollagen I were all attenuated in Sm- and silymarin-treated rats. Moreover, levels of plasma aspartate transaminase activities were reduced by Sm and silymarin treatment. In conclusion, our results show that Sm exerted antifibrotic effects in both HSC-T6 cells and in rats with DMN-induced fibrosis.
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Affiliation(s)
- Yi-Chao Hsu
- Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
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17
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Hsu YC, Chiu YT, Lee CY, Lin YL, Huang YT. Increases in fibrosis-related gene transcripts in livers of dimethylnitrosamine-intoxicated rats. J Biomed Sci 2004; 11:408-17. [PMID: 15067225 DOI: 10.1007/bf02254446] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2003] [Accepted: 12/24/2003] [Indexed: 12/14/2022] Open
Abstract
Fibrosis-related changes in livers of cirrhotic rats induced by dimethylnitrosamine (DMN) have not yet been fully clarified. The aim of this study was to investigate changes in molecular and biochemical markers in DMN-intoxicated rats. DMN was administered to Sprague-Dawley rats for 2 and 5 weeks to induce different degrees of hepatic fibrosis. Liver tissues were assessed for the degree of fibrosis and gene expression. Histological examination of the liver showed a progressive increase in fibrosis scores (1.33 +/- 0.21 and 3.03 +/- 0.29, respectively) and expansion of fibrous septa with collagen-staining fibers in rats after 2 and 5 weeks of DMN administration. Hepatic protein contents of alpha-smooth muscle actin (alpha-SMA) and total collagen were significantly higher in rats administered DMN for both 2 and 5 weeks compared with those in control rats. Hepatic mRNA expressions of alpha-SMA, transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor, tissue inhibitor of metalloproteinase-1, and procollagen I and III were increased in DMN rats after 2 and 5 weeks. Abnormal increases in plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels, plasma and mitochondrial MDA levels, and portal venous pressure were also noted in DMN rats. DMN administration to rats for 2 and 5 weeks induced progressive increases in hepatic fibrosis scores, hepatic mRNA expressions of TGF-beta1 and procollagen I and III genes, plasma levels of ALT and AST, and portal venous pressure, as well as progressive decreases in both liver and body weights. Our results suggest that DMN administration in rats induces biochemical and molecular changes related to fibrogenesis in the liver.
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Affiliation(s)
- Yi-Chao Hsu
- Institute of Traditional Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan, ROC
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18
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George J, Tsutsumi M, Takase S. Expression of hyaluronic acid in N-nitrosodimethylamine induced hepatic fibrosis in rats. Int J Biochem Cell Biol 2004; 36:307-19. [PMID: 14643895 DOI: 10.1016/s1357-2725(03)00253-x] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Hyaluronic acid (HA) plays prominent role in the pathogenesis of liver fibrosis. The mechanism of increased serum and liver HA during hepatic fibrosis was studied in rats. Liver injury was induced by intraperitoneal injections of N-nitrosodimethylamine (NDMA) for 7 consecutive days. A group of animals were sacrificed on everyday during injection and also on days 14 and 21 after the start of NDMA administration. The alpha-smooth muscle actin (alpha-SMA) was stained as a marker for activated stellate cells. Liver HA was studied by histochemical methods and serum HA was monitored by HA binding protein assay. CD44 was stained immunohistochemically. After the start of NDMA administration, necrosis was initiated on day 3 and massive necrosis was observed on days 5 and 7. Fibrosis was developed on day 14 and early cirrhosis was present on day 21. Staining of alpha-SMA demonstrated activated stellate cells from day 3 onwards. Serum HA peaked on day 7 and reduced afterwards. Serial liver sections stained for HA revealed excessive accumulation of HA during NDMA administration. On days 14 and 21, alpha-SMA and HA staining was remarkable in fibrotic and cirrhotic areas. CD44 staining was negative except during necrosis. It is concluded that the early elevation of serum HA is due to the increased synthesis and simultaneous release from the necrotic liver. In latter stages the increase of both serum and liver HA is contributed by the increased synthesis by the activated stellate cells and reduced clearance by the impaired sinusoidal endothelial cells.
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Affiliation(s)
- Joseph George
- Division of Gastroenterology, Department of Internal Medicine, Kanazawa Medical University, Uchinada, Ishikawa,920-0293, Japan.
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19
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Lu G, Shimizu I, Cui X, Itonaga M, Tamaki K, Fukuno H, Inoue H, Honda H, Ito S. Antioxidant and antiapoptotic activities of idoxifene and estradiol in hepatic fibrosis in rats. Life Sci 2004; 74:897-907. [PMID: 14659978 DOI: 10.1016/j.lfs.2003.08.004] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Oxidative stress plays a causative role in the development of hepatic fibrosis and apoptosis. Estradiol (E2) is an antioxidant, and idoxifene is a tissue-specific selective estrogen receptor modulator. We have previously demonstrated that E2 inhibits hepatic fibrosis in a rat model of hepatic fibrosis induced with dimethylnitrosamine (DMN), and suppresses activation of the nuclear factor (NF)-kappaB proinflammatory transcription factor in cultured rat hepatocytes undergoing oxidative stress. This study reports on the antioxidant and antiapoptotic role of idoxifene and E2 in the DMN model of hepatic fibrosis. The DMN model rats were administered with idoxifene or E2, and were examined activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and expression of Bcl-2 family proteins in the liver. During the course of hepatofibrogenesis after DMN treatment, serum levels of lactate dehydrogenase (LDH), a biomarker for necrosis, and hepatic levels of malondialdehyde (MDA), an end product of lipid peroxidation, increased rapidly for 3 days. On day 14, serum LDH levels normalized, and hepatic fibrosis developed with increased levels of MDA and collagen and decreased production of SOD and GPx in the liver. Fibrotic liver also showed downregulation of Bcl-2 and Bcl-X(L) expression and upregulation of Bad expression. Idoxifene and E2 suppressed DMN-mediated necrosis, lipid peroxidation, the loss of antioxidant enzyme activity, and proapoptotic status in Bcl-2 family protein expression as well as hepatic fibrosis. These findings indicate that, in addition to their antiinflammatory and antifibrotic action, idoxifene and E2 could enhance antioxidant and antiapoptotic activity in hepatic fibrosis in rats.
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Affiliation(s)
- Guangming Lu
- Department of Digestive and Cardiovascular Medicine, Tokushima University School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan
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20
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Gulubova M. Immunohistochemical localization of collagen type III and type IV, laminin, tenascin and alpha-smooth muscle actin (alphaSMA) in the human liver in peliosis. Pathol Res Pract 2003; 198:803-12. [PMID: 12608657 DOI: 10.1078/0344-0338-00339] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
The expression of collagen types III and IV, laminin, tenascin, and hepatic stellate cells (HSCs) activation marker alphaSMA was evaluated immunohistochemically in the liver of three patients with non-bacilar peliosis. Peliosis was attributed to tuberculosis, endometriosis treated with anabolic androgenic steroids, and to pheochromocytoma. Ultrastructural examination of the lesions of the liver revealed cavities that were sometimes lined with sinusoidal endothelial cells or hepatocytic microvilli. In liver sinusoids around cavities, cystic dilatation of the space of Disse and an abundance of amorphous matrix were observed. At this location, HSCs were transformed into transitional cells or myofibroblasts. Extracellular matrix proteins (ECM) were increased in the dilated sinusoids around cavities perisinusoidally and in the wall of cavities themselves. AlphaSMA was also increased. Ultrastructural immunohistochemistry revealed strong intracellular deposits of collagen type IV, laminin, and alphaSMA in HSCs. Laminin immunoreactivity was also noted in the endocytic vesicles in the cytoplasm of a monocyte. These findings suggest that enhanced ECM accumulation and the transformation of HSCs into myofibroblasts constitute a secondary event in peliosis and an attempt of the liver to restrict and remove sinusoidal dilatation.
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Affiliation(s)
- Maya Gulubova
- Department of General and Clinical Pathology, Faculty of Medicine, Thracian University, Stara Zagora 6000, Bulgaria.
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21
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Huang YT, Hsu YC, Chen CJ, Liu CT, Wei YH. Oxidative-stress-related changes in the livers of bile-duct-ligated rats. J Biomed Sci 2003; 10:170-8. [PMID: 12595753 DOI: 10.1007/bf02256052] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2002] [Accepted: 11/07/2002] [Indexed: 12/16/2022] Open
Abstract
The role of reactive oxygen species in liver fibrogenesis is not yet clarified. The aim of this study was to investigate oxidative-stress-related changes in cirrhotic rats. Cirrhosis was induced by bile duct ligation in Sprague-Dawley rats. Plasma malondialdehyde (MDA), hepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG), hepatic mitochondrial respiratory functions and gene transcripts were measured at 2 and 4 weeks after surgery in bile-duct-ligated (BDL) and sham-operated-operated rats. The results showed progressive increases in the levels of plasma MDA, hepatic 8-OHdG and procollagen I and III mRNA expression, and progressive impairment of hepatic mitochondrial respiratory function in BDL rats at 2 and 4 weeks after ligation compared with sham-operated rats. Moreover, at 4 weeks after ligation, BDL rats exhibited reduced plasma glutathione and vitamin E levels, impaired hepatic mitochondrial electron transport enzyme activities and oxidative phosphorylation function. In addition, hepatic mRNA expression of transforming growth factor-beta1 was increased. Hepatomegaly, abnormal plasma alanine transaminase and aspartate transaminase levels, and portal hypertension were noted in BDL rats. Our results suggest that bile duct ligation in the rat induces mitochondrial dysfunction and biochemical and molecular changes related to oxidative stress in the liver.
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Affiliation(s)
- Yi-Tsau Huang
- Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC.
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22
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Kang WZ, Xie YM, Nie QH, Zhang Y, Hao CQ, Wang JP, Chen WH. Effect of Oxymatrine on experimental liver fibrosis. Shijie Huaren Xiaohua Zazhi 2003; 11:195-198. [DOI: 10.11569/wcjd.v11.i2.195] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the effect of Oxymatrine on liver fibrosis in immunogenic liver fibrosis rat model.
METHODS: Rat liver fibrosis model was induced by human serum albumin (HSA), 60 Wistar rats were randomly divided into 5 groups, control group without any treatment, liver fibrosis model group, oxymatrine preventive group, oxymatrine therapeutic group, and cochicine therapeutic group. The pathological changes of liver were observed by HE and Von-Gieson staining. The expressions of mRNA and proteins of collagen I/III in liver were determined by in situ hybridization and immunohistochemistry.
RESULTS: The liver fibrosis degree and level of mRNA and proteins of collagen I/III in the liver were significantly reduced in the decreasing order in oxymatrine preventive group, oxymatrine therapeutic group, and cochicine therapeutic group.
CONCLUSION: Oxymatrine may inhibit hepatic inflammation and hepatic synthesis of collagen I/III, and thus prevent and inhibit hepatic fibrosis.
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23
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Liu Y, Shimizu I, Omoya T, Ito S, Gu XS, Zuo J. Protective effect of estradiol on hepatocytic oxidative damage. World J Gastroenterol 2002; 8:363-6. [PMID: 11925626 PMCID: PMC4658385 DOI: 10.3748/wjg.v8.i2.363] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To examine the protective effect of estradiol on the cultured hepatocytes under oxidative stress.
METHODS: Hepatocytes of rat were isolated by using perfusion method, and oxidative stress was induced by a serum-free medium and FeNTA. MDA level was determined with TBA method. Cell damage was assessed by LDH assay. Apoptosis of hepatocytes was assessed with cytoflowmetric analysis. Expression of Bcl-xl in cultured hepatocytes was detected by Western blot. The radical-scavenging activity of estradiol was valued by its ability to scavenge the stable free radical of DDPH.
RESULTS: Oxidative stress increased LDH (from 168 ± 25 × 10-6 IU•cell-1 to 780 ± 62 × 10-6 IU•cell-1) and MDA (from 0.28 ± 0.07 × 10-6 nmol·cell-1 to 1.35 ± 0.12 × 10-6 nmol•cell-1) levels in cultured hepatocyte, and estradiol inhibited both LDH and MDA production in a dose dependent manner. In the presence of estradiol 10-6 mol•L-1, 10-7 mol•L-1 and 10-8 mol•L-1, the LDH levels are 410 ± 53 × 10-6 IU•cell-1 (P < 0.01 vs oxidative group), 530 ± 37 × 10-6 IU•cell-1 (P < 0.01 vs oxidative group), 687 ± 42 × 10-6 IU•cell-1 (P < 0.05 vs oxidative group) respectively, and the MDA level are 0.71 ± 0.12 × 10-6 nmol•cell-1 (P < 0.01vs oxidative group), 0.97 ± 0.11 × 10-6 nmol•cell-1 (P < 0.01 vs oxidative group) and 1.27 ± 0.19 × 10-6 nmol•cell-1 respectively. Estradiol suppressed apoptosis of hepatocytes induced by oxidative stress, administration of estradiol (10-6 mol/L)decreased the apoptotic rate of hepatocytes under oxidative stress from 18.6% ± 1.2% to 6.5% ± 2.5%, P < 0.01. Bcl-xl expression was related to the degree of liver cell damage due to oxidative stress, and estradiol showed a protective action.
CONCLUSION: Estradiol protects hepatocytes from oxidative damage by means of its antioxidant activity.
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Affiliation(s)
- Yan Liu
- Department of Biology, Medical School of Fudan University, 130 Dongan Road, Shanghai 210032, China.
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24
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Zhou Y, Shimizu I, Lu G, Itonaga M, Okamura Y, Shono M, Honda H, Inoue S, Muramatsu M, Ito S. Hepatic stellate cells contain the functional estrogen receptor beta but not the estrogen receptor alpha in male and female rats. Biochem Biophys Res Commun 2001; 286:1059-65. [PMID: 11527408 DOI: 10.1006/bbrc.2001.5479] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
In an earlier study, we showed that estradiol (E2) inhibits proliferation and transformation in cultured rat hepatic stellate cells (HSCs) and that the actions of E2 are mediated through estrogen receptors (ERs). This study reports on an investigation of the cellular localization of ER subtypes ERalpha and ERbeta using immunohistochemistry in experimental fibrotic liver rats and of each ER subtype expression in cultured rat HSCs by evaluating the produced mRNA and protein. The results indicate that high levels of ERbeta expression and low or no levels of ERalpha expression were observed in normal and fibrotic livers and in quiescent and activated HSCs from both males and females. The specificity of E2-mediated antiapoptotic induction through the ERbeta was shown by dose-dependent inhibition by the pure ER antagonist ICI 182,780 in HSCs which were undergoing early apoptosis. These findings demonstrate for the first time that rat HSCs possess functional Erbeta, but not Eralpha, to respond directly to E2 exposure.
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MESH Headings
- Animals
- Apoptosis/drug effects
- Binding, Competitive
- Blotting, Western
- Cell Division
- Cells, Cultured
- Dose-Response Relationship, Drug
- Estradiol/analogs & derivatives
- Estradiol/pharmacology
- Estrogen Receptor alpha
- Estrogen Receptor beta
- Female
- Flow Cytometry
- Fulvestrant
- Hepatocytes/chemistry
- Hepatocytes/metabolism
- Immunohistochemistry
- Liver/cytology
- Liver/metabolism
- Male
- Membrane Potentials
- Microscopy, Confocal
- Proto-Oncogene Proteins c-bcl-2/metabolism
- RNA, Messenger/metabolism
- Rats
- Rats, Wistar
- Receptors, Estrogen/antagonists & inhibitors
- Receptors, Estrogen/biosynthesis
- Receptors, Estrogen/chemistry
- Reverse Transcriptase Polymerase Chain Reaction
- Sex Factors
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Affiliation(s)
- Y Zhou
- Second Department of Internal Medicine, Tokushima University School of Medicine, Kuramoto-cho, Tokushima, 770-8503, Japan
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25
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Tada S, Nakamuta M, Enjoji M, Sugimoto R, Iwamoto H, Kato M, Nakashima Y, Nawata H. Pirfenidone inhibits dimethylnitrosamine-induced hepatic fibrosis in rats. Clin Exp Pharmacol Physiol 2001; 28:522-7. [PMID: 11422218 DOI: 10.1046/j.1440-1681.2001.03481.x] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
1. In the present study, we investigated the preventive effects of pirfenidone (PFD), an antifibrotic agent, on experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. 2. Treatment with DMN caused a significant decrease in bodyweight and liver weight. Oral PFD (500 mg/kg daily for 4 weeks) essentially prevented this DMN-induced loss in bodyweight and tended to suppress the loss in liver weight. There were no significant differences in liver weight and serum L-alanine aminotransferase levels between PFD-treated and -untreated groups. Pirfenidone has no major side effects in vivo. 3. Pirfenidone suppressed the induction of hepatic fibrosis determined by histological evaluation and reduced hepatic hydroxyproline levels. Expression of mRNA for type I collagen and transforming growth factor-beta in the liver was also suppressed by PFD treatment. 4. Because hepatic stellate cells (HSC) are the major cellular source of extracellular matrix in hepatic fibrosis, we examined the effects of PFD on type I collagen production in vitro using rat primary HSC cultures. Pirfenidone inhibited collagen production in HSC culture in a dose-dependent manner. 5. These results demonstrate that the inhibitory effects of PFD against hepatic fibrosis may be due, at least in part, to blockade of collagen production by HSC and suggest that PFD may be potentially useful in the prevention of the development of hepatic fibrosis.
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Affiliation(s)
- S Tada
- Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-12 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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26
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Tada S, Iwamoto H, Nakamuta M, Sugimoto R, Enjoji M, Nakashima Y, Nawata H. A selective ROCK inhibitor, Y27632, prevents dimethylnitrosamine-induced hepatic fibrosis in rats. J Hepatol 2001; 34:529-36. [PMID: 11394652 DOI: 10.1016/s0168-8278(00)00059-3] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
BACKGROUND p160ROCK is a direct Rho target which mediates Rho-induced assembly of focal adhesions and stress fibers. We previously reported that Rho signaling pathways are involved in the activation of hepatic stellate cells (HSC) in vitro. The aim of the present study was to test the hypothesis that an inhibitor specific for p160ROCK (Y27632) could prevent experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. METHODS Y27632 was given orally at 30 mg/kg daily for 4 weeks after the first injection of DMN. The degree of fibrosis was evaluated by image analysis and also by measurements of collagen and hydroxyproline content in the liver. The expression of alpha-smooth muscle actin (alpha-SMA) in the liver and in the primary cultured HSC was also evaluated. Semi-quantitative RT-PCR was performed to evaluate the expression of type I collagen mRNA in the liver. RESULTS Y27632 treatment significantly decreased the occurrence of DMN-induced hepatic fibrosis and reduced the collagen and hydroxyproline content and alpha-SMA expression in the liver. The expression of alpha-SMA in HSC was also suppressed in vitro. CONCLUSIONS These findings indicate that inhibitors of the Rho-ROCK pathway might be useful therapeutically in hepatic fibrosis.
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Affiliation(s)
- S Tada
- Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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Liu HL, Li XH, Wang DY, Yang SP. Matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 expression in fibrotic rat liver. World J Gastroenterol 2000; 6:881-884. [PMID: 11819713 PMCID: PMC4728279 DOI: 10.3748/wjg.v6.i6.881] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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George J, Chandrakasan G. Biochemical abnormalities during the progression of hepatic fibrosis induced by dimethylnitrosamine. Clin Biochem 2000; 33:563-70. [PMID: 11124342 DOI: 10.1016/s0009-9120(00)00170-3] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
OBJECTIVES The pathogenesis of hepatic fibrosis is accompanied with several biochemical and metabolic abnormalities. To obtain more information about the alteration of biochemical and metabolic parameters during alcoholic liver fibrosis, we have monitored the changes of certain important biochemical compounds in experimentally induced hepatic fibrosis. DESIGN AND METHODS The liver injury was induced in adult male albino rats by using dimethylnitrosamine (DMN) in doses of 1 mg/100 g body weight. Total collagen, total protein, cholesterol, lipid peroxides, glucose, urea, and inorganic phosphorus were monitored in liver and blood/serum samples on Days 0, 7, 14, and 21 after the start of DMN administration. Serum insulin levels were assayed by radioimmunoassay. The serum and urinary levels of hydroxyproline, uric acid, and creatinine were also monitored. RESULTS The total collagen content in the liver was increased about 4-fold by Day 21 after the start of DMN administration. A significant increase was observed in lipid peroxide levels in both liver and blood samples. Although inorganic phosphorus level decreased in both serum and liver tissue, cholesterol was lowered only in the serum. Increased serum insulin level with impaired glucose tolerance was observed after 21 days. Serum hydroxyproline level increased throughout after the start of DMN administration. The urinary excretion of hydroxyproline was also significantly increased with a striking elevation on Day 7. Elevated uric acid levels were recorded in serum and urine samples during the latter periods of DMN treatment. No alteration was observed in blood urea and creatinine levels. CONCLUSIONS The results of the present investigation demonstrated important alterations in metabolic parameters and biochemical abnormalities during experimentally induced liver damage. All alterations are compatible with the deterioration of liver functions during the pathogenesis of hepatic fibrosis.
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Affiliation(s)
- J George
- Department of Biochemistry, Central Leather Research Institute, Adyar, Madras 600-020, India.
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Zhang LP, Takahara T, Yata Y, Furui K, Jin B, Kawada N, Watanabe A. Increased expression of plasminogen activator and plasminogen activator inhibitor during liver fibrogenesis of rats: role of stellate cells. J Hepatol 1999; 31:703-11. [PMID: 10551395 DOI: 10.1016/s0168-8278(99)80351-1] [Citation(s) in RCA: 91] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND/AIMS Plasminogen activators and plasminogen activator inhibitors are important regulators of the balance between the proteolytic and antiproteolytic activities that determine extracellular matrix turnover. We examined the expression of plasminogen activator-plasmin system components in experimental liver fibrosis of rats. METHODS Liver fibrosis was produced in rats by injecting carbon tetrachloride for 6 to 12 weeks. Gene expression for plasminogen activator inhibitor-1 (PAI-1), urokinase and tissue plasminogen activators (uPA and tPA), urokinase plasminogen activator receptor (uPAR), and transforming growth factor-beta1 (TGF-beta1) was examined by Northern analysis. Western analysis was performed to detect protein expression of PAI-1, uPA and uPAR. An immunohistochemical study was performed to detect the localization of PAI-1. Additionally, primary cultured liver cells were examined by Northern and Western analyses for this protein with or without prior incubation with TGF-beta1. RESULTS At 6 weeks, when fibrosis had occurred, uPA and uPAR mRNAs had increased 2.8-fold and 1.8-fold, respectively; PAI-1 and tPA mRNA levels were unchanged. At the cirrhotic stage (9 to 12 weeks), mRNA levels for PAI-1, uPA, uPAR and tPA were all increased. Western analysis also showed increased uPA and uPAR expressions in fibrotic liver, and increased PAI-1, uPA and uPAR expressions in cirrhotic liver. PAI-1 protein was also demonstrated immunohistochemically along sinusoids, vessels, and bile duct cells of normal and fibrotic liver. In liver cell cultures, Kupffer cells, hepatocytes, and especially stellate cells, expressed PAI-1. Expression was enhanced in stellate cells cultured from fibrotic or cirrhotic liver or stimulated in vitro with TGF-beta1. CONCLUSION Though increased uPA and uPAR may act on matrix degradation in fibrotic liver, increased PAI-1 together with uPA, uPAR and tPA are associated with overall inhibition of matrix degradation in cirrhotic liver. Hepatic stellate cells are an important source of PAI-1 during liver fibrosis.
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MESH Headings
- Animals
- Blotting, Northern
- Blotting, Western
- Cells, Cultured
- Immunohistochemistry
- Liver/metabolism
- Liver/pathology
- Liver Cirrhosis, Experimental/etiology
- Liver Cirrhosis, Experimental/metabolism
- Liver Cirrhosis, Experimental/pathology
- Male
- Plasminogen Activator Inhibitor 1/biosynthesis
- Plasminogen Activator Inhibitor 1/genetics
- Plasminogen Activator Inhibitor 1/metabolism
- Plasminogen Activators/metabolism
- Plasminogen Inactivators/metabolism
- RNA, Messenger/metabolism
- Rats
- Rats, Wistar
- Receptors, Cell Surface/genetics
- Receptors, Cell Surface/metabolism
- Receptors, Urokinase Plasminogen Activator
- Transforming Growth Factor beta/genetics
- Urokinase-Type Plasminogen Activator/genetics
- Urokinase-Type Plasminogen Activator/metabolism
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Affiliation(s)
- L P Zhang
- Third Department of Internal Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Japan
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Yasuda M, Shimizu I, Shiba M, Ito S. Suppressive effects of estradiol on dimethylnitrosamine-induced fibrosis of the liver in rats. Hepatology 1999; 29:719-27. [PMID: 10051473 DOI: 10.1002/hep.510290307] [Citation(s) in RCA: 191] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
As a model for the analysis of the fibrosuppressive role of estradiol, hepatic fibrosis was induced in male and female rats by the administration of a single dose of dimethylnitrosamine (DMN). The fibrotic response of the male liver after DMN treatment was significantly stronger than that of the female liver. In the male DMN model, estradiol reduced hepatic mRNA for type I and III procollagens and the tissue inhibitor of metalloproteinase-1 (TIMP-1), as well as deposition of type I and III collagen protein total hepatic collagen and malondialdehyde (MDA), a product of lipid peroxidation. Concomitant administration of a neutralizing antibody against rat estradiol enhanced fibrogenesis, as judged by the same parameters. Ovariectomy in the female model had a fibrogenic effect, inducing the hepatic expression of both types of procollagen and TIMP-1; in addition, the number of alpha-smooth muscle actin (alpha-SMA)-positive cells in the liver increased; estradiol replacement was fibrosuppressive in the castrated-female model. In rat hepatic stellate cells incubated in primary culture with estradiol, cell number, type I collagen production, and alpha-SMA expression were all reduced. These findings suggest that estradiol suppressed the induction of hepatic fibrosis, and may in part underlie the more rapid progression in males of hepatic fibrosis and its complications.
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Affiliation(s)
- M Yasuda
- Second Department of Internal Medicine, School of Medicine, University of Tokushima, Tokushima, Japan
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Shimizu I, Ma YR, Mizobuchi Y, Liu F, Miura T, Nakai Y, Yasuda M, Shiba M, Horie T, Amagaya S, Kawada N, Hori H, Ito S. Effects of Sho-saiko-to, a Japanese herbal medicine, on hepatic fibrosis in rats. Hepatology 1999; 29:149-60. [PMID: 9862861 DOI: 10.1002/hep.510290108] [Citation(s) in RCA: 169] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
It has been shown that lipid peroxidation is associated with hepatic fibrosis and stellate cell activation. Sho-saiko-to (TJ-9) is an herbal medicine, which is commonly used to treat chronic hepatitis in Japan, although the mechanism by which TJ-9 protects against hepatic fibrosis is not known. As a result, we assayed the preventive and therapeutic effects of TJ-9 on experimental hepatic fibrosis, induced in rats by dimethylnitrosamine (DMN) or pig serum (PS), and on rat stellate cells and hepatocytes in primary culture, and assessed the antioxidative activities and the active components of TJ-9. Male Wistar rats were given a single intraperitoneal injection of 40 mg/kg DMN or 0.5 mL PS twice weekly for 10 weeks. In each model, rats were fed a basal diet throughout, or the same diet, which also contained 1.5% TJ-9, for 2 weeks before treatment or for the last 2 weeks of treatment. TJ-9 suppressed the induction of hepatic fibrosis, increased hepatic retinoids, and reduced the hepatic levels of collagen and malondialdehyde (MDA), a production of lipid peroxidation. Immunohistochemical examination showed that TJ-9 reduced the deposition of type I collagen and the number of alpha-smooth muscle actin (alpha-SMA) positive-stellate cells in the liver and inhibited, not only lipid peroxidation in cultured rat hepatocytes that were undergoing oxidative stress, but also the production of type I collagen, alpha-SMA expression, cell proliferation, and oxidative burst in cultured rat stellate cells. In addition, TJ-9 inhibited Fe2+/adenosine 5'-diphosphate-induced lipid peroxidation in rat liver mitochondria in a dose-dependent manner and showed radical scavenging activity. Among the active components of TJ-9, baicalin and baicalein were found to be mainly responsible for the antioxidative activity. These findings suggest that Sho-saiko-to (TJ-9) functions as a potent antifibrosuppressant by inhibition of lipid peroxidation in hepatocytes and stellate cells in vivo.
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Affiliation(s)
- I Shimizu
- Second Department of Internal Medicine, School of Medicine, University of Tokushima, Tokushima,
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