Activation of hepatic stellate cells is associated with cytokine expression in thioacetamide-induced hepatic fibrosis in mice.
ABSTRACT The pathophysiological mechanisms of thioacetamide (TAA)-induced hepatic fibrogenesis are not yet fully understood. In particular, the role of hepatic stellate cells (HSCs) remains unclear. We therefore examined proliferation and transdifferentiation of HSC as well as the underlying molecular mechanisms in TAA-induced fibrosis. Hepatic fibrogenesis was induced in mice by addition of TAA to drinking water. Liver damage was determined by assessment of alanine aminotransferase and aspartate aminotransferase levels, and measurement of collagen deposition. Additionally, expression patterns of alpha-smooth muscle actin, glial fibrillary acidic protein (GFAP, specific hepatic biomarker for HSC), cysteine- and glycine-rich protein 2 (CRP2, specific marker of HSC transdifferentiation), tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-9 (MMP-9), interleukins (IL-1beta, IL-6), platelet-derived growth factors (PDGF-B, PDGF-D) , tumor necrosis factor (TNF)-alpha, and (transforming growth factor (TGF)-beta1 were assessed by real-time PCR. Transcription of GFAP and CRP2 were transiently upregulated during TAA-induced fibrogenesis (punctum maxima (p.m.) week 10 for GFAP and week 14 for CRP2). Similar transient expression patterns were demonstrated for IL-1beta, IL-6, TGF-beta1, and PDGF-B (p.m. week 12) whereas TNF-alpha and PDGF-D continuously increased with ongoing liver injury. In particular, not only neutrophil granulocytes, but also macrophages and leukocytes served as a major source for MMP-9 expression. GFAP and CRP2 expression patterns demonstrated transiently increased HSC-activation during TAA-induced hepatic fibrogenesis. The rate of increase of transcription of GFAP correlated best with PDGF-B, whereas CRP2 levels correlated with PDGF-B, PDGF-D, and IL-1beta expression. This study demonstrates for the first time that transiently increased activation patterns of HSC are observed in toxically induced hepatic fibrosis. Thus, TAA in drinking water is an effective and elegant model to induce reproducible states of liver fibrosis without parenchymal damage in mice.
Article: Role of apoptosis-inducing factor, proline dehydrogenase, and NADPH oxidase in apoptosis and oxidative stress.[show abstract] [hide abstract]
ABSTRACT: Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF), proline dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In the mitochondria, AIF enhances mitochondrial bioenergetics and complex I activity/assembly to help maintain proper cellular redox homeostasis. After translocating to the nucleus, AIF forms a chromatin degrading complex with other proteins, such as cyclophilin A. AIF translocation from the mitochondria to the nucleus is triggered by oxidative stress, implicating AIF as a mitochondrial redox sensor. Proline dehydrogenase is a membrane-associated flavoenzyme in the mitochondrion that catalyzes the rate-limiting step of proline oxidation. Upregulation of proline dehydrogenase by the tumor suppressor, p53, leads to enhanced mitochondrial reactive oxygen species that induce the intrinsic apoptotic pathway. NADPH oxidases are a group of enzymes that generate reactive oxygen species for oxidative stress and signaling purposes. Upon activation, NADPH oxidase 2 generates a burst of superoxide in neutrophils that leads to killing of microbes during phagocytosis. NADPH oxidases also participate in redox signaling that involves hydrogen peroxide-mediated activation of different pathways regulating cell proliferation and cell death. Potential therapeutic strategies for each enzyme are also highlighted.Cell Health and Cytoskeleton 02/2012; 2012(4):11-27.
Article: Metabolomic analysis of dynamic response and drug resistance of gastric cancer cells to 5-fluorouracil.[show abstract] [hide abstract]
ABSTRACT: Metabolomics has developed as an important new tool in cancer research. It is expected to lead to the discovery of biomarker candidates for cancer diagnosis and treatment. The current study aimed to perform a comprehensive metabolomic analysis of the intracellular dynamic responses of human gastric cancer cells to 5-fluorouracil (5-FU), referencing the mechanisms of drug action and drug resistance. Small metabolites in gastric cancer cells and 5-FU-resistant cells were measured by liquid chromatography-mass spectrometry. Candidates for drug targets were selected according to the presence or absence of resistance, before and after 5-FU treatment. In addition, the gene expression of each candidate was assessed by reverse transcription-polymerase chain reaction. The number of metabolites in cancer cells dramatically changed during short-term treatment with 5-FU. Particularly, proline was reduced to one-third of its original level and glutamate was increased by a factor of 3 after 3 h of treatment. The metabolic production of glutamate from proline proceeds by proline dehydrogenase (PRODH), producing superoxide. After 5-FU treatment, PRODH mRNA expression was upregulated 2-fold and production of superoxide was increased by a factor of 3. In 5-FU-resistant cells, proline and glutamate levels were less affected than in non-resistant cells, and PRODH mRNA expression and superoxide generation were not increased following treatment. In conclusion, the authors identified a candidate biomarker, PRODH, for drug effects using a meta-bolomic approach, a result that was confirmed by conventional methods. In the future, metabolomics will play an important role in the field of cancer research.Oncology Reports 12/2012; · 1.84 Impact Factor
Article: Transplanted human amniotic membrane-derived mesenchymal stem cells ameliorate carbon tetrachloride-induced liver cirrhosis in mouse.[show abstract] [hide abstract]
ABSTRACT: Human amniotic membrane-derived mesenchymal stem cells (hAMCs) have the potential to reduce heart and lung fibrosis, but whether could reduce liver fibrosis remains largely unknown. Hepatic cirrhosis model was established by infusion of CCl₄ (1 ml/kg body weight twice a week for 8 weeks) in immunocompetent C57Bl/6J mice. hAMCs, isolated from term delivered placenta, were infused into the spleen at 4 weeks after mice were challenged with CCl₄. Control mice received only saline infusion. Animals were sacrificed at 4 weeks post-transplantation. Blood analysis was performed to evaluate alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Histological analysis of the livers for fibrosis, hepatic stellate cells activation, hepatocyte apoptosis, proliferation and senescence were performed. The donor cell engraftment was assessed using immunofluorescence and polymerase chain reaction. The areas of hepatic fibrosis were reduced (6.2%±2.1 vs. control 9.6%±1.7, p<0.05) and liver function parameters (ALT 539.6±545.1 U/dl, AST 589.7±342.8 U/dl,vs. control ALT 139.1±138.3 U/dl, p<0.05 and AST 212.3±110.7 U/dl, p<0.01) were markedly ameliorated in the hAMCs group compared to control group. The transplantation of hAMCs into liver-fibrotic mice suppressed activation of hepatic stellate cells, decreased hepatocyte apoptosis and promoted liver regeneration. More interesting, hepatocyte senescence was depressed significantly in hAMCs group compared to control group. Immunofluorescence and polymerase chain reaction revealed that hAMCs engraftment into host livers and expressed the hepatocyte-specific markers, human albumin and α-fetoproteinran. The transplantation of hAMCs significantly decreased the fibrosis formation and progression of CCl₄-induced cirrhosis, providing a new approach for the treatment of fibrotic liver disease.PLoS ONE 01/2011; 6(2):e16789. · 4.09 Impact Factor