Zinc pretreatment prevents hepatic stellate cells from cadmium-produced oxidative damage

Pretreatment with zinc produces tolerance to several cadmium toxic effects. This study was performed to further elucidate the mechanism of zinc-induced tolerance to cadmium cytotoxicity in a rat hepatic stellate cell line (CFSC-2G). Twenty four hours after seeding, cells were treated with 60 micromol/L ZnCl2 for 24 h. Following zinc pretreatment, cells were exposed to 3 micromol/L and 5 micromol/L CdCl2 for an additional 24 h. The toxicity of cadmium was significantly reduced in the zinc-pretreated cells. Zinc pretreatment produced a decrease in lipid peroxidation damage of cadmium-treated cells. Glutathione cell content diminished 33% and 43% as a result of 3 micromol/L and 5 micromol/ L CdCl2 treatment, respectively. Cell pretreatment with zinc recovered glutathione content at control cells level. Catalase and glutathione peroxidase activities were also recovered to control values with zinc pretreatment. Cadmium (5 micromol/L) was able to induce 39% the expression of alpha1 collagen (I) gene after 1 h treatment, while zinc pretreatment prevented this cadmium profibrogenic effect. We also examined the production of heat shock protein 70 (Hsp70) as a cellular response to oxidative stress produced by cadmium. By Western blot analysis, a 1.3 and 3 times increment in Hsp70, with 3 micromol/L and 5 micromol/L CdCl2 treatment, respectively, was observed. Zinc pretreatment prevented the production of Hsp70. Metallothionein-II (MT-II) gene expression was induced by cadmium, but the induction was unaffected with zinc pretreatment. These data suggest that zinc-induced protection against the cytotoxicity of cadmium in stellate cells may be related to the maintenance of normal redox balance inside the cell.     

Chromosomal terminal methylation status is associated with gut microbiotic alterations

Molecular and Cellular Biochemistry - We explored the association of fecal bacterial species and somatic telomere changes in patients with chronic disease. The results showed that the length of the...     

Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.     

15d-Deoxy-Delta12,14-prostaglandin J2 modulates collagen type I synthesis in human hepatic stellate cells by inducing oxidative stress

15 deoxy-Delta(12,14)-prostaglandin(2) (15d-PGJ(2)) is known to inhibit the proliferation of hepatic stellate cells (HSCs), major cellular components that cause hepatic fibrosis, in vitro. It also induces oxidative stress, which results in hepatic myofibroblast death. On the other hand, oxidative stress generally induces HSC proliferation and collagen synthesis in vitro, and liver fibrogenesis in vivo. In this study, we evaluated the effects of 15d-PGJ(2) at various concentrations on the viability and collagen synthesis of HSCs. 15d-PGJ(2) increased intracellular reactive oxygen species (ROS), and reduced the viability of human HSCs at concentrations 5 microM by inducing apoptotic cell death. In addition, the antioxidants alpha-tocopherol and N-acetylcysteine (NAC) blocked 15d-PGJ(2)-induced HSC death. Collagen I synthesis was increased 1.5-fold by 0.5 microM 15d-PGJ(2) treatment, but was reduced to 30% of the control level by 10 microM 15d-PGJ(2), and NAC pretreatment prevented these changes in collagen production by 15d-PGJ(2). We conclude that 15d-PGJ(2) may either induce or prevent hepatic fibrogenesis depending on its concentration.     

Leptin facilitates proliferation of hepatic stellate cells through up-regulation of platelet-derived growth factor receptor

In the present study, we investigated the effect of leptin on proliferation of hepatic stellate cells (HSCs) in vitro. Proliferation of 3-day cultured rat HSCs was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the nuclei. The percentages of BrdU-positive cells were increased in the presence of PDGF-BB (5 ng/ml) for 8h as expected. Co-incubation with leptin (10-100 nM) potentiates this PDGF-dependent increase in BrdU positive cells in a dose-dependent manner. Messenger RNA for PDGF receptor alpha and beta subunits was increased almost 2- to 3-fold by incubation with leptin for 6h. Further, pre-incubation with leptin for 6h enhanced PDGF-induced increases in phospho-p44/42 MAP kinase and phospho-Akt levels in a dose-dependent manner. In the same condition, however, leptin per se did not increase phospho-STAT 3 and phospho-p44/42 MAP kinase levels. Instead, leptin increased phospho-Akt levels in HSCs within 30 min, suggesting that the phosphatidylinositol 3 kinase (PI3K)/Akt pathway is involved in the mechanism by which leptin accelerates the proliferation of HSCs. In conclusion, the present study clearly indicated that leptin potentiates PDGF-dependent proliferative responses of HSCs in vitro.     

Addressing liver fibrosis with liposomes targeted to hepatic stellate cells

Liver fibrosis is a chronic disease that results from hepatitis B and C infections, alcohol abuse or metabolic and genetic disorders. Ultimately, progression of fibrosis leads to cirrhosis, a stage of the disease characterized by failure of the normal liver functions. Currently, the treatment of liver fibrosis is mainly based on the removal of the underlying cause of the disease and liver transplantation, which is the only treatment for patients with advanced fibrosis. Hepatic stellate cells (HSC) are considered to be key players in the development of liver fibrosis. Chronically activated HSC produces large amounts of extracellular matrix and enhance fibrosis by secreting a broad spectrum of cytokines that exert pro-fibrotic actions in other cells, and in an autocrine manner perpetuate their own activation. Therefore, therapeutic interventions that inhibit activation of HSC and its pro-fibrotic activities are currently under investigation worldwide. In the present study we applied targeted liposomes as drug carriers to HSC in the fibrotic liver and explored the potential of these liposomes in antifibrotic therapies. Moreover, we investigated effects of bioactive compounds delivered by these liposomes on the progression of liver fibrosis. To our knowledge, this is the first study demonstrating that lipid-based drug carriers can be selectively delivered to HSC in the fibrotic liver. By incorporating the bioactive lipid DLPC, these liposomes can modulate different processes such as inflammation and fibrogenesis in the fibrotic liver. This dual functionality of liposomes as a drug carrier system with intrinsic biological effects may be exploited in new approaches to treat liver fibrosis.     

硫化氢在大鼠肝星状细胞氧应激中对α-SMA与细胞周期影响的实验研究

目的:探讨硫化氢在大鼠肝星状细胞氧应激中对α-SMA与细胞周期的影响.方法:将大鼠肝星状细胞株HSC-T6分为8组:空白组(B组,HSC-T6);对照组(C组,B组+500μ mol/L Fe-NTA);NaHS组(N1:C组+20μ mol/LNaHS;N2:C组+100μ mol/LNaHS;N3:C组+200μ mol/L NaHS);格列苯脲组(G1:C组+20umol/LGLBN;G2:C组+200umol/L GLBN;G3:C组+700umol/L GLBN).用免疫组化的方法定性测定α-SMA,用流式细胞仪测定细胞周期.结果:24h的细胞DNA含量S期对照组和空白组有明显差异(41%vs58.6%,P＜0.05),给与NaHS后,细胞DNA含量S期N1、N2和N3组与对照组均有明显差异(62.0%,66.8%,72.8%vs58.6%均P＜0.05),给与GLBN处理后,细胞DNA含量S期G1、G2和G3组与对照组均有明显差异(38.6%,34.8%,32.5%vs58.6%均P＜0.05),各项指标结果与给与NaHS处理后结果相对应,各组与相对应组比较均有明显差异(P＜0.05).24h的免疫组化照片显示对照组和空白组有明显差异,给与GLBN后,G2组与G3组与对照组有明显差异,给与NaHS后,N1,N2,N3组均与对照组,G1,G2,G3组有显著差异.结论:氧应激下硫化氢对HSC细胞具有保护作用,可抑制肝纤维化的发生发展.     

Progression of hepatic stellate cell activation is associated with the level of oxidative stress rather than cytokines during CCl4-induced fibrogenesis

In order to identify a fibrogenic factor associated with the potential of hepatic stellate cells (HSC) activation that arises during the CCl4-induced fibrogenic process, the relationship between the activation of HSC and levels of several fibrogenic factors were investigated. After isolation of HSC from the liver at different stages of CCl4 intoxication, the activation of HSC was assessed by the expression of alpha-smooth muscle actin. Levels of cytokines and oxidative stress in liver homogenates and plasma were measured by enzyme linked immunosorbent assay and the colorimetric method. In primary culture, HSC isolated from a rat liver were gradually activated in a time-dependent manner according to CCl4 administration. The progression of HSC activation was closely correlated with parameters related to oxidative stress in liver homogenates rather than the tissue levels of several cytokines. Also, the levels of antioxidants and arginase activity were inversely correlated with HSC activation. In plasma, the levels of oxidative stress and cytokines in CCl4-treated rat livers were not associated with the activation of HSC found during the CCl4-induced fibrogenic process. The relationship between HSC activation and oxidative stress was also confirmed through several factor-treated HSC cultures. In conclusion, the activation of HSC was accelerated according to CCl4 administration, and the progression of HSC activation is absolutely related to the oxidative stress. These results show that enhanced oxidative stress is an important signal for activation of HSC in experimental liver fibrogenesis.     

Syncoilin is an intermediate filament protein in activated hepatic stellate cells

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl₄- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.     

Low density lipoprotein receptor-related protein 1 modulates the proliferation and migration of human hepatic stellate cells

Human hepatic stellate cells (HHSCs) proliferation and migration play a key role in the pathogenesis of liver inflammation and fibrogenesis. Low density lipoprotein receptor‐related protein (LRP1) is an endocytic receptor involved in intracellular signal transduction. The aim of this work was to analyse the role of low density lipoprotein receptor‐related protein (LRP1) in HHSCs proliferation and migration and the mechanisms involved. Human LRP1 deficient‐HHSCs were generated by nucleofecting the line HHSCs with siRNA anti‐LRP1. HHSCs DNA synthesis was measured by [³H]‐thymidine incorporation and cell cycle progression by flow cytometry after annexin V and iodure propidium staining. Cell migration was assessed using a wound repair model system. LRP1 expression and extracellular matrix‐regulated kinase (ERK1,2) phosphorylation were analysed by Western blot analysis. Transforming growth factor‐β (TGF‐β) extracellular levels were analysed by ELISA. siRNA‐antiLRP1 treatment almost completely inhibited LRP1 mRNA and protein expression. LRP1 deficient HHSCs showed higher proliferative response (172 ± 19 vs. 93 ± 8 [³H]‐thymidine incorporation; 78.68% vs. 82.69% in G0/G1, 21.32% vs. 17.30% in G2/S) and higher migration rates than control HHSCs. LRP1 deficient cells showed higher levels of phosphorylated ERK1,2. TGF‐β extracellular levels were threefold higher in LRP1‐deficient than in control HHSCs cells. These results demonstrate that LRP1 regulates HHSCs proliferation and migration through modulation of ERK1,2 phosphorylation and TGF‐β extracellular levels. These results suggest that HHSCs‐LRP1 may play a key role in the modulation of factors determining hepatic fibrosis. J. Cell. Physiol. 227: 3528–3533, 2012. © 2012 Wiley Periodicals, Inc.     

Targeted delivery of a triplex-forming oligonucleotide to hepatic stellate cells

Liver fibrosis is characterized by abnormal accumulation of extracellular matrix (ECM), namely, fibrillar collagens in the hepatic stellate cells (HSCs). Earlier, we developed an antigene approach, using a type alpha1(I) collagen gene promoter specific triplex-forming oligonucleotide (TFO) to inhibit collagen gene expression. In this paper, to enhance overall delivery of TFOs to the liver and more specifically to HSCs, we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) by phosphorylating p-nitrophenyl-alpha-d-mannopyranoside, reducing its nitro group, and reacting it with thiophosgene to produce p-isothiocyanatophenyl-6-phospho-alpha-d-mannopyranoside (itcM6P) for conjugation with BSA. (33)P-TFO was conjugated with M6P-BSA via a disulfide bond, and the stability of the (M6P)(20)-BSA-TFO conjugate was determined. Following tail vein injection into rats, (M6P)(20)-BSA-(33)P-TFO rapidly cleared from the circulation and accumulated mainly in the liver. Almost 66% of the injected (M6P)(20)-BSA-(33)P-TFO accumulated in the liver at 30 min postinjection, which was significantly higher than that deposited after injection of (33)P-TFO. A large proportion of the injected (M6P)(20)-BSA-(33)P-TFO was taken up by the HSCs as evidenced by determination of radioactivity in the digested liver cells upon liver perfusion and separation on a Nycodenz gradient. Therefore, this TFO conjugate may be used for the treatment of liver fibrosis.     

FBXO31 modulates activation of hepatic stellate cells and liver fibrogenesis by promoting ubiquitination of Smad7

Liver fibrosis is a critical pathological process in the early stage of many liver diseases, including hepatic cirrhosis and liver cancer. However, the molecular mechanism is not fully revealed. In this study, we investigated the role of F-box protein 31 (FBXO31) in liver fibrosis. We found FBXO31 upregulated in carbon tetrachloride (CCl₄) induced liver fibrosis and in activated hepatic stellate cells, induced by transforming growth factor-β (TGF-β). The enforced expression of FBXO31 caused enhanced proliferation and increased expression of α-smooth muscle actin (α-SMA) and Col-1 in HSC-T6 cells. Conversely, suppression of FBXO31 resulted in inhibition of proliferation and decreased accumulation of α-SMA and Col-1 in HSC-T6 cells. In addition, upregulation of FBXO31 in HSC-T6 cells decreased accumulation of Smad7, the negative regulator of the TGF-β/smad signaling pathway, and suppression of the FBXO31 increased accumulation of Smad7. Immunofluorescence staining showed FBXO31 colocalized with Smad7 in HSC-T6 cells and in liver tissues of BALB/c mice treated with CCl₄. Immunoprecipitation demonstrated FBXO31 interacted with Smad7. Moreover, FBXO31 enhanced ubiquitination of Smad7. In conclusion, FBXO31 modulates activation of HSCs and liver fibrogenesis by promoting ubiquitination of Smad7.     

Maintenance of hepatocyte functions in coculture with hepatic stellate cells

Hepatic stellate cells (HSCs) are a type of nonparenchymal liver cells (NPCs) and are present in the perisinusoidal space of Disse. Hepatocytes were cocultured with HSCs isolated from the NPC fraction with the aim of maintaining differentiated liver functions in vitro. Hepatocytes inoculated directly onto the HSC layer (Co-mix) exhibited lower activity of albumin secretion and higher DNA synthesis activity than hepatocytes of the monoculture control. On the contrary, hepatocytes cocultured with HSCs but separated by a semipermeable membrane (Co-sep) maintained the activities of albumin secretion and urea synthesis. The soluble factor(s) secreted from HSCs had the maintenance effect. Subcultured HSCs were activated to myofibroblast-like cells (MFBs) and decreased the maintenance effect on hepatocyte function. However, the MFBs were found to resume the ability to maintain the hepatocyte function by cultivation on type I collagen. The coculture of hepatocytes and HSCS/MFB could be applied to the development of bioartificial liver support system and liver regenerative medicine.     

A class of imidazolium salts is anti-oxidative and anti-fibrotic in hepatic stellate cells

A class of imidazolium salts (IMSs) is routinely used in organic synthetic chemistry as precursors to generate N-heterocyclic carbenes (NHCs) with catalytic activity. However, their biological properties are largely unknown. The current study investigates the biological activity of a typical NHC precursor DBZIM and its trimer TDBZIM in hepatic stellate cells (HSCs), which is an in vitro model for studying liver fibrosis. The results show that HSCs treated with IMSs have an enhanced GSH/GSSG ratio and a reduced level of reactive oxygen species (ROS), which may consequently contribute to the attenuation in gene expression of fibrogenic molecules such as smooth muscle actin-α (SMAA), transforming growth factor-beta 1 (TGF-β1), procollagen αI(I) and fibronectin. Further, the in vivo experiments demonstrate that DBZIM is an anti-fibrotic agent in a mouse model of liver fibrosis. These findings suggest that the versatile IMSs could be a potential source for developing novel therapeutics to treat liver fibrosis and other fibrogenic disorders caused by oxidative stress and TGF-β1 mal-signalling.     

Warm ischemia-induced alterations in oxidative and inflammatory proteins in hepatic Kupffer cells in rats

The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 +/- 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 +/- 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 +/- 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 +/- 0.19; p < 0.001) and catalase (1.23 +/- 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options.     

Hypoxia-inducible factor-1alpha regulates autophagy to activate hepatic stellate cells

The role of autophagy in Hif-1α modulated activation of hepatic stellate cells was illustrated in current work. Autophagy markers were determined in livers of Schistosoma japonicum infected mice and hypoxia or LPS treated human hepatic stellate cell, LX-2 cells. The action of Hif-1 to autophagy was defined as increase of autophagy markers was significantly suppressed in Hif-1α siRNA transfected cells upon hypoxia or LPS stimulation. The function of autophagy in activation of LX-2 cells was assessed as increase of activation markers was blocked using autophagy inhibitors under hypoxia and LPS stimulation. Conclusively, Hif-1α regulates activation of hepatic stellate cell by modulating autophagy.