u-0126 and Liver-Cirrhosis

Obstructive sleep apnea syndrome (OSAS) is a common and complex disorder that is associated with liver injury. Moreover, previous studies have revealed that chronic intermittent hypoxia (CIH) is associated with the development of non‑alcoholic fatty liver disease and hepatic fibrosis. However, the underlying molecular mechanisms remain largely unknown. The present study aimed to investigate whether chronic intermittent hypoxia induced hepatic fibrosis, in addition to determining its underlying mechanisms, in CIH model rats using immunohistochemistry, western blotting and reverse transcription‑quantitative PCR. The present results suggested that CIH caused hepatic fibrosis and increased the expression levels of interleukin (IL)‑1β, IL‑8, monocyte chemotactic‑1, tumor necrosis factor‑α, intercellular adhesion molecule‑1 and vascular cell adhesion molecule‑1 in the liver; these conditions could be reversed by Toll‑like receptor 4 (TLR4) short hairpin RNA lentivirus treatment. Moreover, immunohistochemistry and western blotting results indicated that TLR4 and NF‑κB expression levels were significantly increased in the CIH and CIH‑TLR4 empty vector lentivirus group. However, protein expression levels of TLR4, NF‑κB, inhibitor of NF‑κB and phosphorylated‑mitogen‑activated protein kinase (MAPK)‑1 in the hypoxia/reoxygenation group were significantly higher compared with the control group (P<0.05), and these results were reversed by the MAPK inhibitor U0126 in vitro. Collectively, the present preliminary results suggested that inflammation and the TLR4/NF‑κB/MAPK signaling pathway may be involved in CIH‑induced liver fibrosis.

Topics: Animals; Butadienes; Cell Line; Disease Models, Animal; Enzyme Inhibitors; Gene Silencing; Hepatic Stellate Cells; Hypoxia; Inflammation; Liver Cirrhosis; Male; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Rats; Rats, Sprague-Dawley; Signal Transduction; Sleep Apnea, Obstructive; Toll-Like Receptor 4

Immune tolerance is considered the only way to manage liver transplantation (LT). The current study hypothesized that galectin-1 via the activation of hepatic stellate cells (HSCs) is capable of inducing immune tolerance in LT.. Lentiviral-mediated gene knockdown and overexpression of galectin-1 were conducted in HSC-T6 cells. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to determine galectin-1 expression. LT was performed in 20 C57BL/J6 mice and 20 C3H mice. T-cells were assigned into control, Galectin-1 shRNA, Galectin-1 OE, Galectin-1 OE SB431542, Galectin-1 OE Sulforaphane, Galectin-1 OE Y27632, and Galectin-1 OE UO126 groups. CFSE, flow cytometry, and ELISA were respectively employed to detect T-cell proliferation, CD4+/ CD8+ ratio and IL-2, IL-10 and TGF-β levels. After establishing mouse models of immune tolerance and acute rejection, immunohistochemistry, TUNEL, and immunofluorescence assay were performed to determine CD3+ expression, apoptosis, α-SMA, and desmin. Mouse models of CCl4-induced liver fibrosis were established, followed by assigning the control1 and CCl4 groups. ELISA was used to determine ALT, AST, TBIL and Hyp levels. A total of 3 C57BL/J6 mice (donor) and 6 C3H mice (recipient) were grouped into the control2 and UO126 groups, followed by ELISA detection for IL-2, IL-10 and TGF-β.. In T-cells, galectin-1 shRNA increased cell proliferation and IL-2 levels with reduced IL-10 and TGF-β levels, while the Galectin-1 OE and Galectin-1 OE UO126 groups revealed the opposite results. Galectin-1 overexpression elevated the ratio of the CD4+ to CD8+ T-cells. The acute rejection group exhibited enhanced desmin expression and reduced α-SMA expression. Compared with the immune tolerance group, the acute rejection group displayed higher galectin-1 expression, a positive expression rate of CD3+ T-cells, and an increased apoptosis rate. Compared with the control1 group, the CCl4 group exhibited higher galectin-1 expression, ALT, AST, TBIL, and Hyp levels, α-SMA expression and CD4+/CD8+ T-cell ratio, in addition to decreased expression of desmin. Compared with the control2 group, UO126 increased galectin-1 expressions, IL-10 and TGF-β levels and reduced IL-2 levels with inactivated HSCs.. The findings of the current study indicated that the overexpression of galectin-1 promoted the activation of HSCs, which reduced the inflammatory response by exerting immunosuppressive effects and accordingly contributed to immune tolerance in LT.

Topics: Actins; Animals; Butadienes; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line; Cell Proliferation; Cytokines; Galectin 1; Hepatic Stellate Cells; Immune Tolerance; Liver Cirrhosis; Liver Transplantation; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Nitriles; RNA Interference; RNA, Small Interfering; Signal Transduction

Previously, we suggested that IGFBPrP1 played a major role in hepatic stellate cell (HSC) activation, yet the molecular mechanism of IGFBPrP1 in hepatic fibrosis is unclear. The ERK pathway is involved in activation of HSCs. This study investigated the involvement of the ERK1/2 pathway in IGFBPrP1-induced liver inflammation and fibrosis.. An adenoviral vector encoding IGFBPrP1 (AdIGFBPrP1) was constructed. Rats received AdIGFBPrP1 or CAd (vector control) via their tail vein injection. One hour prior to adenoviral injections, rats were intraperitoneally administrated with 10 mg/kg U0126 (a specific MEK/ERK1/2 inhibitor) or DMSO (vehicle control). At weeks 2 or 4 post-gene transduction, serum samples were obtained and the levels of liver enzymes and hydroxyproline were determined. Liver tissue were histologically evaluated for inflammation and fibrosis. The expression of α-SMA and ECM were evaluated by qRT-PCR and western blotting.. After transduction, IGFBPrP1 expression significantly increased in livers and transduced cells. MEK/ERK1/2 inhibition administration of AdIGFBPrP1-treated rats and cells significantly blocked AdIGFBPrP1-induced activation of ERK1/2. U0126 significantly down-regulated the number of F4/80-positive cells and CD3-positive cells (markers of liver inflammation), the expression of α-SMA and the concentration of ECM components in vivo. In addition, α-SMA and TGF-β1 levels in AdIGFBPrP1 HSCs were markedly inhibited by a MEK/ERK1/2 inhibitor, indicating that HSC activation was inhibited.. These findings suggest that IGFBPrP1 acts as an initiator of liver fibrosis by inducing inflammation, HSC activation and ECM deposition through the ERK1/2 pathway.

Topics: Actins; Animals; Butadienes; CD3 Complex; Cells, Cultured; Enzyme Inhibitors; Extracellular Matrix Proteins; Genetic Vectors; Hepatic Stellate Cells; Hepatitis; Insulin-Like Growth Factor Binding Proteins; Kupffer Cells; Liver; Liver Cirrhosis; Lymphocyte Count; Male; MAP Kinase Signaling System; Nitriles; Rats; Rats, Sprague-Dawley; RNA, Messenger; T-Lymphocytes; Transduction, Genetic; Transforming Growth Factor beta1

RNA-binding proteins (RBPs) play a major role in the control of messenger RNA (mRNA) turnover and translation rates. We examined the role of the RBP, human antigen R (HuR), during cholestatic liver injury and hepatic stellate cell (HSC) activation. HuR silencing attenuated fibrosis development in vivo after BDL, reducing liver damage, oxidative stress, inflammation, and collagen and alpha smooth muscle actin (α-SMA) expression. HuR expression increased in activated HSCs from bile duct ligation mice and during HSC activation in vitro, and HuR silencing markedly reduced HSC activation. HuR regulated platelet-derived growth factor (PDGF)-induced proliferation and migration and controlled the expression of several mRNAs involved in these processes (e.g., Actin, matrix metalloproteinase 9, and cyclin D1 and B1). These functions of HuR were linked to its abundance and cytoplasmic localization, controlled by PDGF, by extracellular signal-regulated kinases (ERK) and phosphatidylinositol 3-kinase activation as well as ERK/LKB1 (liver kinase B1) activation, respectively. More important, we identified the tumor suppressor, LKB1, as a novel downstream target of PDGF-induced ERK activation in HSCs. HuR also controlled transforming growth factor beta (TGF-β)-induced profibrogenic actions by regulating the expression of TGF-β, α-SMA, and p21. This was likely the result of an increased cytoplasmic localization of HuR, controlled by TGF-β-induced p38 mitogen-activated protein kinase activation. Finally, we found that HuR and LKB1 (Ser428) levels were highly expressed in activated HSCs in human cirrhotic samples.. Our results show that HuR is important for the pathogenesis of liver fibrosis development in the cholestatic injury model, for HSC activation, and for the response of activated HSC to PDGF and TGF-β.

Topics: Actins; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Antigens, Surface; Butadienes; Carbon Tetrachloride; Cell Movement; Cell Proliferation; Common Bile Duct; ELAV Proteins; ELAV-Like Protein 1; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Gene Silencing; Hepatic Stellate Cells; Humans; Ligation; Liver Cirrhosis; Mice; Nitriles; Phosphatidylinositol 3-Kinase; Phosphorylation; Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Rats; RNA-Binding Proteins; RNA, Messenger; Transforming Growth Factor beta

Pentoxifylline (PTX), which is a xanthine derivative, is a well-known suppressor of tumor necrosis factor-alpha (TNF-alpha) production in inflammatory cells and has also been shown to inhibit collagen synthesis in hepatic stellate cells (HSCs) in vitro. The present study aimed to evaluate the effects of PTX on proliferation in HSCs as mediated by the Raf/MEK/extracellular-signal-regulated kinase (ERK) signaling pathway. The rat hepatic stellate cell line T6 and activated primary rat HSCs were used in this study. The proliferation rate of the cells treated with 1 mM PTX significantly decreased compared with that of the control in T6 cells (78.3 ± 6.03% at 12 h, 61.0 ± 7.55% at 24 h, and 44.7 ± 2.08% at 48 h, p < 0.05). PTX (1 mM) also decreased the fraction of the HSC population in the S and G2/M-phases of the cell cycle in primary activated rat HSCs. The Raf-1 inhibitor GW5074 and the ERK inhibitor U0126 had inhibitory effects that were similar to those of PTX on HSC proliferation. In addition, PTX inhibited the phosphorylation of Raf-1 (p-Raf-1) and ERK (p-ERK) in a dose- and time-dependent manner in HSCs. These data provide evidence that PTX suppresses HSC proliferation via the Raf/MEK/ERK pathway.

Topics: Animals; Apoptosis; Butadienes; Cell Line; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Hepatic Stellate Cells; Indoles; Liver Cirrhosis; Male; MAP Kinase Signaling System; Nitriles; Pentoxifylline; Phenols; Phosphodiesterase Inhibitors; raf Kinases; Rats; Rats, Sprague-Dawley

Liver fibrogenesis is dependent upon transdifferentiation of hepatic stellate cells to a profibrogenic phenotype. Prooxidant stress purportedly stimulates both an antioxidant response and myofibroblastic transdifferentiation with fibrogenic gene expression; however, mechanisms by which oxidative stress mediates stellate cell activation remain unclear. To this end, stellate cells were treated with tert-butylhydroquinone (tBHQ), a known inducer of antioxidant response genes. As anticipated, tBHQ induced expression of antioxidant response element (ARE)-regulated genes via the transcription factor Nrf2. Further, tBHQ promoted transdifferentiation of quiescent stellate cells cultured on Engelbreth-Holm-Swarm extracellular matrix. Pretreatment of cultured stellate cells with a phosphatidylinositol 3-kinase (PI3K) inhibitor blocked tBHQ-mediated ARE-dependent gene induction as well as stellate cell transdifferentiation. In contrast, extracellular signal-regulated kinase, which was demonstrated to be prominently phosphorylated following tBHQ treatment, was not found to affect either induction of the antioxidant response nor stellate cell transdifferentiation. These data implicate involvement of PI3K pathways in tBHQ-mediated stellate cell activation and demonstrate a requirement for PI3K in the antioxidant response of hepatic stellate cells.

Topics: Animals; Antioxidants; Butadienes; Cell Differentiation; Cells, Cultured; Chromones; Enzyme Induction; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression Regulation; Hydroquinones; Liver; Liver Cirrhosis; Morpholines; NF-E2-Related Factor 2; Nitriles; Oxidative Stress; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Rats; Rats, Sprague-Dawley; Response Elements; Transcriptional Activation