Ascorbic acid supplementation down-regulates the alcohol induced oxidative stress,hepatic stellate cell activation,cytotoxicity and mRNA levels of selected fibrotic genes in guinea pigs

Both oxidative stress and endotoxins mediated immunological reactions play a major role in the progression of alcoholic hepatic fibrosis. Ascorbic acid has been reported to reduce alcohol-induced toxicity and ascorbic acid levels are reduced in alcoholics. Hence, we investigated the hepatoprotective action of ascorbic acid in the reversal of alcohol-induced hepatic fibrosis in male guinea pigs (n = 36), and it was compared with the animals abstenting from alcohol treatment. In comparison with the alcohol abstention group, there was a reduction in the activities of toxicity markers and levels of lipid and protein peroxidation products, expression of α-SMA, caspase-3 activity and mRNA levels of CYP2E1, TGF-β₁, TNF-α and α₁(I) collagen in liver of the ascorbic acid-supplemented group. The ascorbic acid content in liver was significantly reduced in the alcohol-treated guinea pigs. But it was reversed to normal level in the ascorbic acid-supplemented group. The anti-fibrotic action of ascorbic acid in the rapid regression of alcoholic liver fibrosis may be attributed to decrease in the oxidative stress, hepatic stellate cells activation, cytotoxicity and mRNA expression of fibrotic genes CYP2E1, TGF-β₁, TNF-α and α₁ (I) collagen in hepatic tissues.     

Relationship between anti-fibrotic effect of Panax notoginseng saponins and serum cytokines in rat hepatic fibrosis

The aim of this study was to investigate the relationship between anti-fibrotic effect of Panax notoginseng saponins (PNS) and serum cytokines in rat hepatic fibrosis. Hepatic fibrosis induced by carbon tetrachloride (CCl₄) was studied in animal models using SD rats. Liver index, serum alanine amino transferase (ALT), aspartate amino transferase (AST), transforming growth factor-β1 (TGF-β1), tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) were measured, respectively. Liver index and the degree of liver fibrosis were also determined. Our results showed that the levels of ALT, AST and liver index in PNS-treated group were markedly lower than those in model group. PNS therapy also significantly attenuated the degree of hepatic fibrosis, collagen area and collagen area percent in liver tissue. Furthermore, the levels of serum TGF-β1, TNF-α and IL-6 were strikingly reduced in PNS-treated group compared with model group while the production of IL-10 was up-regulated. These findings demonstrate that PNS has certain therapeutic effects on hepatic fibrosis probably by immunoregulating the imbalance between pro-fibrotic and anti-fibrotic cytokines.     

抵抗素对NAFLD肝纤维化的影响及可能的体内外机制

应用高脂饮食饲养Wistar大鼠建立非酒精性脂肪肝病(non-alcoholic fatty liver disease,NAFLD)肝纤维化体内模型,选用重组抵抗素作用于肝星状细胞(hepatic stellate cell-t6,HSC-T6)建立体外模型。观察肝组织纤维化的情况;检测体内体外Ⅲ型前胶原(PCⅢ)、透明质酸(HA)、Ⅳ型胶原(CIV)、层黏蛋白(LN)水平;检测肝组织抵抗素mRNA和蛋白的表达水平;检测HSC-T6中转化生长因子β-1(TGF-β1)mRNA及肿瘤坏死因子α(TNF-α)mRNA表达水平。结果显示,随着高脂喂养时间的延长,大鼠肝组织抵抗表达逐渐增加且纤维化程度逐渐加重;随着抵抗素浓度的增加,HSC-T6上清中纤维化指标升高,细胞中TGF-β1mRNA及TNF-αmRNA表达增加。与对照组比较,各指标差异均有显著性(P<0.05或0.01)。上述结果显示抵抗素通过TNF-α、TGF-β1诱导NAFLD肝纤维的发生和发展。     

TGF-β1和FBRS可指示肝纤维化发展程度

为了探讨转化生长因子(TGF-β1)、外周血纤维化蛋白(FBRS)表达水平变化与肝纤维化发生发展的相关性,本研究选取自2015年5月至2017年6月间在我院诊治的慢性病毒性肝炎患者120例,设为肝炎组。采用免疫组化法检测肝组织TGF-β1的表达水平,酶联免疫分析检测血清中TGF-β1的含量,RT-PCR检测外周血单个核细胞FBRS mRNA的表达水平,分析TGF-β1、FBRS与肝纤维化程度的相关性。研究结果表明:随肝纤维化程度的不同,肝组织TGF-β1、血清TGF-β1表达水平、FBRS mRNA表达水平与肝脏胶原含量同步性升高(p<0.05)。进一步的相关分析表明:肝组织TGF-β1水平、FBRS mRNA与肝纤4项检查,即血清Ⅲ型前胶原(PC-Ⅲ)、Ⅳ型胶原片段(Ⅳ-C)、层粘连蛋白(LN)和透明质酸(HA)水平之间均呈正相关。本研究结果初步得出结论,慢性病毒性肝炎肝组织TGF-β1、血清TGF-β1表达水平、外周血FBRS的表达水平与肝组织纤维化程度呈正相关。     

Ascorbic acid is superior to silymarin in the recovery of ethanol-induced inflammatory reactions in hepatocytes of guinea pigs

Both oxidative stress and inflammatory reactions play a major role in alcoholic liver fibrosis. We evaluated the efficacy of ascorbic acid (AA) and silymarin in the regression of alcohol-induced inflammation in hepatocytes of guinea pigs (Cavia porcellus). Animals were administered with ethanol at a daily dose of 4 g/kg body weight (b.wt) for 90 days. On the ninety-first day, ethanol administration was stopped and animals were divided into alcohol abstention group and silymarin- (25 mg/100 g b.wt) and AA- (25 mg/100 g b.wt) supplemented groups and maintained for 30 days. There was a significant increase in the activities of alanine aminotransferase, aspartate aminotransferase, and γ-glutamyl transpeptidase in the serum of the ethanol group. The intracellular reactive oxygen species (ROS) and expressions of cytochrome P4502E1 and nuclear factor κB1, tumor necrosis factor-α, and transforming growth factor-β₁ in hepatocytes were significantly increased in ethanol group. The fibrotic markers α-smooth muscle actin and α ₁(I) collagen and activity of cytotoxicity marker caspase-3 were significantly increased and AA content was significantly reduced in hepatocytes of alcohol-treated guinea pigs. But the AA and silymarin supplementation significantly reduced these changes in comparison with alcohol abstention group. AA could induce greater reduction of inflammatory and fibrotic markers in hepatocytes than silymarin. This indicates that AA is superior to silymarin in inhibiting intracellular ROS generation and thereby reducing the ethanol-induced inflammation in hepatocytes.     

Profibrotic effect of miR-33a with Akt activation in hepatic stellate cells

MicroRNAs (miRNAs) attract more attention in the pathophysiology of liver fibrosis and miR-33a has been previously demonstrated as involved in the regulation of cholesterol and lipid metabolism. Transforming growth factor-beta1 (TGF-β1) is generally accepted to be the main stimulating factor in the hepatic stellate cells (HSCs) activation, which plays an important role in hepatic fibrosis. However, the involvement and underlying mechanism of miR-33a and its role in TGF-β1-induced hepatic fibrogenesis remains unknown. Here, we investigate the role of miR-33a in the activation of immortalized human HSCs, Lx-2 cells. Our findings have shown that the expression of miR-33a with its host gene sterol regulatory element-binding protein 2 (SREBP2) was more highly expressed in activation of Lx-2 cells than in quiescent cells. The expression of miR-33a on TGF-β1-induced HSCs activation may be modulated via the activation of PI3K/Akt pathway. In addition, miR-33a significantly correlated with TGF-β1-induced expression of α1 (I) collagen (Col1A1) and α-SMA in HSCs. Bioinformatics analyses predict that peroxisome proliferator activated receptor-alpha (PPAR-α) is the potential target of miR-33a. We further found that anti-miR-33a significantly increases target gene PPAR-α mRNA and protein level, suggesting that miR-33a involved in HSCs function might be modulated by targeting PPAR-α. Finally, our results indicate that the expression of miR-33a increased with the progression of liver fibrosis. These results suggested that anti-miR-33a inhibit activation and extracellular matrix production, at least in part, via the activation of PI3K/Akt pathway and PPAR-α and anti sense of miR-33a may be a novel potential therapeutic approach for treating hepatic fibrosis in the future.     

Regulation of matrix metalloproteinase-1 and alpha-smooth muscle actin expression by interleukin-1 alpha and tumour necrosis factor alpha in hepatic stellate cells

Hepatic stellate cells (HSCs) are key players in liver fibrosis and regeneration via collagen degradation and synthesis. These phenomena involve inflammatory cytokines released from non-parenchymal liver cells such as Kupffer cells. Although the effects of individual cytokines on many cell types have been investigated in various conditions, such as inflammation and tissue fibrosis, investigating the effect of combined cytokines would further our understanding of the regulatory mechanisms in tissue fibrosis. Here, we report the effect of multiple cytokine combinations on primary HSCs. We first examined the effect of individual cytokines and then the simultaneous exposure of different cytokines, including interleukin-6 (IL-6), IL-1 alpha (IL-1α), platelet-derived growth factor (PDGF), tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β), on matrix metalloproteinase-1 (MMP1) gene expression in primary HSCs. We observed that the combination of all five cytokines induced higher levels of MMP1 gene expression. Of these cytokines, TNF-α and IL-1α were found to be the key cytokines for not only inducing MMP1 expression, but also increasing α-smooth muscle actin gene expression. In conclusion, the combined treatment of TNF-α and IL-1α on HSCs had an enhanced effect on the expression of the fibrotic genes, MMP1 and α-smooth muscle actin, so appears to be an important regulator for tissue regeneration. This finding suggests that stimulation with combined anti-fibrotic cytokines is a potential approach in the development of a novel therapy for the recovery of liver fibrosis.     

Antagonizing TGF-beta induced liver fibrosis by a retinoic acid derivative through regulation of ROS and calcium influx

Transforming growth factor-beta1 (TGF-β1) mediates the regulation of extracellular matrix via reactive oxygen species (ROS) and calcium influx, both are activators of hepatic stellate cells (HSC) which play a critical role in hepatic fibrogenesis. Hence one can use ROS assay as the main screening tool for molecules that might antagonize the process of liver fibrosis. A retinoic acid derivative isolated from the mycelium of Phellinus linteus that down-regulates ROS generation and calcium influx in HSC-T6 cells was thus obtained in our screening process. The retinoic acid derivative also reverses an early liver fibrosis, as assayed by liver contents of hydroxyproline, α-smooth muscle actin (α-SMA), and collagen 1A2, in an early liver fibrosis model we established previously where an inducible expression vector containing a TGF-β gene was hydrodynamically transferred into a testing animal. Retinoic acid derivative thus acts both in vitro and in vivo to prevent liver fibrosis at an early phase.     

Effect of ascorbic acid on heme metabolism in hepatic microsomes

Hepatic microsonal cytochrome P-450 levels are significantly decreased (46–68%) in ascorbic acid-deficient guinea pigs. Studies attempting to elucidate the mechanism responsible for decreased cytochrome P-450 demonstrated that ascorbic acid status did not influence the turnover $\text{(}\text{t}\text{1}\text{2}\text{)}$ or the degradation of hepatic cytochrome P-450 heme. Urinary excretion of Δ-aminolevulinic acid (ALA) and coproporphyrin was significantly decreased (30 and 69% respectively) in ascorbic acid-deficient guinea pigs. Injections (ip) of ALA into ascorbic acid-deficient guinea pigs were not effective in returning cytochrome P-450 levels to values found in ascorbic acid-supplemented guinea pigs. In addition, plasma and hepatic iron and blood heme were related directly, while hepatic copper and plasma copper or ceruloplasmin were related inversely, to the ascorbic-acid status of the guinea pig. These data, along with past investigations on heme synthesis in the ascorbic acid-deficient guinea pig, are consistent with mechanisms proposing that ascorbic acid may influence: 1) apocytochrome P-450 synthesis, 2) binding of heme and apo-cytochrome P-450 to form active cytochrome P-450, and/or 3) incorporation of Fe++ into the heme moiety of cytochrome P-450, perhaps via changes in copper metabolism.     

育阴软肝颗粒剂对肝纤维化大鼠TGF-β1表达的影响

目的：观测育阴软肝颗粒剂对大鼠肝纤维化模型的防治作用及对转化生长因子-β1（TGF-β1）表达的影响。方法：将Wistar大鼠分为6组（n=10）,注射四氯化碳、饲以高脂饲料并饮用20%乙醇6周复制肝纤维化大鼠模型,经6.2~24.8 g/kg育阴软肝颗粒剂干预（qd）6周后,测定肝纤维化大鼠血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）活性、透明质酸（HA）、Ⅲ型前胶原（PCⅢ）、Ⅳ型胶原（C-Ⅳ）及板层素（LN）含量,观测肝组织病理学及肝组织TGF-β1表达的变化,对育阴软肝颗粒剂防治肝纤维作用及机制进行研究。结果：实验第7周,模型组大鼠肝组织出现明显的纤维化病变（P<0.01）;与模型组比较,6.2~24.8g/kg的育阴软肝颗粒剂能明显降低肝指数以及血清ALT、AST活性与HA、PCⅢ、C-Ⅳ、LN含量,缓解肝组织纤维化病理变化,抑制纤维化肝组织TGF-β1的表达（P<0.05,0.01）。结论：育阴软肝颗粒剂对多因素复制肝纤维化大鼠造模具有明显的治疗作用,而抑制TGF-β1的表达可能是其作用机制之一。     

The use of pentoxifylline as adjuvant therapy with praziquantel downregulates profibrogenic cytokines, collagen deposition and oxidative stress in experimental schistosomiasis mansoni

This study investigates the possible use of pentoxifylline (PTX), with antifibrotic and anti-inflammatory properties, as adjuvant in treatment of schistosomal liver fibrosis through determination of some profibrogenic cytokines, oxidative stress and collagen deposition. Animals were classified into seven groups: normal control (i), Schistosoma mansoni-infected untreated (ii), infected treated with praziquantel (PZQ) curative, 1000 mg/kg (iii) or sub curative, 200 mg/kg dose (iv), infected treated with PTX alone (10 mg/kg/day; 5 days/wk) for 8 weeks starting from the 2nd to the 10th week post infection (v), or in addition to curative (vi) or sub curative dose of PZQ (vii). Serum transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), matrix metalloproteinases-2 (MMP-2) and hepatic hydroxyproline (Hyp) content, glutathione related antioxidant enzymes and malondialdehyde (MDA) were determined. Results showed that S. mansoni infection produced remarkable elevations in the serum levels of TGF-β1, TNF-α, MMP-2 and the hepatic contents of Hyp, glutathione reductase (GR), MDA with significant reduction in reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and superoxide dismutase (SOD) when compared with their corresponding normal controls. Treatment of infected mice with PTX in addition to PZQ curative rather than its sub curative dose produced the best results evidenced by complete normalization in the previously mentioned serum and hepatic parameters. Conclusion: PTX could attenuate liver fibrosis in early stages of S. mansoni infection through downregulation of profibrogenic cytokines, oxidative stress and collagen deposition.     

The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats

Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-β1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-β1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague–Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-β1 siRNA 0.125 mg/kg treatment group, TGF-β1 siRNA 0.25 mg/kg treatment group and TGF-β1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin–Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-β1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-β1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-β1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-β1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-β1 siRNA negative control group and the TGF-β1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-β1, type I collagen and type III collagen (P < 0.01). Conclusions: Using siRNA to target TGF-β1 can inhibit the expression of TGF-β1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-β1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.     

Nanofiber-expanded stem cells mitigate liver fibrosis: Experimental study

ObjectivesThis study examines a pretreatment strategy to strengthen the hepatic lineage divergence of mesenchymal stem cells (MSCs).Design and methodsBMSCs were expanded in the presence or absence of nanofiber (NF) and treated with growth factors (GF) prior to transplantation. Thioacetamide (TA) was used for liver fibrosis induction and transplantation of NF-expanded BMSCs was compared biochemically and histologically to the cells expanded without NF scaffold.ResultsThe ultraweb NF caused better proliferation and characterization of MSCs. MSCs transplantation significantly improved liver functions, increased hepatic HGF and Bcl-2 levels, whereas decreased serum fibronectin, hepatic TNF-α and TGF-β1 levels. Hepatic HNF4α, FOXa2, CYP7a1 genes expression were enhanced while β-5-Tub and AFP genes expression were depressed. Histological study documented these results. Differentiated NF-MSCs showed pronounced enhancement of the aforementioned parameters as compared to differentiated MSCs in the absence of NF.Conclusionpretreatment with growth factors in the presence of NF augment homing, repopulation and hepatic differentiation abilities of MSCs and proves to be a promising approach for the treatment of liver fibrosis.     

Plin3 protects against alcoholic liver injury by facilitating lipid export from the endoplasmic reticulum

Hepatic lipid accumulation is the most common pathological characteristic of alcoholic liver disease (ALD). In mammalian cells, excess neutral lipids are stored in lipid droplets (LDs). As a member of perilipin family proteins, Plin3 was recently found to regulate the LD biogenesis. However, the roles and mechanism of Plin3 in ALD progression remain unclear. Herein, we found that alcohol stimulated Plin3 expression in both mouse livers and cultured AML12 mouse hepatic cells, which was accompanied by excess LD accumulation in hepatocytes. The elevations of Plin3 in alcohol-treated hepatocytes paralleled with the levels of both PPARα and γ, and the protein degradation of Plin3 was also reduced after alcohol exposure. Moreover, Plin3 knockdown increased cellular sensitivity to alcohol-induced apoptosis, endoplasmic reticulum (ER) stress, and inflammatory cytokines release, including TNF-α, IL-1, and IL-6β. Notably, alcohol exacerbated triglycerides (TG) accumulation in the ER and caused ER dilation in Plin3-knockdown AML12 cells. Finally, we observed that Plin3 interacted with dynein subunit Dync1i1 and mediated the colocalization of LDs and microtubules, while high concentration of alcohol disrupted microtubules and caused dispersion of excess small LDs in cytoplasm. Summarily, Plin3 promotes lipid export from the ER and reduces ER lipotoxic stress, thereby, protecting against alcoholic liver injury. Moreover, Plin3 could be an adapter protein mediating LD transport by microtubules. This study explored the roles of Plin3 in alcohol-induced hepatic injury, suggesting Plin3 as a potential target for the prevention of ALD progression.     

牛磺酸上调基因1(TUG1)对肝纤维化的作用及其机制*

目的:探讨牛磺酸上调基因1(TUG1)在肝纤维化中的作用机制。方法:按照文献建立TGF-β1(5 ng/ml)刺激的活化肝星状细胞模型和经典的1%DMN(1 ml/kg/d)致大鼠肝纤维化模型,将肝纤维化大鼠和活化肝星状细胞(HSC)均分为模型对照组、阴性对照组(沉默TUG1阴性对照)、siRNA干扰组(TUG1基因沉默组)。实验结束后利用苏木精-伊红(HE)染色检测大鼠肝脏组织病理变化;采用逆转录-聚合酶链反应(RT-PCR)法、蛋白免疫印记(Western blot)分别测定大鼠肝组织及活化肝星状细胞中α-平滑肌肌动蛋白(α-SMA)、TUG1、I型胶原蛋白(collagenI)、基质金属蛋白酶-2(MMP-2)、金属蛋白酶组织抑制因子(TIMP-1)、Smad2、Smad3表达水平。结果:肝组织病理学检查显示,沉默TUG1能够明显缓解肝脏纤维化病理改变,Western blot结果显示,沉默TUG1能够显著降低大鼠肝组织和活化肝星状细胞中TUG1、α-SMA、collagenI、MMP-2、TIMP-1、Smad2、Smad3基因与蛋白表达水平(P＜0.05)。与模型对照组相比,阴性对照组的TUG1、α-SMA的蛋白与基因水平明显升高(P＜0.05)。与模型对照组和阴性对照组相比,siRNA干扰组中TUG1, α-SMA, collagenI, MMP-2, TIMP-1, Smad2 and Smad3的蛋白和基因水平显著降低(P＜0.05),而在模型对照组和阴性对照组中TUG1, α-SMA, collagenI, MMP-2, TIMP-1, Smad2 and Smad3的蛋白和基因表达水平之间差异无显著性。结论:TUG1在肝纤维化组织和活化的肝星状细胞中显著上调,沉默TUG1可能通过抑制转化生长因子-β1(TGF-β1)/Smad信号通路改善1%DMN致大鼠肝纤维化病理损伤,降低活化肝星状细胞中纤维化相关蛋白水平,发挥抗肝纤维化的作用。     

Physalin D attenuates hepatic stellate cell activation and liver fibrosis by blocking TGF-β/Smad and YAP signaling

BackgroundHepatic fibrosis is considered integral to the progression of chronic liver diseases, as it leads to the development of cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) is the dominant event in hepatic fibrogenesis. The transforming growth factor-β1 (TGF-β1) and Yes-associated protein (YAP) pathways play a pivotal role in HSC activation, hepatic fibrosis and cirrhosis progression. Therefore, targeting the TGF-β/Smad and YAP signaling pathways is a promising strategy for antifibrotic therapy.PurposeThe present study investigated the protective effects of Physalin D (PD), a withanolide isolated from Physalis species (Solanaceae), against liver fibrosis and further elucidated the mechanisms involved in vitro and in vivo.Study design/methodsWe conducted a series of experiments using carbon tetrachloride (CCl₄)- and bile duct ligation (BDL)-induced fibrotic mice and cultured LX-2 cells. Serum markers of liver injury, and the morphology, histology and fibrosis of liver tissue were investigated. Western blot assays and quantitative real-time PCR were used to investigate the mechanisms underlying the antifibrotic effects of PD.ResultPD decreased TGF-β1-induced COL1A1 promoter activity. PD inhibited TGF-β1-induced expression of Collagen I and α-smooth muscle actin (α-SMA) in human hepatic stellate LX-2 cells. PD significantly ameliorated hepatic injury, including transaminase activities, histology, collagen deposition and α-SMA, in CCl₄- or BDL-induced mice. Moreover, PD markedly decreased the expression of phosphorylated Smad2/3 in vitro and in vivo. Furthermore, PD significantly decreased YAP protein levels, and YAP knockdown did not further enhance the effects of PD, namely α-SMA inhibition, Collagen I expression and YAP target gene expression in LX-2 cells.ConclusionThese results clearly show that PD ameliorated experimental liver fibrosis by inhibiting the TGF-β/Smad and YAP signaling pathways, indicating that PD has the potential to effectively treat liver fibrosis.     

A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.     

A novel fused 1,2,4-triazine aryl derivative as antioxidant and nonselective antagonist of adenosine A(2A) receptors in ethanol-activated liver stellate cells

It has been detected that hepatic adenosine A(2A) receptors play an active role in the pathogenesis of hepatic fibrosis and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis. In this paper we examined if our new triazine derivative (IMT) can inhibit ethanol-induced activation of HSCs measured as increased α-SMA, collagen synthesis and enhanced oxidative stress in rat liver stellate cells. We also investigated its influence on cytokines (TGF-β, TNF-α) synthesis, MMP-2 and TIMP-1 production and ethanol-induced intracellular signal transduction. Moreover, with using of known adenosine A(2A) receptor agonist (CGS 21680), and antagonist (SCH 58261) we examined if this triazine derivative acts on adenosine receptors. We detected a strong antagonistic action of new triazine derivative (IMT) on ethanol-induced rat liver stellate cells activation, observed as a significant decrease in α-SMA, collagen synthesis, reactive oxygen species production, TGF-β, TNF-α, MMP-2 and TIMP-1 production as well as JNK, p38MAPK, NFκB, IκB, Smad3 phosphorylation. Moreover, IMT strongly inhibited activation of stellate cells by known selective agonist of adenosine A(2A) receptor (CGS 21680). When known A(2A) receptor antagonist (SCH 58261) was used together with IMT this effect was not spectacular. Additionally, only slight enhancement of inhibition was observed when cells were pretreated both IMT with SCH 58261, hence we suppose that IMT acts as nonselective antagonist of A(2A) receptors, and, besides its antioxidant activity, also by this way inhibited ethanol-induced stellate cell activation.     

Cytochrome P4502E1, oxidative stress, JNK, and autophagy in acute alcohol-induced fatty liver

Binge alcohol drinking induces hepatic steatosis. Recent studies showed that chronic ethanol-induced fatty liver was, at least in part, CYP2E1 dependent. The mechanism of acute alcohol-induced steatosis and whether CYP2E1 plays any role are still unclear. Increasing oxidative stress by alcohol can activate the JNK MAP kinase signaling pathway, suggesting that JNK might be a target for prevention of alcohol-induced steatosis. We used CYP2E1 knockout (KO) mice, a JNK inhibitor, and JNK1 or JNK2 knockout mice to test the role of CYP2E1, JNK, and the individual role of JNK1 and JNK2 in acute alcohol-induced steatosis. In wild-type (WT) mice, acute alcohol activates CYP2E1 and increases oxidative stress, which reciprocally increases activation of the JNK signaling pathway. Acute alcohol-induced fatty liver and oxidative stress were blunted in CYP2E1 KO mice and by the JNK inhibitor in WT mice. The antioxidant N-acetylcysteine decreased the acute alcohol-induced oxidative stress, the activation of JNK, and the steatosis but not the activation of CYP2E1. Acute alcohol decreased autophagy and increased expression of SREBP, effects blocked by the JNK inhibitor. Acute alcohol-induced fatty liver was the same in JNK1 and JNK2 KO mice as in WT mice; thus either JNK1 or JNK2 per se is sufficient for induction of steatosis by acute alcohol. The results show that acute alcohol elevation of CYP2E1, oxidative stress, and activation of JNK interact to lower autophagy and increase lipogenic SREBP resulting in fatty liver.