α-Lipoic acid inhibits liver fibrosis through the attenuation of ROS-triggered signaling in hepatic stellate cells activated by PDGF and TGF-β

Reactive oxygen species (ROS) have been implicated in hepatic stellate cell activation and liver fibrosis. We previously reported that α-lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA) inhibited toxicant-induced inflammation and ROS generation. In the present study, we further examined the effects of LA/DHLA on thioacetamide (TAA)-induced liver fibrosis in rats and the possible underlying mechanisms in hepatic stellate cells in vitro. We found that co-administration of LA to rats chronically treated with TAA inhibited the development of liver cirrhosis, as indicated by reductions in cirrhosis incidence, hepatic fibrosis, and AST/ALT activities. We also found that DHLA inhibited TGF-β/PDGF-stimulated HSC-T6 activation and ROS generation. These effects could be mediated by the MAPK and PI3K/Akt pathways. According to our current results, LA may have a beneficial role in the treatment of chronic liver diseases caused by ongoing hepatic damage.     

TGF-β/Smad信号转导通路与肝纤维化研究进展

肝纤维化是慢性肝病向肝硬化发展的中间过程,以胶原蛋白为主的细胞外基质（extracellular matrix,ECM）合成与降解失衡,导致过多的胶原在肝内沉积,是多条细胞信号转导通路和一系列细胞信息分子网络共同控制的结果。转化生长因子β（TGF-β）是肝纤维化病变过程中最关键的细胞因子之一。其中TGF-β/Smad信号转导通路是TGF-β信号转导调节肝星状细胞（HSC）活化促进ECM生成的主要途径。本文就TGF-β/Smad信号转导通路在肝纤维化过程中的作用及其研究进展予以综述。     

Small extracellular vesicles encapsulating lefty1 mRNA inhibit hepatic fibrosis

Liver fibrosis is the deposition of extracellular matrix (ECM) in the liver caused by persistent chronic injury, which can lead to more serious diseases such as cirrhosis or cancer. Blocking the effect of transforming growth factor β1 (TGF-β1), one of the most important cytokines in liver fibrosis, may be one of the effective ways to inhibit liver fibrosis. As a kind of natural nano-scale vesicles, small extracellular vesicles (sEvs) have displayed excellent delivery vehicle properties. Herein, we prepared hepatic stellate cell (HSC)-derived sEvs loading left-right determination factor 1 (lefty1) mRNA (sEvLs) and we wanted to verify whether they can inhibit fibrosis by blocking the TGF-β1 signaling pathway. The results showed that sEvLs had effective cell uptake and reduced activation of HSCs. Rats that were injected with CCl₄ by intraperitoneal injection for 6 weeks exhibited obvious symptoms of liver fibrosis and were treated with systemically administered sEvLs and free sEvs for 4 weeks. Rats injected with olive oil alone served as sham controls. Administration of sEvLs significantly reduced the area of fibrosis compared with free sEvs. We demonstrated that sEvLs inhibited HSCs activation and ECM production, and promote ECM degradation by downregulating α-smooth muscle actin (α-SMA), collagen I, tissue inhibitor of metalloproteinase (TIMP) -1 and upregulating matrix metalloprotease (MMP) -1. In summary, as an endogenous delivery vehicle, sEvs could deliver mRNA to attenuate hepatic fibrosis by blocking the TGF-β/Smad signaling pathway.     

Valeriana jatamansi partially reverses liver cirrhosis and tissue hyperproliferative response in rat

Valeriana jatamansi (family, Valerianaceae) is a traditional medicinal herb in the Indian subcontinent. This study provides experimental evidence indicating the therapeutic effect of the extract prepared from the dried rhizome of the herb in an animal model of liver cirrhosis and on cell proliferation. Liver cirrhosis was induced in rats by thioacetamide (0.03% in drinking water for 16 weeks). After inducing liver cirrhosis, rats were administered the extract orally for 9 weeks. Treatment was found to partially reverse the elevated levels of alkaline phosphatase, γ-glutamyl transferase and selected biochemical markers of hepatic injury including drug-metabolizing enzymes. Histopathology of the hepatic tissue confirmed the therapeutic effect of the extract which corroborated with the biochemical changes. The extract is also reported to ameliorate hepatic cell proliferation in rats injected with thioacetamide. The study has implications in finding a treatment for liver cirrhosis in humans.     

Magnolol Attenuates Concanavalin A‐induced Hepatic Fibrosis,Inhibits CD4+ T Helper 17 (Th17) Cell Differentiation and Suppresses Hepatic Stellate Cell Activation: Blockade of Smad3/Smad4 Signalling

Magnolol is a pharmacological biphenolic compound extracted from Chinese herb Magnolia officinalis, which displays anti‐inflammatory and antioxidant effects. This study was aimed at exploring the potential effect of magnolol on immune‐related liver fibrosis. Herein, BALB/c mice were injected with concanavalin A (ConA, 8 mg/kg/week) up to 6 weeks to establish hepatic fibrosis, and magnolol (10, 20, 30 mg/kg/day) was given to these mice orally throughout the whole experiment. We found that magnolol preserved liver function and attenuated liver fibrotic injury in vivo. In response to ConA stimulation, the CD4⁺ T cells preferred to polarizing towards CD4⁺ T helper 17 (Th17) cells in liver. Magnolol was observed to inhibit Th17 cell differentiation in ConA‐treated liver in addition to suppressing interleukin (IL)‐17A generation. Hepatic stellate cells were activated in fibrotic liver as demonstrated by increased alpha smooth muscle actin (α‐SMA) and desmin. More transforming growth factor (TGF)‐β1 and activin A were secreted into the serum. Magnolol suppressed this abnormal HSC activation. Furthermore, the phosphorylation of Smad3 in its linker area (Thr179, Ser 204/208/213) was inhibited by magnolol. In vitro, the recombinant IL‐17A plus TGF‐β1 or activin A induced activation of human LX2 HSCs and promoted their collagen production. Smad3/Smad4 signalling pathway was activated in LX2 cells exposed to the fibrotic stimuli, as illustrated by the up‐regulated phospho‐Smad3 and the enhanced interaction between Smad3 and Smad4. These alterations were suppressed by magnolol. Collectively, our study reveals a novel antifibrotic effect of magnolol on Th17 cell‐mediated fibrosis.     

Adenosine A(2A) receptors play a role in the pathogenesis of hepatic cirrhosis

1. Adenosine is a potent endogenous regulator of inflammation and tissue repair. Adenosine, which is released from injured and hypoxic tissue or in response to toxins and medications, may induce pulmonary fibrosis in mice, presumably via interaction with a specific adenosine receptor. We therefore determined whether adenosine and its receptors contribute to the pathogenesis of hepatic fibrosis. 2. As in other tissues and cell types, adenosine is released in vitro in response to the fibrogenic stimuli ethanol (40 mg dl(-1)) and methotrexate (100 nM). 3. Adenosine A(2A) receptors are expressed on rat and human hepatic stellate cell lines and adenosine A(2A) receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl(4)) (0.05 ml in oil, 50 : 50 v : v, subcutaneously) and thioacetamide (100 mg kg(-1) in PBS, intraperitoneally) released more adenosine than those from untreated mice when cultured ex vivo. 4. Adenosine A(2A) receptor-deficient, but not wild-type or A(3) receptor-deficient, mice are protected from development of hepatic fibrosis following CCl(4) or thioacetamide exposure. 5. Similarly, caffeine (50 mg kg(-1) day(-1), po), a nonselective adenosine receptor antagonist, and ZM241385 (25 mg kg(-1) bid), a more selective antagonist of the adenosine A(2A) receptor, diminished hepatic fibrosis in wild-type mice exposed to either CCl(4) or thioacetamide. 6. These results demonstrate 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.     

Association of thioacetamide and ethanol treatment: dolichol and retinol in isolated rat liver cells

Our aim was to study the distribution of dolichol, dolichol isoprenoids, and retinol in hepatocytes, Kupffer, sinusoidal endothelial and two subfractions of hepatic stellate cells, --Ito-1 and Ito-2--, after chronic treatment of rats for 2 and 4 months with a low dosage of thioacetamide associated with ethanol. Each type of cell responded differently to the two hepatotoxins. Overall, ethanol rarely affected the action of thioacetamide. Some new information emerges with regard to these hepatotoxins in comparison with the effects exerted by each of the drugs separately: treatment with thioacetamide plus ethanol determined an early decrease in dolichol in Kupffer cells (about 13% and 50% after 2 and 4 months, respectively). Moreover, after liver damage, a load of vitamin A evidenced altered percentages of the form of dolichol with eighteen isoprene units; these percentages were modified by all treatments in all cell types. The results confirm that dolichol is the preferred target of oxidative stress and suggest a relationship between dolichol and retinol metabolisms, and a possible new role of dolichol precursors, of prenyltransferases and of retinol metabolites in liver pathology.     

瘦素在肝脏疾病中的作用和分子机制

瘦素是由ob基因编码产生的分子量为 16ku的物质 ,以游离或结合形式存在于血浆中。瘦素在多种肝脏疾病的病理机制中发挥重要的作用 ,其通过与细胞表面的瘦素受体(leptinreceptor,OB R)结合而激活肝星状细胞及Kuffer细胞 ,从而调节脂肪肝、酒精性及非酒精性肝硬化中炎症因子及纤维化因子的表达。该文就瘦素的生物学活性 ,瘦素在脂肪肝、肝炎、肝硬化等多种肝脏疾病中的作用及其可能的分子机制作一综述     

Rebaudioside A administration prevents experimental liver fibrosis: an in vivo and in vitro study of the mechanisms of action involved

Rebaudioside A (Reb A) is a diterpenoid isolated from the leaves of Stevia rebaudiana (Bertoni) that has been shown to possess pharmacological activity, including anti‐inflammatory and antioxidant properties. However, the ability of Reb A to prevent liver injury has not been evaluated. Therefore, we aimed to study the potential of Reb A (20 mg/kg; two times daily intraperitoneally) to prevent liver injury induced by thioacetamide (TAA) administration (200 mg/kg; three times per week intraperitoneally). In addition, cocultures were incubated with either lipopolysaccharide or ethanol. Antifibrotic, antioxidant and immunological responses were evaluated. Chronic TAA administration produced considerable liver damage and distorted the liver parenchyma with the presence of prominent thick bands of collagen. In addition, TAA upregulated the expression of α‐smooth muscle actin, transforming growth factor‐β1, metalloproteinases 9, 2 and 13, and nuclear factor kappaB and downregulated nuclear erythroid factor 2. Reb A administration prevented all of these changes. In cocultured cells, Reb A prevented the upregulation of genes implicated in fibrotic and inflammatory processes when cells were exposed to ethanol and lipopolysaccharide. Altogether, our results suggest that Reb A prevents liver damage by blocking oxidative processes via upregulation of nuclear erythroid factor 2, exerts immunomodulatory effects by downregulating the nuclear factor‐κB system and acts as an antifibrotic agent by maintaining collagen content.     

Oridonin ameliorates carbon tetrachloride-induced liver fibrosis in mice through inhibition of the NLRP3 inflammasome

Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) and accumulation of the extracellular matrix. There are limitations in the current therapies for liver fibrosis. Recently, oridonin was shown to induce apoptosis in HSCs. Thus, we aimed to determine the roles of oridonin in chronic liver injury and fibrosis. Liver fibrosis was induced by CCl₄ in mice injected intraperitoneally with oridonin for 6 weeks. The administration of oridonin significantly attenuated liver injury and reduced ALT levels. In addition, Sirius Red staining and the expression of α-smooth muscle actin (α-SMA) were significantly reduced by oridonin in murine livers with fibrosis. The expression of NLRP3, caspase-1, and IL-1β was downregulated with the oridonin treatment. Furthermore, the expression of F4/80 in liver tissues was also decreased by oridonin treatment. These results demonstrate that oridonin ameliorates chronic liver injury and fibrosis. Mechanically, oridonin may inhibit the activity of the NLRP3 inflammasome and inflammation in the liver. These results highlight the potential of oridonin as a therapeutic agent for liver fibrosis.     

β-Arrestin2 deficiency attenuates oxidative stress in mouse hepatic fibrosis through modulation of NOX4

Hepatic fibrosis is a disease characterized by excessive deposition of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is responsible for most of ECM production. Oxidative stress and reactive oxygen species (ROS) may be important factors leading to liver fibrosis. NADPH oxidase 4 (NOX4) is the main source of ROS in hepatic fibrosis, but the mechanism by which NOX4 regulates oxidative stress is not fully understood. β-Arrestin2 is a multifunctional scaffold protein that regulates receptor endocytosis, signaling and trafficking. In this study, we investigated whether β-arrestin2 regulated oxidative stress in hepatic fibrosis. Both β-arrestin2 knockout (Arrb2 KO) mice and wild-type mice were intraperitoneally injected with carbon tetrachloride (CCl₄) to induce hepatic fibrosis. Arrb2 KO mice showed significantly attenuated liver fibrosis, decreased ROS levels and NOX4 expression, and reduced collagen levels in their livers. In vitro, NOX4 knockdown significantly inhibited ROS production, and decreased expression of alpha-smooth muscle actin in angiotensin II-stimulated human HSC cell line LX-2. Through overexpression or depletion of β-arrestin2 in LX-2 cells, we revealed that decreased β-arrestin2 inhibited ROS levels and NOX4 expression, and reduced collagen production; it also inhibited activation of ERK and JNK signaling pathways. These results demonstrate that β-arrestin2 deficiency protects against liver fibrosis by downregulating ROS production through NOX4. This effect appears to be mediated by ERK and JNK signaling pathways. Thus, targeted inhibition of β-arrestin2 might reduce oxidative stress and inhibit the progression of liver fibrosis.     

Effect of S-adenosyl-L-methionine on the activation, proliferation and contraction of hepatic stellate cells

Inhibition of hepatic stellate cell activation is an important clinical aspect for the control of liver inflammation, fibrosis and cirrhosis. S-adenosyl-L-methionine (SAM), an intermediate product of L-methionine metabolism, is a precursor of glutathione and an endogenous methyl donor. Although the hepato-protective action of SAM has been reported in several animal models, the effect of SAM on the function of hepatic stellate cells has not been elucidated. Using a primary-culture model of hepatic stellate cells, we found that SAM blunts the activation process as indicated by the suppression of expression of collagen alpha1(I) and smooth muscle alpha-actin. SAM also hampers the DNA synthesis of hepatic stellate cells stimulated with a dimer of platelet-derived growth factor-B via the inhibition of phosphorylation of PDGF receptor-beta and down-stream signaling pathways. SAM additionally inhibits the contraction of hepatic stellate cells by disturbing the formation of F-actin stress fibers and phosphorylated myosin light chains. Thus, SAM regulates the activation of hepatic stellate cells and may clinically contribute to therapy targeted at human liver fibrosis.     

Alcohol consumption and hepatic fibrosis affect the fatty acid composition of red blood cells and their susceptibility to lipid peroxidation

Erythrocytes from alcoholics with and without liver cirrhosis and from rats treated either with ethanol or thioacetamide, the latter treatment resulting in hepatic fibrosis, were analysed for their membrane fatty acid composition and their susceptibility to lipid peroxidation. Red cells containing less arachidonic acid than controls, as found in alcoholics with liver cirrhosis, were less susceptible to lipid peroxidation than controls. This observation was confirmed by experiments with rat erythrocytes obtained from animals with hepatic fibrosis. However, red cells containing less linoleic acid than controls, as found in alcoholics without liver cirrhosis, exhibited a normal degree of lipid peroxidation upon oxidant stress induced by hydrogen peroxide. The results demonstrated that in red cells only fatty acids with four double bonds seem to be involved in membrane peroxidation reactions under the condition chosen. This observation might be of relevance for in vivo aging of red cells.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Systematic review: hepatic fibrosis - regression with therapy

Background Hepatic fibrosis occurs in response to chronic liver injury, regardless of the cause. An impressive amount of knowledge concerning the pathogenesis and treatment of liver fibrosis has emerged over the past few years. The hallmark of this event is the activation of the hepatic stellate cell. The latter event causes accumulation of extracellular matrix and formation of scar, leading to deterioration in hepatic function. Aim To assess chronic liver injury, many invasive and non‐invasive methods have been suggested. Methods Although transient elastography, image analysis of fractal geometry and fibrotest with actitest have been used in clinical practice, liver biopsy remains the recommended choice, especially when histological staging of fibrosis or response to treatment is needed. Conclusions The recent advances in anti‐viral therapy have resulted in many reports on fibrosis and even on cirrhosis regression, especially early and in young people. A number of new agents have been suggested for the treatment of fibrosis, with promising results in animals; however, their efficacy in humans remains to be elucidated. The investigation of heterogeneity and plasticity of hepatic stellate cells is a topic of scientific interest and may result in improvements in patient management.     

Isochlorogenic acid A attenuates progression of liver fibrosis through regulating HMGB1/TLR4/NF-κB signaling pathways

OBJECTIVE Liver fibrosis is a chronic damage process related to the further progression of hepatic cirrhosis and has yet no truly effective treatment is available. This study aimed to investigate the effects of isochlorogenic acid A(ICQA) on liver fibrosis induced by carbon tetrachloride(CCl4) and clarify the underlying mechanism. METHODS Rats were treated with CCl4 for eight weeks in order to induce liver fibrosis and simultaneously orally administered with ICQA(10, 20 and 40 mg·kg~(-1)). RESULTS ICQA had significant protective effect on liver injury, inflammation, and fibrosis in rats. Meanwhile, ICQA prevented the activation of hepatic stellate cells(HSC) as indicated by inhibiting the overexpression of a-smooth muscle actin(a-SMA). In addition, reduced fibrosis was found to be associated with decreased protein expression of high-mobility group box 1(HMGB1) and toll like receptor(TLR)4. Moreover, ICQA supressed the cytoplasmic translocation of HMGB1 in rat liver. Further investigations indicated that ICQA treatment significantly attenuated nuclear translocation of the nuclear factor-κB(NF-κB) p65 and inhibited degradation of IkB a expression in the liver of rats with liver fibrosis. CONCLUSION ICQA has hepatoprotective and anti-fibrotic effects in rats with liver fibrosis through modulating the HMGB1/TLR4/NF-κB signaling pathways.     

Protective effect of isochlorogenic acid B on liver fibrosis in non‐alcoholic steatohepatitis of mice

Liver fibrosis is a common symptom of non‐alcoholic steatohepatitis (NASH) and a worldwide clinical issue. The miR‐122/HIF‐1α signalling pathway is believed to play an important role in the genesis of progressive fibrosis. Isochlorogenic acid B (ICAB), naturally isolated from Laggera alata, is verified to have antioxidative and hepatoprotective properties. The aim of this study was to investigate the effect of ICAB on liver fibrosis in NASH and its potential protective mechanisms. NASH was induced in a mouse model with a methionine‐ and choline‐deficient (MCD) diet for 4 weeks, and ICAB was orally administered every day at three doses (5, 10 and 20 mg/kg). Pathological results indicated that ICAB significantly improved the pathological lesions of liver fibrosis. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hepatic hydroxyproline (Hyp), cholesterol (CHO) and triglyceride (TG) were also significantly decreased by ICAB. In addition, ICAB inhibited hepatic stellate cells (HSCs) activation and the expressions of hepatic genes involved in liver fibrosis including LOX, TGF‐β1, MCP‐1, COL1α1 and TIMP‐1. ICAB also attenuated liver oxidative stress through Nrf2 signalling pathway. What is more, the decreased levels of miR‐122 and over‐expression of hepatic HIF‐1α could be reversed by ICAB treatment. These results simultaneously confirmed that ICAB had a significant protective effect on fibrosis in NASH by inhibiting oxidative stress via Nrf2 and suppressing multiple profibrogenic factors through miR‐122/HIF‐1α signalling pathway.     

肝硬化患者肝组织中IGFBPrP1与TGF-β_1/Smad3信号通路的相关性研究

目的探讨肝硬化患者肝组织中胰岛素样生长因子结合蛋白相关蛋白1(IGFBPrP1)与转化生长因子(TGF)-β1/Smad3信号通路的可能关系。方法31例肝组织蜡块来自2002年1月至2007年12月山西医科大学第一医院病理科。其中肝硬化组16例,对照组15例为手术切除肝血管瘤外正常肝组织。蜡块5μm厚连续切片,行苏木素-伊红(HE)及Masson染色,免疫组织化学染色观察肝组织中IGFBPrP1、TGF-β1及Smad3的表达,收集肝硬化组16例患者的血清透明质酸(HA)实验室检查结果。结果HE及Masson染色结果显示对照组肝细胞形态正常、排列整齐,肝小叶结构完整;肝硬化组小叶结构被破坏,形成假小叶。肝硬化患者肝组织IGF-BPrP1、TGF-β1、Smad3的表达比正常对照组明显升高,且肝组织IGFBPrP1的表达分别与肝组织胶原纤维面积、TGF-β1、Smad3及血清HA呈正相关。结论IGFBPrP1在肝硬化的发生发展中起重要作用,且该作用可能与TGF-β1/Smad3信号转导通路有关。     

Modification of chemokine pathways and immune cell infiltration as a novel therapeutic approach in liver inflammation and fibrosis

Despite increasing knowledge about molecular pathways in pathogenesis of chronic liver disease, selective therapeutic options are scarce, especially in advanced diseases characterized by scarring of the liver (termed fibrosis) or even complete cirrhosis. Sustained hepatic inflammation as a result to various types of injury (e.g., hepatitis C, nonalcoholic steatohepatitis) is generally accepted to represent the key prerequisite for fibrogenesis. Liver inflammation is characterized by an activation of distinct chemokine pathways in the liver and the circulation allowing distinct immune cell populations to enter the liver via sinusoids and postsinusoidal venules. Recent investigations have shed light on the intimate interactions between the fibrogenic hepatic stellate cell (HSC) and infiltrating immune cells, which fundamentally drive liver scarring. Experimental fibrosis and inflammation models have demonstrated that disruption of chemokine pathways such as CCL2 (MCP-1) or its receptor CCR2, CCL5 (RANTES) or CCR1 / CCR5 and others may efficiently prevent collagen deposition, by targeting monocytes and macrophages, T-cell populations or NKT cells. However, immigration of certain mononuclear cells may even be beneficial in the course of fibrosis. Infiltrating NK cells and monocyte-derived macrophage subsets can promote resolution of extracellular matrix. This emphasizes that hepatic fibrosis is not a unidirectional process, but can be reverted up to a certain point. The present review aims at summarizing the contribution of immune cell infiltration as well as related chemokine systems to experimental liver fibrosis and will discuss possible therapeutic applications in humans, with a special emphasis on the monocyte/macrophage lineage and their related chemokine pathways.     

Enhancement by estradiol 3-benzoate in thioacetamide-induced liver cirrhosis of rats.

As part of an investigation on the role of estrogen in liver disease, we tested the effects of estradiol-3-benzoate (EB) in the thioacetamide (TAA)-induced rat liver cirrhosis model. Male F344 rats (n = 100) were divided into six groups. Animals of groups 1-4 received TAA (0.03% in drinking water) for 12 weeks, and groups 5 and 6 served as controls without TAA. For the exposure period, EB pellets were implanted subcutaneously to give doses of 0 (groups 1 and 5), 1 (group 2), 10 (group 3), and 100 mug (groups 4 and 6) simultaneously. All animals were sacrificed at week 12. Significant increase of liver cirrhosis, liver weight, collagen content, and lipid peroxidation in the livers was evident in groups 3 and 4 (p < 0.05) compared with group 1. Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was significantly elevated in group 4 (p < 0.01), along with expression of alpha-smooth muscle actin (alpha-SMA) and stellate cell activation-associated protein (STAP), as determined by RT-PCR analysis (p < 0.01). However, there were no differences in liver weight, collagen content, lipid peroxidation, 8-OHdG formation, and alpha-SMA and STAP mRNA expression between groups 5 and 6. We conclude that EB treatment enhances TAA-induced cirrhosis, associated with increase of oxidative stress and activation of hepatic stellate cells.