Role of melatonin in the oxidative damage prevention at different times of hepatic regeneration

The process of regenerating liver is the result of a balance between stimulating factors and inhibitors of hepatocyte proliferation. Melatonin and its metabolites have been found to protect tissues against oxidative damage generated by a variety of toxic agents and metabolic processes. Furthermore, studies in liver of rats showed a decrease in the liver mitochondrial hydroxylation of drugs returning to the normal state after the administration of antioxidants. This study was designed to determine, in experimental animals, whether the administration of an antioxidant agent such as melatonin could prevent cells events leading to tissue injury and hepatic dysfunction after partial hepatectomy (PH). Biliary flow (BF), oxidative stress in hepatic tissue and Na⁺/K⁺ATPase activities in whole plasma membrane were determined. PH decreased the Na⁺/K⁺ATPase activity. PH significantly reduced the BF (36%) and promoted oxidative stress with an increase of lipoperoxidation and decrease of glutathione peroxidase and catalase activities. Treatment with melatonin prevented the decrease of BF in rats with hepatectomy and normalized the Na⁺/K⁺ATPase activity. Moreover, melatonin markedly attenuated oxidative stress produced by PH. This may be the results of the higher efficacy of melatonin in scavenging various free radicals and also because of its ability in stimulating the antioxidant enzymes. We suggest that oxidative stress before and during liver regeneration has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage and the impairment in liver transport function induced by PH and that melatonin could modulate the degree of oxidative stress and through it prevent the alterations in liver function carrier. Copyright © 2012 John Wiley & Sons, Ltd.     

Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis

Transforming growth factor (TGF) β(1) plays a critical role in liver fibrosis. Previous studies demonstrated embryonic liver fodrin (ELF), a β-spectrin was involved in TGF-β/Smad signalling pathway as Smad3/4 adaptor. Here we investigate the role of ELF in pathogenesis of liver cirrhosis. In carbon tetrachloride (CCl(4))-induced mice model of liver cirrhosis, ELF is up-regulated in activated hepatic stellate cells (HSCs), and down-regulated in regenerative hepatocytes of cirrhotic nodules. In activated HSCs in vitro, reduction of ELF expression mediated by siRNA leads to the inhibition of HSC activation and procollagen I expression. BrdU assay demonstrates that down-regulation of ELF expression does not inhibit proliferation of activated HSCs in vitro. Immunostaining of cytokeratin 19 and Ki67 indicates that regenerative hepatocytes in cirrhotic liver are derived from hepatic progenitor cells (HPC). Further study reveals that HPC expansion occurs as an initial phase, before the reduction of ELF expression in regenerative hepatocytes. Regenerative hepatocytes in cirrhotic liver show the change in proliferative activity and expression pattern of proteins involved in G1/S transition, which suggests the deregulation of cell cycle in regenerative hepatocytes. Finally, we find that ELF participates in TGF-β/Smad signal in activated HSCs and hepatocytes through regulating the localization of Smad3/4. These data reveal that ELF is involved in HSC activation and the formation of regenerative nodules derived from HPC in cirrhotic liver.     

The effect of melatonin on glycoprotein levels and oxidative liver injury in experimental diabetes

In this present study, the duration of melatonin (Mel) administered to diabetic rats was prolonged so as to examine its effects on the biochemical liver parameters of diabetic rats. In the experiment, Male Sprague Dawley rats were divided randomly into five groups; the control, diabetic + Mel, diabetic, diabetic + insulin, and diabetic + Mel + insulin. Diabetes mellitus was induced by administration of a single dose of streptozotocin (60 mg/kg) intraperitoneally and rats were given vehicle as a solvent for Mel every day for 12 weeks. In the diabetic + Mel group, diabetic rats were administered Mel (10 mg/kg/day) for 12 weeks to treat diabetes. The diabetic + insulin group were diabetic rats given insulin (6 U/kg) subcutaneously for 12 weeks. The diabetic + Mel + insulin rats received insulin and Mel at the same dose and time. At the end of the experiment, the animals were decapitated and liver tissues were taken. The protective effect of Mel on liver tissue of diabetic rats was investigated, total antioxidant status, total oxidant status, reactive oxygen species, oxidative stress index, adenosine deaminase, xanthine oxidase, paraoxonase 1, sodium/potassium ATPase, myeloperoxidase, γ-glutamyl transferase, sorbitol dehydrogenase, tumor necrosis factor-alpha, homocysteine, nitric oxide, glucose-6-phosphate dehydrogenase, and glycoprotein levels were determined in liver tissues. Treatment with Mel and/or insulin has been found to have a protective effect on biochemical parameters. The results showed that administration of Mel to diabetic rats prevented the distortion of the studied biochemical parameters of liver tissues.     

Corrections by melatonin of liver mitochondrial disorders under diabetes and acute intoxication in rats

The aim of the present work was to investigate the mechanisms of oxidative damage of the liver mitochondria under diabetes and intoxication in rats as well as to evaluate the possibility of corrections of mitochondrial disorders by pharmacological doses of melatonin. The experimental (30 days) streptozotocin‐induced diabetes mellitus caused a significant damage of the respiratory activity in rat liver mitochondria. In the case of succinate as a respiratory substrate, the ADP‐stimulated respiration rate V₃ considerably decreased (by 25%, p < 0·05) as well as the acceptor control ratio (ACR) V₃/V₂ markedly diminished (by 25%, p < 0·01). We observed a decrease of the ADP‐stimulated respiration rate V₃ by 35% (p < 0·05), with glutamate as substrate. In this case, ACR also decreased (by 20%, p < 0·05). Surprisingly, the phosphorylation coefficient ADP/O did not change under diabetic liver damage. Acute rat carbon tetrachloride‐induced intoxication resulted in considerable decrease of the phosphorylation coefficient because of uncoupling of the oxidation and phosphorylation processes in the liver mitochondria. The melatonin administration during diabetes (10 mg·kg^‐1 body weight, 30 days, daily) showed a considerable protective effect on the liver mitochondrial function, reversing the decreased respiration rate V₃ and the diminished ACR to the control values both for succinate‐dependent respiration and for glutamate‐dependent respiration. The melatonin administration to intoxicated animals (10 mg·kg⁻¹ body weight, three times) partially increased the rate of succinate‐dependent respiration coupled with phosphorylation. The impairment of mitochondrial respiratory plays a key role in the development of liver injury under diabetes and intoxication. Melatonin might be considered as an effector that regulates the mitochondrial function under diabetes. Copyright © 2011 John Wiley & Sons, Ltd.     

CXCL6‐EGFR‐induced Kupffer cells secrete TGF‐β1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C‐MYC/EZH2 pathway in liver fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP‐2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF‐β in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC‐T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF‐β showed increased expression of α‐SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C‐MYC/EZH2 pathway by enhancing the SMAD3‐BRD4 interaction and promoting direct binding of BRD4 to the C‐MYC promoter and CMY‐C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl₄‐induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF‐β in serum and the expression of α‐SMA, SMAD3, BRD4, C‐MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF‐β by KCs and thereby activating HSCs.     

HSCs play a distinct role in different phases of oval cell‐mediated liver regeneration

Hepatic stem cell niche plays an important role in hepatic oval cell‐mediated liver regeneration. As a component of hepatic stem cell niche, the role of hepatic stellate cells (HSCs) in oval cell proliferation needs further studies. In the present study, we isolated HSCs from rats at indicated time point after partial hepatectomy (PH) in 2‐acetylaminofluorene/PH oval cell proliferation model. Conditional medium (CM) from HSCs were collected to detect their effects on proliferation and the mitogen‐activated protein kinase pathway activation of two oval cell lines. We found that CM collected from HSCs at early phase of liver regeneration (4 and 9 days group) contained high levels of hepatocyte growth factor (HGF) and stimulated oval cell proliferation via extracellular signal‐regulated kinase and p38 pathway. CM collected from HSCs at terminal phase of liver regeneration (12 and 15 days group) contained high levels of transforming growth factor (TGF)‐β1, which suppressed DNA synthesis of oval cells. The shift between these two distinct effects depended on the balance between HGF and TGF‐β1 secreted by HSCs. Our study demonstrated that HSCs acted as a positive regulator at the early phase and a negative regulator at the terminal phase of the oval cell‐mediated liver regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

肝纤维化的表观遗传学研究进展

肝纤维化是常见的慢性进行性肝病,是慢性肝病发展到肝硬化阶段的必经阶段,却有逆转的可能性。肝纤维化的中心环节是肝星状细胞的激活。关于肝星状细胞的激活,除了经典的肝纤维化通路,不断有新的通路和机制出现,包括自噬、内质网应激、氧化应激、胆固醇代谢和表观遗传,这些都揭示了肝星状细胞的活化机制。表观遗传包括DNA甲基化、组蛋白修饰和调节性非编码RNAs,这些机制也参与调节肝星状细胞活化和肝纤维化发生,对表观遗传和肝纤维化治疗之间的关系研究具有重要意义。     

Disruption of myofibroblastic Notch signaling attenuates liver fibrosis by modulating fibrosis progression and regression

The phenotypic transformation of hepatic myofibroblasts (MFs) is involved in the whole process of the progression and regression of liver fibrosis. Notch signaling has been demonstrated to modulate the fibrosis. In this study, we found that Notch signaling in MFs was overactivated and suppressed with the progression and regression of hepatic fibrosis respectively, by detecting Notch signaling readouts in MFs. Moreover, we inactivated Notch signaling specifically in MFs with Sm22α^CreER-RBPj^flox/flox mice (RBPj^MF-KO), and identified that MFs-specific down-regulation of Notch signaling significantly alleviated CCl₄-induced liver fibrosis during the progression and regression. During the progression of liver fibrosis, MFs-specific blockade of Notch signaling inhibited the activation of HSCs to MFs and increases the expression of MMPs to reduce the deposition of ECM. During the regression of fibrosis, blocking Notch signaling in MFs increased the expression of HGF to promote proliferation in hepatocytes and up-regulated the expression of pro-apoptotic factors, Ngfr and Septin4, to induce apoptosis of MFs, thereby accelerating the reversal of fibrosis. Collectively, the MFs-specific disruption of Notch signaling attenuates liver fibrosis by modulating fibrosis progression and regression, which suggests a promising therapeutic strategy for liver fibrosis.     

IRE1α/NOX4 signaling pathway mediates ROS‐dependent activation of hepatic stellate cells in NaAsO2‐induced liver fibrosis

Liver fibrosis is a severe health problem worldwide, and it is characterized by the activation of hepatic stellate cells (HSCs) and excessive deposition of collagen. Prolonged arsenic exposure can induce HSCs activation and liver fibrosis. In the present study, the results showed that chronic NaAsO₂ ingestion could result in liver fibrosis and oxidative stress in Sprague–Dawley rats, along with representative collagen deposition and HSCs activation. In addition, the inositol‐requiring enzyme 1α (IRE1α)–endoplasmic reticulum (ER)‐stress pathway was activated, and the activity of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) was upregulated in rat livers. Simultaneously, the excessive production of reactive oxygen species (ROS) could induce HSCs activation, and NOX4 played an important role in generating ROS in vitro. Moreover, ER stress occurred with HSCs activation at the same time under NaAsO₂ exposure, and during ER stress, the IRE1α pathway was responsible for NOX4 activation. Therefore, inhibition of IRE1α activation could attenuate the HSCs activation induced by NaAsO₂. In conclusion, the present study manifested that inorganic arsenic exposure could activate HSCs through IRE1α/NOX4‐mediated ROS generation.     

Intercellular communication among liver cells in the perisinusoidal space of the injured liver: Pathophysiology and therapeutic directions

Hepatic stellate cells (HSCs) in the perisinusoidal space are surrounded by hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and other resident immune cells. In the normal liver, HSCs communicate with these cells to maintain normal liver functions. However, after chronic liver injury, injured hepatocytes release several proinflammatory mediators, reactive oxygen species, and damage-associated molecular patterns into the perisinusoidal space. Consequently, such alteration activates quiescent HSCs to acquire a myofibroblast-like phenotype and express high amounts of transforming growth factor-β1, angiopoietins, vascular endothelial growth factors, interleukins 6 and 8, fibril forming collagens, laminin, and E-cadherin. These phenotypic and functional transdifferentiation lead to hepatic fibrosis with a typical abnormal extracellular matrix synthesis and disorganization of the perisinusoidal space of the injured liver. Those changes provide a favorable environment that regulates tumor cell proliferation, migration, adhesion, and survival in the perisinusoidal space. Such tumor cells by releasing transforming growth factor-β1 and other cytokines, will, in turn, activate and deeply interact with HSCs via a bidirectional loop. Furthermore, hepatocellular carcinoma-derived mediators convert HSCs and macrophages into protumorigenic cell populations. Thus, the perisinusoidal space serves as a critical hub for activating HSCs and their interactions with other cell types, which cause a variety of liver diseases such as hepatic inflammation, fibrosis, cirrhosis, and their complications, such as portal hypertension and hepatocellular carcinoma. Therefore, targeting the crosstalk between activated HSCs and tumor cells/immune cells in the tumor microenvironment may also support a promising therapeutic strategy.     

MiR‐122 modification enhances the therapeutic efficacy of adipose tissue‐derived mesenchymal stem cells against liver fibrosis

Mesenchymal stem cell (MSC) transplantation alone may be insufficient for treatment of liver fibrosis because of complicated histopathological changes in the liver. Given that miR‐122 plays an essential role in liver fibrosis by negatively regulating the proliferation and transactivation of hepatic stellate cells (HSCs), this study investigated whether miR‐122 modification can improve the therapeutic efficacy of adipose tissue‐derived MSCs in treating liver fibrosis. MiR‐122‐modified AMSCs (AMSC‐122) were constructed through lentivirus‐mediated transfer of pre‐miR‐122. MiR‐122‐modified AMSCs expressed high level of miR‐122, while they retained their phenotype and differentiation potential as naïve AMSCs. AMSC‐122 more effectively suppressed the proliferation of and collagen maturation in HSCs than scramble miRNA‐modified AMSCs. In addition, AMSC‐derived exosomes mediated the miR‐122 communication between AMSCs and HSCs, further affecting the expression levels of miR‐122 target genes, such as insulin‐like growth factor receptor 1 (IGF1R), Cyclin G(1) (CCNG1) and prolyl‐4‐hydroxylase α1 (P4HA1), which are involved in proliferation of and collagen maturation in HSCs. Moreover, miR‐122 modification enhanced the therapeutic efficacy of AMSCs in the treatment of carbon tetrachloride (CCl₄)‐induced liver fibrosis by suppressing the activation of HSCs and alleviating collagen deposition. Results demonstrate that miR‐122 modification improves the therapeutic efficacy of AMSCs through exosome‐mediated miR‐122 communication; thus, miR‐122 modification is a new potential strategy for treatment of liver fibrosis.     

补骨脂素对肝星状细胞(HSC-T6)增殖、氧化应激及Ⅰ型胶原分泌的影响

通过研究植物雌激素香豆素补骨脂素对体外培养的大鼠肝星状细胞HSC-T6增殖及相关因子表达的影响,为补骨脂素治疗肝纤维化提供实验依据。常规培养肝星状细胞HSC-T6,采用0.1 mmol/L的H2O2制造HSC-T6氧化应激的模型。分别用MTT法检测肝星状细胞增殖、放射免疫法检测细胞上清液中超氧化物歧化酶(SOD),丙二醛(MDA),还原性谷胱甘肽(GSH),谷胱甘肽过氧化物酶(GSH-Px)的活性和含量,ELISA法测定Ⅰ型胶原的分泌。结果表明:与正常对照组组比较,补骨脂素在浓度为10μmol/L,1μmol/L,0.1μmol/L,均呈现出抑制HSC-T6增殖的作用(P<0.05),且最佳作用时间为48 h(P<0.05);与模型组比较,补骨脂素各个浓度组能够提高SOD和GSH-Px的活性(P<0.05),并降低细胞上清液中MDA和GSH的含量(P<0.05);与模型组比较,补骨脂素各个浓度组在作用48 h后,细胞上清液中的Ⅰ型胶原的表达量均降低(P<0.05)。因此,作为植物雌激素的一种,补骨脂素能有效的抑制HSC-T6的增殖及抗HSC-T6氧化应激,很可能成为雌激素的替代品在治疗肝纤维化中。     

Comparison of the effects of melatonin and pentoxifylline on carbon tetrachloride-induced liver toxicity in mice

The purpose of the study was to determine whether along and in combination melatonin (MLT) and pentoxlfylline (PTX) exerted beneficial effects on histopathological changes and changes in oxidant and antioxidant systems in liver caused by CCl₄-induced liver toxicity in mice. Mice were randomly divided into six groups: control, olive oil, toxicity, MLT, PTX, PTX+MLT. MLT 10 mg/kg/day, PTX 50 mg/kg/day, and the same individual doses in MLT+PTX combination were given intraperitoneally to mice for 7 day. CCl₄ 0.8 mg/kg/day was administered on the 4th, 5th, and 6th days of therapy in all groups except the control and olive oil groups. In the toxicity group, increased concentrations of malondialdehyde (MDA) and lipid hydroperoxides (LOOH) and decreased glutathione peroxidase (GSH-Px) and catalase (CAT) activities were found compared to the control and olive oil groups (p < 0.05). Compared to the toxicity group, both the PTX group and the PTX+MLT group had decreased MDA and LOOH levels, whereas MLT reduced only LOOH levels (p < 0.01). MLT, PTX and MLT+PTX increased the GSH-Px and CAT activities compared to the toxicity group (p < 0.05). MLT increased CAT activity compared to PTX and MLT+PTX (p < 0.05). Superoxide dismutase enzyme activity did not change in any group (p < 0.05). Histopatholically, ballooning, degeneration, apoptosis, and bridging necrosis were seen in the toxicity group. MLT, PTX and MLT+PTX decreased the apoptosis and bridging necrosis (p < 0.01), and PTX and MLT+PTX decreased balloon degeneration compared to the toxicity group (p < 0.01). These results indicate that administration of PTX and MLT alone and in combination before onset of liver toxicity might prevent the oxidative damage by reducing oxidative stress and increasing antioxidant enzyme levels.     

Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells

BackgroundIntravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs.MethodsHuman AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects.ResultsHSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen–G1, a hAEC-derived factor, significantly decreased TGF-β1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL.ConclusionsHuman AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.     

Cells in the liver microenvironment regulate the process of liver metastasis

The research of liver metastasis is a developing field. The ability of tumor cells to invade the liver depends on the complicated interactions between metastatic cells and local subpopulations in the liver (including Kupffer cells, hepatic stellate cells, liver sinusoidal endothelial cells, and immune-related cells). These interactions are mainly mediated by intercellular adhesion and the release of cytokines. Cell populations in the liver microenvironment can play a dual role in the progression of liver metastasis through different mechanisms. At the same time, we can see the participation of liver parenchymal cells and nonparenchymal cells in the process of liver metastasis of different tumors. Therefore, the purpose of this article is to summarize the relationship between cellular components of liver microenvironment and metastasis and emphasize the importance of different cells in the occurrence or potential regression of liver metastasis.     

Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and is involved in pathological angiogenesis associated with chronic liver diseases. However, the precise mechanisms underlying PlGF signalling contributing to liver fibrosis and angiogenesis remain largely unexplored. This study aimed to assess the effect of reducing PlGF expression using small interfering RNA (siRNA) on experimental liver fibrosis and angiogenesis, and to elucidate the underlying molecular mechanisms. Fibrosis was induced in mice by carbon tetrachloride (CCl₄) for 8 weeks, and mice were treated with PlGF siRNA or non‐targeting control siRNA starting two weeks after initiating CCl₄ injections. The results showed that PlGF was highly expressed in cirrhotic human and mice livers; which mainly distributed in activated hepatic stellate cells (HSCs). PlGF silencing robustly reduced liver inflammation, fibrosis, intrahepatic macrophage recruitment, and inhibited the activation of HSCs in vivo. Moreover, PlGF siRNA‐treated fibrotic mice showed diminished hepatic microvessel density and angiogenic factors, such as hypoxia‐inducible factor‐1α (HIF‐1α), VEGF and VEGF receptor‐1. Moreover, down‐regulation of PlGF with siRNA in HSCs inhibited the activation and proliferation of HSCs. Mechanistically, overexpression of PlGF in activated HSCs was induced by hypoxia dependent on HIF‐1α, and PlGF induces HSC activation and proliferation via activation the phosphatidylinositol 3‐kinase (PI3K)/Akt signalling pathways. These findings indicate that PlGF plays an important role in liver fibrosis‐associated angiogenesis and that blockage of PlGF could be an effective strategy for chronic liver disease.     

Ursolic acid ameliorates CCl4‐induced liver fibrosis through the NOXs/ROS pathway

Liver fibrosis is a reversible wound‐healing response that occurs after liver injury. NADPH oxidases (NOXs) and reactive oxygen species (ROS) which are expressed in hepatocytes (HCs), hepatic stellate cells (HSCs), and Kupffer cells (KCs) play an important role in the development of hepatic fibrosis. In in vitro studies, we had shown that ursolic acid (UA) could reverse liver fibrosis by inhibiting the activation of NOX‐mediated fibrotic signaling networks in HSCs. In this study, we verified that UA could alleviate CCl4‐induced liver fibrosis by reducing the expression of NOXs/ROS in HCs, HSCs, KCs. Meanwhile, the phagocytic index α and clearance index K which represent phagocytosis of KCs were unchanged. Together, all our data demonstrated that UA induced the proliferation of HCs, promoted apoptosis in HSCs, and prevented activation of KCs in vivo by reducing the expression of NOXs/ROS in HCs, HSCs, KCs. Besides, UA had no effect on the host defense function.