Scopoletin and umbelliferone protect hepatocytes against palmitate- and bile acid-induced cell death by reducing endoplasmic reticulum stress and oxidative stress

BACKGROUND: The number of patients with non-alcoholic fatty liver disease (NAFLD) is rapidly increasing due to the growing epidemic of obesity. Non-alcoholic steatohepatitis (NASH), the inflammatory stage of NAFLD, is characterized by lipid accumulation in hepatocytes, chronic inflammation and hepatocyte cell death. Scopoletin and umbelliferone are coumarin-like molecules and have antioxidant, anti-cancer and anti-inflammatory effects. Cytoprotective effects of these compounds have not been described in hepatocytes and the mechanisms of the beneficial effects of scopoletin and umbelliferone are unknown. AIM: To investigate whether scopoletin and/or umbelliferone protect hepatocytes against palmitate-induced cell death. For comparison, we also tested the cytoprotective effect of scopoletin and umbelliferone against bile acid-induced cell death. METHODS: Primary rat hepatocytes were exposed to palmitate (1 mmol/L) or the hydrophobic bile acid glycochenodeoxycholic acid (GCDCA; 50 μmol/L). Apoptosis was assessed by caspase-3 activity assay, necrosis by Sytox green assay, mRNA levels by qPCR, protein levels by Western blot and production of reactive oxygen species (ROS) by fluorescence assay. RESULTS: Both scopoletin and umbelliferone protected against palmitate and GCDCA-induced cell death. Both palmitate and GCDCA induced the expression of ER stress markers. Scopoletin and umbelliferone decreased palmitate- and GCDCA-induced expression of ER stress markers, phosphorylation of the cell death signaling intermediate JNK as well as ROS production. CONCLUSION: Scopoletin and umbelliferone protect against palmitate and bile acid-induced cell death of hepatocytes by inhibition of ER stress and ROS generation and decreasing phosphorylation of JNK. Scopoletin and umbelliferone may hold promise as a therapeutic modality for the treatment of NAFLD

Role of Oxidative Stress in the Pathogenesis of Non-Alcoholic Fatty Liver Disease:Implications for Prevention and Therapy

Oxidative stress (OxS) is considered a major factor in the pathophysiology of inflammatory chronic liver diseases, including non-alcoholic liver disease (NAFLD). Chronic impairment of lipid metabolism is closely related to alterations of the oxidant/antioxidant balance, which affect metabolism-related organelles, leading to cellular lipotoxicity, lipid peroxidation, chronic endoplasmic reticulum (ER) stress, and mitochondrial dysfunction. Increased OxS also triggers hepatocytes stress pathways, leading to inflammation and fibrogenesis, contributing to the progression of non-alcoholic steatohepatitis (NASH). The antioxidant response, regulated by the Nrf2/ARE pathway, is a key component in this process and counteracts oxidative stress-induced damage, contributing to the restoration of normal lipid metabolism. Therefore, modulation of the antioxidant response emerges as an interesting target to prevent NAFLD development and progression. This review highlights the link between disturbed lipid metabolism and oxidative stress in the context of NAFLD. In addition, emerging potential therapies based on antioxidant effects and their likely molecular targets are discussed

Responses of retinal and brain microvasculature to streptozotocin induced diabetes revealed by global expression profiling

This study aims to determine the effects of diabetes in the retinal and brain microvasculature through gene expression profiling. Twelve male Wistar rats were randomly divided into two groups: streptozotocin-induced diabetic rats and time-matched nondiabetic rats. The retinal microvessels (RMVs) and brain microvessels (BMVs) were mechanically isolated from individual rats. Differentially expressed genes (DEGs) in diabetic and nondiabetic microvessels were identified by cDNA microarrays analysis. In RMVs, we identified 43 DEGs, of which 20 were upregulated while 23 were downregulated by diabetes. In BMVs, 35 genes DEGs were identified, of which 22 were upregulated and 13 were downregulated by diabetes. Altered expression of the Nars, Gars, Mars, Iars, Yars, Bcl2, Nqo1, NR4A3, Gpd1, Stc1, Tsc22d3, Tnfrsf21 mRNA as observed in the microarray analyses, was confirmed by quantitative RT-PCR. The aminoacyl-tRNA synthetases (aaRSs) pathway in RMVs was significantly overrepresented as compared to BMVs. Our study demonstrates for the first time that in the brain microvasculature multiple compensatory mechanisms exists, serving to protect brain tissue from diabetic insults, whereas these mechanisms are not activated in the retinal microvasculature. This provides new insights as to why brain microvasculature is less susceptible to diabetes.</p

Hesperetin protects against palmitate-induced cellular toxicity via induction of GRP78 in hepatocytes

Lipotoxicity plays a critical role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Hesperetin, a flavonoid derivative, has anti-oxidant, anti-inflammatory and cytoprotective properties. In the present study, we aim to examine whether hesperetin protects against palmitate-induced lipotoxic cell death and to investigate the underlying mechanisms in hepatocytes. Primary rat hepatocytes and HepG2 cells were pretreated with hesperetin for 30 min and then exposed to palmitate (1.0 mmol/L in primary rat hepatocytes; 0.5 mmol/L in HepG2 cells) in the presence or absence of hesperetin. Necrotic cell death was measured via Sytox green nuclei staining and quantified by LDH release assay. Apoptotic cell death was determined by caspase 3/7 activity and the protein level of cleaved-PARP. The unfolded protein response (UPR) was assessed by measuring the expression of GRP78, sXBP1, ATF4 and CHOP. Results show that hesperetin (50 μmol/L and 100 μmol/L) protected against palmitate-induced cell death and inhibited palmitate-induced endoplasmic reticulum (ER) stress in both primary rat hepatocytes and HepG2 cells. Hesperetin (100 μmol/L) significantly activated sXBP1/GRP78 signaling, whereas a high concentration of hesperetin (200 μmol/L) activated p-eIF2α and caused hepatic cell death. Importantly, GRP78 knockdown via siRNA abolished the protective effects of hesperetin in HepG2 cells. In conclusion, hesperetin protected against palmitate-induced hepatic cell death via activation of the sXBP1/GRP78 signaling pathway, thus inhibiting palmitate-induced ER stress. Moreover, high concentrations of hesperetin induce ER stress and subsequently cause cell death in hepatocytes

How does hepatic lipid accumulation lead to lipotoxicity in non-alcoholic fatty liver disease?

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD), characterized as excess lipid accumulation in the liver which is not due to alcohol use, has emerged as one of the major health problems around the world. The dysregulated lipid metabolism creates a lipotoxic environment which promotes the development of NAFLD, especially the progression from simple steatosis (NAFL) to non-alcoholic steatohepatitis (NASH). PURPOSEAND AIM: This review focuses on the mechanisms of lipid accumulation in the liver, with an emphasis on the metabolic fate of free fatty acids (FFAs) in NAFLD and presents an update on the relevant cellular processes/mechanisms that are involved in lipotoxicity. The changes in the levels of various lipid species that result from the imbalance between lipolysis/lipid uptake/lipogenesis and lipid oxidation/secretion can cause organellar dysfunction, e.g. ER stress, mitochondrial dysfunction, lysosomal dysfunction, JNK activation, secretion of extracellular vesicles (EVs) and aggravate (or be exacerbated by) hypoxia which ultimately lead to cell death. The aim of this review is to provide an overview of how abnormal lipid metabolism leads to lipotoxicity and the cellular mechanisms of lipotoxicity in the context of NAFLD

Hepatic Stellate Cell Senescence in Liver Fibrosis:Characteristics, Mechanisms and Perspectives

Myofibroblasts play an important role in fibrogenesis. Hepatic stellate cells are the main precursors of myofibroblasts. Cellular senescence is the terminal cell fate in which proliferating cells undergo irreversible cell cycle arrest. Senescent hepatic stellate cells were identified in liver fibrosis. Senescent hepatic stellate cells display decreased collagen production and proliferation. Therefore, induction of senescence could be a protective mechanism against progression of liver fibrosis and the concept of therapy-induced senescence has been proposed to treat liver fibrosis. In this review, characteristics of senescent hepatic stellate cells and the essential signaling pathways involved in senescence are reviewed. Furthermore, the potential impact of senescent hepatic stellate cells on other liver cell types are discussed. Senescent cells are cleared by the immune system. The persistence of senescent cells can remodel the microenvironment and interact with inflammatory cells to induce aging-related dysfunction. Therefore, senolytics, a class of compounds that selectively induce death of senescent cells, were introduced as treatment to remove senescent cells and consequently decrease the disadvantageous effects of persisting senescent cells. The effects of senescent hepatic stellate cells in liver fibrosis need further investigation

Hydrogen sulfide stimulates activation of hepatic stellate cells through increased cellular bio-energetics

Hepatic fibrosis is caused by chronic inflammation and characterized as the excessive accumulation of extra cellular matrix (ECM) by activated hepatic stellate cells (HSCs). Gasotransmitters like NO and CO are known to modulate inflammation and fibrosis, however, little is known about the role of the gasotransmitter hydrogen sulfide (H2S) in liver fibrogenesis and stellate cell activation. Endogenous H2S is produced by the enzymes cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and 3-mercaptopyruvate sulfur transferase (MPST) [1]. The aim of this study was to elucidate the role of endogenously produced and/or exogenously administered H2S on rat hepatic stellate cell activation and fibrogenesis. Primary rat HSCs were culture-activated for 7 days and treated with different H2S releasing donors (slow releasing donor GYY4137, fast releasing donor NaHS) or inhibitors of the H2S producing enzymes CTH and CBS (DL-PAG, AOAA). The main message of our study is that mRNA and protein expression level of H2S synthesizing enzymes are low in HSCs compared to hepatocytes and Kupffer cells. However, H2S promotes hepatic stellate cell activation. This conclusion is based on the fact that production of H2S and mRNA and protein expression of its producing enzyme CTH are increased during hepatic stellate cell activation. Furthermore, exogenous H2S increased HSC proliferation while inhibitors of endogenous H2S production reduce proliferation and fibrotic makers of HSCs. The effect of H2S on stellate cell activation correlated with increased cellular bioenergetics. Our results indicate that the H2S generation in hepatic stellate cells is a target for anti-fibrotic intervention and that systemic interventions with H2S should take into account cell-specific effects of H2S.</p

Extracellular Vesicles in NAFLD/ALD:From Pathobiology to Therapy

In recent years, knowledge on the biology and pathobiology of extracellular vesicles (EVs) has exploded. EVs are submicron membrane-bound structures secreted from different cell types containing a wide variety of bioactive molecules (e.g., proteins, lipids, and nucleic acids (coding and non-coding RNA) and mitochondrial DNA). EVs have important functions in cell-to-cell communication and are found in a wide variety of tissues and body fluids. Better delineation of EV structures and advances in the isolation and characterization of their cargo have allowed the diagnostic and therapeutic implications of these particles to be explored. In the field of liver diseases, EVs are emerging as key players in the pathogenesis of both nonalcoholic liver disease (NAFLD) and alcoholic liver disease (ALD), the most prevalent liver diseases worldwide, and their complications, including development of hepatocellular carcinoma. In these diseases, stressed/damaged hepatocytes release large quantities of EVs that contribute to the occurrence of inflammation, fibrogenesis, and angiogenesis, which are key pathobiological processes in liver disease progression. Moreover, the specific molecular signatures of released EVs in biofluids have allowed EVs to be considered as promising candidates to serve as disease biomarkers. Additionally, different experimental studies have shown that EVs may have potential for therapeutic use as a liver-specific delivery method of different agents, taking advantage of their hepatocellular uptake through interactions with specific receptors. In this review, we focused on the most recent findings concerning the role of EVs as new structures mediating autocrine and paracrine intercellular communication in both ALD and NAFLD, as well as their potential use as biomarkers of disease severity and progression. Emerging therapeutic applications of EVs in these liver diseases were also examined, along with the potential for successful transition from bench to clinic

Hepatitis C virus core or NS3/4A protein expression preconditions hepatocytes against oxidative stress and endoplasmic reticulum stress

OBJECTIVES: The occurrence of oxidative stress and endoplasmic reticulum (ER) stress in hepatitis C virus (HCV) infection has been demonstrated and play an important role in liver injury. During viral infection, hepatocytes must handle not only the replication of the virus, but also inflammatory signals generating oxidative stress and damage. Although several mechanisms exist to overcome cellular stress, little attention has been given to the adaptive response of hepatocytes during exposure to multiple noxious triggers.METHODS: In the present study, Huh-7 cells and hepatocytes expressing HCV Core or NS3/4A proteins, both inducers of oxidative and ER stress, were additionally challenged with the superoxide anion generator menadione to mimic external oxidative stress. The production of reactive oxygen species (ROS) as well as the response to oxidative stress and ER stress were investigated.RESULTS: We demonstrate that hepatocytes diminish oxidative stress through a reduction in ROS production, ER-stress markers (HSPA5 [GRP78], sXBP1) and apoptosis (caspase-3 activity) despite external oxidative stress. Interestingly, the level of the autophagy substrate protein p62 was downregulated together with HCV Core degradation, suggesting that hepatocytes can overcome excess oxidative stress through autophagic degradation of one of the stressors, thereby increasing cell survival. Duscussion: In conclusion, hepatocytes exposed to direct and indirect oxidative stress inducers are able to cope with cellular stress associated with viral hepatitis and thus promote cell survival.</p

Hepatitis C Virus Proteins Core and NS5A Are Highly Sensitive to Oxidative Stress-Induced Degradation after eIF2α/ATF4 Pathway Activation

Hepatitis C virus (HCV) infection is accompanied by increased oxidative stress and endoplasmic reticulum stress as a consequence of viral replication, production of viral proteins, and pro-inflammatory signals. To overcome the cellular stress, hepatocytes have developed several adaptive mechanisms like anti-oxidant response, activation of Unfolded Protein Response and autophagy to achieve cell survival. These adaptive mechanisms could both improve or inhibit viral replication, however, little is known in this regard. In this study, we investigate the mechanisms by which hepatocyte-like (Huh7) cells adapt to cellular stress in the context of HCV protein overexpression and oxidative stress. Huh7 cells stably expressing individual HCV (Core, NS3/4A and NS5A) proteins were treated with the superoxide anion donor menadione to induce oxidative stress. Production of reactive oxygen species and activation of caspase 3 were quantified. The activation of the eIF2α/ATF4 pathway and changes in the steady state levels of the autophagy-related proteins LC3 and p62 were determined either by quantitative polymerase chain reaction (qPCR) or Western blotting. Huh7 cells expressing Core or NS5A demonstrated reduced oxidative stress and apoptosis. In addition, phosphorylation of eIF2α and increased ATF4 and CHOP expression was observed with subsequent HCV Core and NS5A protein degradation. In line with these results, in liver biopsies from patients with hepatitis C, the expression of ATF4 and CHOP was confirmed. HCV Core and NS5A protein degradation was reversed by antioxidant treatment or silencing of the autophagy adaptor protein p62. We demonstrated that hepatocyte-like cells expressing HCV proteins and additionally exposed to oxidative stress adapt to cellular stress through eIF2a/ATF4 activation and selective degradation of HCV pro-oxidant proteins Core and NS5A. This selective degradation is dependent on p62 and results in increased resistance to apoptotic cell death induced by oxidative stress. This mechanism may provide a new key for the study of HCV pathology and lead to novel clinically applicable therapeutic interventions