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

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

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

Protective effect of metformin against palmitate-induced hepatic cell death

Lipotoxicity causes hepatic cell death and therefore plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metformin, a first-line anti-diabetic drug, has shown a potential protective effect against NAFLD. However, the underlying mechanism is still not clear. In this study, we aim to understand the molecular mechanism of the protective effect of metformin in NAFLD, focusing on lipotoxicity. Cell death was studied in HepG2 cells and primary rat hepatocytes exposed to palmitate and metformin. Metformin ameliorated palmitate-induced necrosis and apoptosis (decreased caspase-3/7 activity by 52% and 57% respectively) in HepG2 cells. Metformin also reduced palmitate-induced necrosis in primary rat hepatocytes (P < 0.05). The protective effect of metformin is not due to reducing intracellular lipid content or activation of AMPK signaling pathways. Metformin and a low concentration (0.1 μmol/L) of rotenone showed moderate inhibition on mitochondrial respiration indicated by reduced basal and maximal mitochondrial respiration and proton leak in HepG2 cells. Moreover, metformin and rotenone (0.1 μmol/L) preserved mitochondrial membrane potential in both HepG2 cells and primary rat hepatocytes. In addition, metformin and rotenone (0.1 μmol/L) also reduces reactive oxygen species (ROS) production and increase superoxide dismutase 2 (SOD2) expression. Our results establish that metformin AMPK-independently protects against PA-induced hepatic cell death by moderate inhibition of the mitochondrial respiratory chain, recovering mitochondrial function, decreasing cellular ROS production, and inducing SOD2 expression, indicating that metformin may have beneficial actions beyond its glucose-lowering effect and also suggests that mitochondrial complex І may be a therapeutic target in NAFLD

Arginase 1 expression is increased during hepatic stellate cell activation and facilitates collagen synthesis

Activation of hepatic stellate cells (HSC) is a key event in the initiation of liver fibrosis. Activated HSCs proliferate and secrete excessive amounts of extracellular matrix (ECM), disturbing liver architecture and function, leading to fibrosis and eventually cirrhosis. Collagen is the most abundant constituent of ECM and proline is the most abundant amino acid of collagen. Arginine is the precursor in the biosynthetic pathway of proline. Arginine is the exclusive substrate of both nitric oxide synthase (NOS) and arginase. NOS is an M1 (proinflammatory) marker of macrophage polarization whereas arginase-1 (Arg1) is an M2 (profibrogenic) marker of macrophage polarization. Differential expression of NOS and Arg1 has not been studied in HSCs yet. To identify the expression profile of arginine catabolic enzymes during HSC activation and to investigate their role in HSC activation, primary rat HSCs were cultured-activated for 7 days and expression of iNOS and Arg1 were investigated. Nor-NOHA was used as a specific and reversible arginase inhibitor. During HSC activation, iNOS expression decreased whereas Arg1 expression increased. Inhibition of Arg1 in activated HSCs efficiently inhibited collagen production but not cell proliferation. HSC activation is accompanied by a switch of arginine catabolism from iNOS to Arg1. Inhibition of Arg1 decreases collagen synthesis. Therefore, we conclude that Arg1 can be a therapeutic target for the inhibition of liver fibrogenesis.</p

Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes:A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors

Background: Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism including the classical protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). Aim: Investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Methods: Rat hepatocytes were exposed to 400 µmol/L diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by qPCR and Western blot, respectively. The cAMP-elevating agent forskolin, the pan-phosphodiesterase inhibitor IBMX and EPAC inhibitors CE3F4 and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Results: Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition, but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP while both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. Conclusion: cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. Significance Statement Our study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, we propose the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in DILI. Both EPAC1 and EPAC2, but not PKA, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of drug-induced liver injury (DILI) and liver injury involving mitochondrial dysfunction

Hydroxyurea attenuates hepatic stellate cell proliferation in vitro and liver fibrogenesis in vivo

Liver fibrosis results from excessive proliferation of, and collagen production by hepatic stellate cells (HSCs) that is caused by chronic liver injury. No drugs are available to cure liver fibrosis. Hydroxyurea is an anti-proliferative drug that is used in benign and malignant disorders. Here, we studied the effect of hydroxyurea on primary HSCs and its anti-fibrotic effect in the CCl 4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl 4 or vehicle was administered to C57BL/6/J mice for 4 weeks, with or without hydroxyurea (100 mg/kg/day) co-treatment. We used real-time cell proliferation analysis, Oil Red O (lipid droplet) staining, immunohistochemistry, Acridine Orange staining (apoptosis), Sytox green staining (necrosis), RT-qPCR, ELISA, and Western Blotting for analysis. Hydroxyurea dose-dependently suppressed lipid droplet-loss and mRNA levels of Col1α1 and Acta2 in transdifferentiating HSCs. In fully-activated HSCs, hydroxyurea dose-dependently attenuated PCNA protein levels and BrdU incorporation, but did not reverse Col1α1 and Acta2 mRNA expression. Hydroxyurea did not induce apoptosis or necrosis in HSCs or hepatocytes. Hydroxyurea suppressed accumulation of desmin-positive HSCs and hepatic collagen deposition after CCl 4 treatment. CCl 4 -induced regenerative hepatocyte proliferation, Col1α1 and Acta2 mRNA expression and α-SMA protein levels were not affected. This study demonstrates that hydroxyurea inhibits HSC proliferation in vitro and attenuates early development of liver fibrosis in vivo, while preserving hepatocyte regeneration after toxic insults by CCl 4. Thus, hydroxyurea may have therapeutic value against liver fibrosis. </p