Extracellular vesicles derived from liver sinusoidal endothelial cells inhibit the activation of hepatic stellate cells and Kupffer cells in vitro.

Liver sinusoidal endothelial cells (LSECs) play a crucial role in maintaining liver microcirculation and exchange of nutrients in the liver and are thought to be involved in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). The activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) has been considered to be responsible for the onset of liver fibrosis and the aggravation of liver injury. However, the paracrine regulatory effects of LSECs in the development of MASLD, in particular the role of LSEC-derived extracellular vesicles (EVs) remains unclear. Therefore, the aim of the present study was to investigate the influence of LSEC-derived EVs on HSCs and KCs. Primary rat LSECs, HSCs and KCs were isolated from male Wistar rats. LSEC-derived EVs were isolated from conditioned medium by ultracentrifugation and analyzed by nanoparticle tracking analysis, and expression of specific markers. LSEC-derived EVs reduced the expression of activation markers in activated HSCs but did not affect quiescent HSCs. Also, LSEC-derived EVs suppressed proliferation of activated HSCs activation, as assessed by Xcelligence and BrdU assay. LSEC-derived EVs also increased the expression of inflammatory genes in HSCs that normally are lowly expression during their activation. In contrast, EVs decreased the expression of inflammatory genes in activated KCs. In summary, our results suggest that LSEC-derived EVs may attenuate the fibrogenic phenotype of activated HSCs and the inflammatory phenotype of KCs. Our results show promise for LSEC-derived EVs as therapeutic moieties to treat MASLD. In addition, these EVs might prove of diagnostic value.

The Coumarin-Derivative Esculetin Protects against Lipotoxicity in Primary Rat Hepatocytes via Attenuating JNK-Mediated Oxidative Stress and Attenuates Free Fatty Acid-Induced Lipid Accumulation.

Coumarin derivates have been proposed as a potential treatment for metabolic-dysfunction-associated fatty liver disease (MAFLD). However, the mechanisms underlying their beneficial effects remain unclear. In the present study, we explored the potential of the coumarin derivate esculetin in MAFLD, focusing on hepatocyte lipotoxicity and lipid accumulation. Primary cultures of rat hepatocytes were exposed to palmitic acid (PA) and palmitic acid plus oleic acid (OA/PA) as models of lipotoxicity and lipid accumulation, respectively. Esculetin significantly reduced oxidative stress in PA-treated hepatocytes, as shown by decreased total reactive oxygen species (ROS) and mitochondrial superoxide production and elevated expression of antioxidant genes, including Nrf2 and Gpx1. In addition, esculetin protects against PA-induced necrosis. Esculetin also improved lipid metabolism in primary hepatocytes exposed to nonlipotoxic OA/PA by decreasing the expression of the lipogenesis-related gene Srebp1c and increasing the expression of the fatty acid ß-oxidation-related gene Ppar-α. Moreover, esculetin attenuated lipid accumulation in OA/PA-treated hepatocytes. The protective effects of esculetin against lipotoxicity and lipid accumulation were shown to be dependent on the inhibition of JNK and the activation of AMPK, respectively. We conclude that esculetin is a promising compound to target lipotoxicity and lipid accumulation in the treatment of MAFLD.

Hepatic stellate cells induce an inflammatory phenotype in Kupffer cells via the release of extracellular vesicles.

Liver fibrosis is the response of the liver to chronic liver inflammation. The communication between the resident liver macrophages (Kupffer cells [KCs]) and hepatic stellate cells (HSCs) has been mainly viewed as one-directional: from KCs to HSCs with KCs promoting fibrogenesis. However, recent studies indicated that HSCs may function as a hub of intercellular communications. Therefore, the aim of the present study was to investigate the role of HSCs on the inflammatory phenotype of KCs. Primary rat HSCs and KCs were isolated from male Wistar rats. HSCs-derived conditioned medium (CM) was harvested from different time intervals (Day 0-2: CM-D2 and Day 5-7: CM-D7) during the activation of HSCs. Extracellular vesicles (EVs) were isolated from CM by ultracentrifugation and evaluated by nanoparticle tracking analysis and western blot analysis. M1 and M2 markers of inflammation were measured by quantitative PCR and macrophage function by assessing phagocytic capacity. CM-D2 significantly induced the inflammatory phenotype in KCs, but not CM-D7. Neither CM-D2 nor CM-D7 affected the phagocytosis of KCs. Importantly, the proinflammatory effect of HSCs-derived CM is mediated via EVs released from HSCs since EVs isolated from CM mimicked the effect of CM, whereas EV-depleted CM lost its ability to induce a proinflammatory phenotype in KCs. In addition, when the activation of HSCs was inhibited, HSCs produced less EVs. Furthermore, the proinflammatory effects of CM and EVs are related to activating Toll-like receptor 4 (TLR4) in KCs. In conclusion, HSCs at an early stage of activation induce a proinflammatory phenotype in KCs via the release of EVs. This effect is absent in CM derived from HSCs at a later stage of activation and is dependent on the activation of TLR4 signaling pathway.

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 CCl4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl4 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 CCl4 treatment. CCl4 -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 CCl4. Thus, hydroxyurea may have therapeutic value against liver fibrosis.

Protective effects of caffeine against palmitate-induced lipid toxicity in primary rat hepatocytes is associated with modulation of adenosine receptor A1 signaling.

BACKGROUND: Epidemiological evidence has shown an association between coffee consumption and reduced risk for chronic liver diseases, including metabolic-dysfunction-associated liver disease (MALFD). Lipotoxicity is a key cause of hepatocyte injury during MAFLD. The coffee component caffeine is known to modulate adenosine receptor signaling via the antagonism of adenosine receptors. The involvement of these receptors in the prevention of hepatic lipotoxicity has not yet been explored. The aim of this study was to explore whether caffeine protects against palmitate-induced lipotoxicity by modulating adenosine receptor signaling. METHODS: Primary hepatocytes were isolated from male rats. Hepatocytes were treated with palmitate with or without caffeine or 1,7DMX. Lipotoxicity was verified using Sytox viability staining and mitochondrial JC-10 staining. PKA activation was verified by Western blotting. Selective (ant)agonists of A1AR (DPCPX and CPA, respectively) and A2AR (istradefyline and regadenoson, respectively), the AMPK inhibitor compound C, and the Protein Kinase A (PKA) inhibitor Rp8CTP were used. Lipid accumulation was verified by ORO and BODIPY 453/50 staining. RESULTS: Caffeine and its metabolite 1,7DMX prevented palmitate-induced toxicity in hepatocytes. The A1AR antagonist DPCPX also prevented lipotoxicity, whereas both the inhibition of PKA and the A1AR agonist CPA (partially) abolished the protective effect. Caffeine and DPCPX increased lipid droplet formation only in palmitate-treated hepatocytes and decreased mitochondrial ROS production. CONCLUSIONS: The protective effect of caffeine against palmitate lipotoxicity was shown to be dependent on A1AR receptor and PKA activation. Antagonism of A1AR also protects against lipotoxicity. Targeting A1AR receptor may be a potential therapeutic intervention with which to treat MAFLD.

Branched Chain Amino Acids Are Associated with Physical Performance in Patients with End-Stage Liver Disease.

Decreased circulating branched chain amino acids (BCAA) represent a prominent change in amino acid profiles in patients with end-stage liver disease (ESLD). These alterations are considered to contribute to sarcopenia and hepatic encephalopathy and may relate to poor prognosis. Here, we cross-sectionally analyzed the association between plasma BCAA levels and the severity of ESLD and muscle function in participants of the liver transplant subgroup of TransplantLines, enrolled between January 2017 and January 2020. Plasma BCAA levels were measured by nuclear magnetic resonance spectroscopy. Physical performance was analyzed with a hand grip strength test, 4 m walking test, sit-to-stand test, timed up and go test, standing balance test and clinical frailty scale. We included 92 patients (65% men). The Child Pugh Turcotte classification was significantly higher in the lowest sex-stratified BCAA tertile compared to the highest tertile (p = 0.015). The times for the sit-to-stand (r = -0.352, p < 0.05) and timed up and go tests (r = -0.472, p < 0.01) were inversely correlated with total BCAA levels. In conclusion, lower circulating BCAA are associated with the severity of liver disease and impaired muscle function. This suggests that BCAA may represent a useful prognostic marker in the staging of liver disease severity.

Studying Hepatic Stellate Cell Senescence.

Hepatic stellate cells (HSCs) are the key effector cells in liver fibrosis. They are the main producers of excessive amounts of extracellular matrix components during fibrogenesis and therefore a potential target for the treatment of liver fibrosis. Induction of senescence in HSCs may be a promising strategy to slow down, stop, or even reverse fibrogenesis. Senescence is a complex and heterogeneous process linked to fibrosis and cancer, but the exact mechanism and relevant markers can be cell-type dependent. Therefore, many markers of senescence have been proposed, and many methods to detect senescence have been developed. In this chapter, we review relevant methods and biomarkers to detect cellular senescence in hepatic stellate cells.

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.

Intestinal BMP-9 locally upregulates FGF19 and is down-regulated in obese patients with diabetes.

Bone morphogenetic protein (BMP)-9, a member of the TGFß-family of cytokines, is believed to be mainly produced in the liver. The serum levels of BMP-9 were reported to be reduced in newly diagnosed diabetic patients and BMP-9 overexpression ameliorated steatosis in the high fat diet-induced obesity mouse model. Furthermore, injection of BMP-9 in mice enhanced expression of fibroblast growth factor (FGF)21. However, whether BMP-9 also regulates the expression of the related FGF19 is not clear. Because both FGF21 and 19 were described to protect the liver from steatosis, we have further investigated the role of BMP-9 in this context. We first analyzed BMP-9 levels in the serum of streptozotocin (STZ)-induced diabetic rats (a model of type I diabetes) and confirmed that BMP-9 serum levels decrease during diabetes. Microarray analyses of RNA samples from hepatic and intestinal tissue from BMP-9 KO- and wild-type mice (C57/Bl6 background) pointed to basal expression of BMP-9 in both organs and revealed a down-regulation of hepatic Fgf21 and intestinal Fgf19 in the KO mice. Next, we analyzed BMP-9 levels in a cohort of obese patients with or without diabetes. Serum BMP-9 levels did not correlate with diabetes, but hepatic BMP-9 mRNA expression negatively correlated with steatosis in those patients that did not yet develop diabetes. Likewise, hepatic BMP-9 expression also negatively correlated with serum LPS levels. In situ hybridization analyses confirmed intestinal BMP-9 expression. Intestinal (but not hepatic) BMP-9 mRNA levels were decreased with diabetes and positively correlated with intestinal E-Cadherin expression. In vitro studies using organoids demonstrated that BMP-9 directly induces FGF19 in gut but not hepatocyte organoids, whereas no evidence of a direct induction of hepatic FGF21 by BMP-9 was found. Consistent with the in vitro data, a correlation between intestinal BMP-9 and FGF19 mRNA expression was seen in the patients' samples. In summary, our data confirm that BMP-9 is involved in diabetes development in humans and in the control of the FGF-axis. More importantly, our data imply that not only hepatic but also intestinal BMP-9 associates with diabetes and steatosis development and controls FGF19 expression. The data support the conclusion that increased levels of BMP-9 would most likely be beneficial under pre-steatotic conditions, making supplementation of BMP-9 an interesting new approach for future therapies aiming at prevention of the development of a metabolic syndrome and liver steatosis.

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.

Molecular and Antioxidant Characterization of Opuntia robusta Fruit Extract and Its Protective Effect against Diclofenac-Induced Acute Liver Injury in an In Vivo Rat Model.

A molecular characterization of the main phytochemicals and antioxidant activity of Opuntia robusta (OR) fruit extract was carried out, as well as an evaluation of its hepatoprotective effect against diclofenac (DF)-induced acute liver injury was evaluated. Phenols, flavonoids and betalains were quantified, and antioxidant characterization was performed by means of the ABTS•+, DPPH and FRAP assays. UPLC-QTOF-MS/MS was used to identify the main biocompounds present in OR fruit extract was carried out via. In the in vivo model, groups of rats were treated prophylactically with the OR fruit extract, betanin and N-acteylcysteine followed by a single dose of DF. Biochemical markers of oxidative stress (MDA and GSH) and relative gene expression of the inducible antioxidant response (Nrf2, Sod2, Hmox1, Nqo1 and Gclc), cell death (Casp3) and DNA repair (Gadd45a) were analyzed. Western blot analysis was performed to measure protein levels of Nrf2 and immunohistochemical analysis was used to assess caspase-3 activity in the experimental groups. In our study, the OR fruit extract showed strong antioxidant and cytoprotective capacity due to the presence of bioactive compounds, such as betalain and phenols. We conclude that OR fruit extract or selected components can be used clinically to support patients with acute liver injury.

Differential effects of oleate on vascular endothelial and liver sinusoidal endothelial cells reveal its toxic features in vitro.

Several fatty acids, in particular saturated fatty acids like palmitic acid, cause lipotoxicity in the context of non-alcoholic fatty liver disease . Unsaturated fatty acids (e.g. oleic acid) protect against lipotoxicity in hepatocytes. However, the effect of oleic acid on other liver cell types, in particular liver sinusoidal endothelial cells (LSECs), is unknown. Human umbilical vein endothelial cells (HUVECs) are often used as a substitute for LSECs, however, because of the unique phenotype of LSECs, HUVECs cannot represent the same biological features as LSECs. In this study, we investigate the effects of oleate and palmitate (the sodium salts of oleic acid and palmitic acid) on primary rat LSECs in comparison to their effects on HUVECs. Oleate induces necrotic cell death in LSECs, but not in HUVECs. Necrotic cell death of LSECs can be prevented by supplementation of 2-stearoylglycerol, which promotes cellular triglyceride (TG) synthesis. Repressing TG synthesis, by knocking down DGAT1 renders HUVECs sensitive to oleate-induced necrotic death. Mechanistically, oleate causes a sharp drop of intracellular ATP level and impairs mitochondrial respiration in LSECs. The combination of oleate and palmitate reverses the toxic effect of oleate in both LSECs and HUVECs. These results indicate that oleate is toxic and its toxicity can be attenuated by stimulating TG synthesis. The toxicity of oleate is characterized by mitochondrial dysfunction and necrotic cell death. Moreover, HUVECs are not suitable as a substitute model for LSECs.

Hepatoprotective Effect of Opuntia robusta Fruit Biocomponents in a Rat Model of Thioacetamide-Induced Liver Fibrosis.

Liver fibrosis is a chronic disease associated with oxidative stress that has a great impact on the population mortality. Due to their antioxidant capacity, we evaluated the protective effect of Opuntia robusta fruit (Or) on liver fibrosis. A nutraceutical characterization of Or was performed and a model of fibrosis was induced with thioacetamide (TAA) in Wistar rats. Aminotransferases, reduced glutathione (GSH) and histopathology were evaluated. Or contained 436.5 ± 57 mg of Betacyanins equivalents/L., 793 mg of catechin equivalents (CAE)/100 g for flavonoids, 1118 mg of gallic acid equivalents (GAE)/100 g for total phenols, 141.14 mg/100 g for vitamin C and 429.9 µg/100 g for vitamin E. The antioxidant capacity of Or was: 2.27 mmol of Trolox® equivalents (TE)/L (DPPH), 62.2 ± 5.0 µmol TE/g (ABTS•+), 80.2 ± 11.7 µmol TE/g (FRAP), 247.9 ± 15.6 µmol TE/g (AAPH) and 15.0% of H2O2 elimination. An increase (p < 0.05) of aminotransferases and a decrease (p < 0.05) of hepatic GSH was observed in the TAA group compared to the control and the concomitant groups. Histopathology showed changes in the normal architecture of the liver treated with TAA compared to the concomitant treatments. Or contains bioactive components with antioxidant capacity, which can reduce fibrotic liver damage.

Modulation of Oxidative Stress-Induced Senescence during Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease is characterized by disturbed lipid metabolism and increased oxidative stress. These conditions lead to the activation of different cellular response mechanisms, including senescence. Cellular senescence constitutes an important response to injury in the liver. Recent findings show that chronic oxidative stress can induce senescence, and this might be a driving mechanism for NAFLD progression, aggravating the disturbance of lipid metabolism, organelle dysfunction, pro-inflammatory response and hepatocellular damage. In this context, the modulation of cellular senescence can be beneficial to ameliorate oxidative stress-related damage during NAFLD progression. This review focuses on the role of oxidative stress and senescence in the mechanisms leading to NAFLD and discusses the possibilities to modulate senescence as a therapeutic strategy in the treatment of NAFLD.

A Potential Role for Bile Acid Signaling in Celiac Disease-Associated Fatty Liver.

Celiac disease (CeD) is a chronic autoimmune disorder characterized by an intolerance to storage proteins of many grains. CeD is frequently associated with liver damage and steatosis. Bile acid (BA) signaling has been identified as an important mediator in gut-liver interaction and the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Here, we aimed to analyze BA signaling and liver injury in CeD patients. Therefore, we analyzed data of 20 CeD patients on a gluten-free diet compared to 20 healthy controls (HC). We furthermore analyzed transaminase levels, markers of cell death, BA, and fatty acid metabolism. Hepatic steatosis was determined via transient elastography, by MRI and non-invasive scores. In CeD, we observed an increase of the apoptosis marker M30 and more hepatic steatosis as compared to HC. Fibroblast growth factor 19 (FGF19) was repressed in CeD, while low levels were associated with steatosis, especially in patients with high levels of anti-tissue transglutaminase antibodies (anti-tTG). When comparing anti-tTG-positive CeD patients to individuals without detectable anti-tTG levels, hepatic steatosis was accentuated. CeD patients with significant sonographic steatosis (defined by CAP ≥ 283 db/m) were exclusively anti-tTG-positive. In summary, our results suggest that even in CeD patients in clinical remission under gluten-free diet, alterations in gut-liver axis, especially BA signaling, might contribute to steatotic liver injury and should be further addressed in future studies and clinical practice.

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.

α-1 Adrenergic receptor antagonist doxazosin reverses hepatic stellate cells activation via induction of senescence.

BACKGROUND: Activated hepatic stellate cells (aHSCs) are the main effector cells during liver fibrogenesis. α-1 adrenergic antagonist doxazosin (DX) was shown to be anti-fibrotic in an in vivo model of liver fibrosis (LF), but the mechanism remains to be elucidated. Recent studies suggest that reversion of LF can be achieved by inducing cellular senescence characterized by irreversible cell-cycle arrest and acquisition of the senescence-associated secretory phenotype (SASP). AIM: To elucidate the mechanism of the anti-fibrotic effect of DX and determine whether it induces senescence. METHODS: Primary culture-activated rat HSCs were used. mRNA and protein expression were measured by qPCR and Western blot, respectively. Cell proliferation was assessed by BrdU incorporation and xCelligence analysis. TGF-ß was used for maximal HSC activation. Norepinephrine (NE), PMA and m-3M3FBS were used to activate alpha-1 adrenergic signaling. RESULTS: Expression of Col1α1 was significantly decreased by DX (10 µmol/L) at mRNA (-30 %) and protein level (-50 %) in TGF-ß treated aHSCs. DX significantly reduced aHSCs proliferation and increased expression of senescence and SASP markers. PMA and m-3M3FBS reversed the effect of DX on senescence markers. CONCLUSION: Doxazosin reverses the fibrogenic phenotype of aHSCs and induces the senescence phenotype.

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.

Metformin protects against diclofenac-induced toxicity in primary rat hepatocytes by preserving mitochondrial integrity via a pathway involving EPAC.

BACKGROUND AND PURPOSE: It has been shown that the antidiabetic drug metformin protects hepatocytes against toxicity by various stressors. Chronic or excessive consumption of diclofenac (DF) - a pain-relieving drug, leads to drug-induced liver injury via a mechanism involving mitochondrial damage and ultimately apoptotic death of hepatocytes. However, whether metformin protects against DF-induced toxicity is unknown. Recently, it was also shown that cAMP elevation is protective against DF-induced apoptotic death in hepatocytes, a protective effect primarily involving the downstream cAMP effector EPAC and preservation of mitochondrial function. This study therefore aimed at investigating whether metformin protects against DF-induced toxicity via cAMP-EPACs. EXPERIMENTAL APPROACH: Primary rat hepatocytes were exposed to 400 µmol/L DF. CE3F4 or ESI-O5 were used as EPAC-1 or 2 inhibitors respectively. Apoptosis was measured by caspase-3 activity and necrosis by Sytox green staining. Seahorse X96 assay was used to determine mitochondrial function. Mitochondrial reactive oxygen species (ROS) production was measured using MitoSox, mitochondrial MnSOD expression was determined by immunostaining and mitochondrial morphology (fusion and fission ratio) by 3D refractive index imaging. KEY RESULTS: Metformin (1 mmol/L) was protective against DF-induced apoptosis in hepatocytes. This protective effect was EPAC-dependent (mainly EPAC-2). Metformin restored mitochondrial morphology in an EPAC-independent manner. DF-induced mitochondrial dysfunction which was demonstrated by decreased oxygen consumption rate, an increased ROS production and a reduced MnSOD level, were all reversed by metformin in an EPAC-dependent manner. CONCLUSION AND IMPLICATIONS: Metformin protects hepatocytes against DF-induced toxicity via cAMP-dependent EPAC-2.

Anti-TNFα treatment in Crohn's disease: Impact on hepatic steatosis, gut-derived hormones and metabolic status.

BACKGROUND AND AIMS: An association between Crohn's disease (CD) and hepatic steatosis has been reported. However, the underlying mechanisms of steatosis progression in CD are not clear. Among the most effective CD treatments are agents that inhibit Tumor-Necrosis-Factor (TNF) activity, yet it is unclear why anti-TNFα agents would affect steatosis in CD. Recent studies suggest that microbiome can affect both, CD and steatosis pathogenesis. Therefore, we here analysed a potential relationship between anti-TNF treatment and hepatic steatosis in CD, focusing on the gut-liver axis. METHODS: This cross-sectional study evaluated patients with established CD, with and without anti-TNFα treatment, analysing serum markers of liver injury, measurement of transient elastography, controlled attenuation parameter (CAP) and MRI for fat detection. Changes in lipid and metabolic profiles were assessed by serum and stool lipidomics and metabolimics. Additionally, we analysed gut microbiota composition and mediators of bile acid (BA) signalling via stool and serum analysis. RESULTS: Patients on anti-TNFα treatment had less hepatic steatosis as assessed by CAP and MRI. Serum FGF19 levels were significantly higher in patients on anti-TNFα therapy and associate with reduced steatosis and increased bowel motility. Neutral lipids including triglycerides were reduced in the serum of patients on anti-TNF treatment. Bacteria involved in BA metabolism and FGF19 regulation, including Firmicutes, showed group-specific alterations with low levels in patients without anti-TNFα treatment. Low abundance of Firmicutes was associated with higher triglyceride levels. CONCLUSIONS: Anti-TNFα treatment is associated with reduced steatosis, lower triglyceride levels, alterations in FXR-signalling (eg FGF19) and microbiota composition in CD.