ABSTRACT
AIMS: The activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis, however non-alcoholic fatty liver disease (NAFLD) associated liver fibrogenesis have been poorly understood. We aimed to determine the significance of mineralocorticoid receptor (MR)/osteopontin (OPN)/high-mobility group box-1 (HMGB1) axis in this setting. MAIN METHODS: Liver specimens were collected from NAFLD patients and murine NAFLD models established with 12-week high fat diet (HFD) for analysis of both upstream signals of MR and intrahepatic MR/OPN/HMGB1 axis. The in vitro cell model of NAFLD-associated liver fibrogenesis was established by treating LX-2 (a cell line of human HSCs) with free fatty acids (FFA). The effects of MR signaling were evaluated using with ALD (MR activator) or eplerenone (Ep, MR antagonist). Moreover, the in vitro loss- and gain- of function approaches were applied to confirm the upstream and downstream relationships of mediators contained in the intracellular MR/OPN/HMGB1 axis of LX-2. KEY FINDINGS: In NAFLD condition, both human and mouse liver tissue samples demonstrated a significant up-regulation of MR/OPN/HMGB1 axis simultaneously with enhanced expression of pro-fibrogenic markers, including ACTA2, TIMP1, TGFB1 and COL1A1. Besides, enhanced production of serum aldosterone (ALD) was also observed in mouse NAFLD models. Moreover, the in vitro data demonstrated MR play an essential role in FFA-induced HSCs fibrogenesis. Meanwhile, MR acts as the upstream effector mediator of OPN and shares downstream HMGB1 with OPN. SIGNIFICANCE: The MR/OPN/HMGB1 axis could be therapeutically targeted to treat NAFLD associated hepatic fibrogenesis.
Subject(s)
HMGB1 Protein/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Osteopontin/metabolism , Receptors, Mineralocorticoid/metabolism , Adult , Animals , Female , Humans , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Middle Aged , Signal Transduction/physiologyABSTRACT
AIMS: The activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis progression. Phospholipase D (PLD) enzymes participate in multiple cellular activities. However, whether and how PLD regulates HSCs activation remain elusive. MAIN METHODS: The expression of intrahepatic PLD1 and PLD2 was determined in CCl4-induced mouse liver fibrosis models by western blot and immunohistochemistry. Cell model of liver fibrogenesis was constructed using rat HSCs line (HSC-T6) treated with recombinant transforming growth factor ß1 (TGFß1). Fibrogenesis was evaluated on the aspects of proliferation, expression of pro-fibrogenic markers and migration. The effects mediated by PLD1-mTOR axis on TGFß1-induced fibrogenesis were evaluated using HSC-T6 treated with small-molecular PLD1 inhibitors, PLD1-SiRNA, rapamycin (mTOR inhibitor) and MHY1485 (mTOR activator). KEY FINDINGS: Significant increase of PLD1, not PLD2 was documented in CCl4-induced cirrhotic compared to normal liver tissues. Suppression of PLD1 activities by PLD inhibitors or down-regulation of PLD1 expression in HSC-T6 could significantly restrain TGFß1-induced fibrogenesis, as reflected by decreased cell proliferation and reduced expression of pro-fibrogenic markers. Besides, either PLD1 inhibitor or PLD1-SiRNA significantly inhibited mTOR activity of HSC-T6. Moreover, PLD1 inhibitors not only exhibited similar effects with rapamycin in TGFß1-induced fibrogenesis, but also blunted MHY1485 enhanced cell proliferation of HSC-T6. SIGNIFICANCE: The PLD1-mTOR axis of HSCs could be therapeutically targeted in advanced liver fibrosis.
Subject(s)
Hepatic Stellate Cells/metabolism , Liver Cirrhosis/physiopathology , Phospholipase D/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Cell Line , Cell Proliferation/physiology , Disease Models, Animal , Disease Progression , Male , Mice , Mice, Inbred C57BL , RatsABSTRACT
Accumulating evidence links osteopontin (OPN), a pro-fibrogenic extracellular matrix protein, to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In this study, liver tissues isolated from non-alcoholic steatohepatitis (NASH) patients expressed higher OPN than those of controls. However, the exact mechanism(s) for this phenomenon is yet to be clarified. Autophagy is the natural, regulated degradation and recycling of a cell's dysfunctional components, in order to maintain homeostasis. Increasing evidence supports that autophagy can constitute an effective Defence mechanism against NAFLD conditions. Herein, we constructed NAFLD mice model by high-fat (HF) and methionine-choline-deficient (MCD) diet and found that OPN is upregulated in livers of NAFLD mice. Besides, secreted OPN inhibited autophagosome-lysosome fusion via binding with its receptors integrin αVß3 and αVß5 in HepG2 cells supplemented with free fatty acids (FFA) and the livers of NAFLD mice. Silencing of OPN attenuated autophagy impairment and reduced lipid accumulation, while supplementation of OPN exhibited the opposite effect. Furthermore, treatment with anti-OPN Ab significantly attenuated steatosis as well as autophagy impairment in the liver. Our findings indicated that OPN plays a vital role in the pathogenesis of the development of NAFLD via autophagy impairment, which might represent a potential new therapeutic target for the treatment of NAFLD.