Melatonin inhibits autophagy and endoplasmic reticulum stress in mice with carbon tetrachloride-induced fibrosis Abstract: This study aimed to investigate whether inhibition of autophagy and endoplasmic reticulum (ER stress) associates with the antifibrogenic effect of melatonin in mice treated with carbon tetrachloride (CCl 4 ). Mice received CCl 4 5 lL/g body wt i.p. twice a week for 4 wk or 6 wk. Melatonin was given at 5 or 10 mg/kg/day i.p, beginning 2 wk after the start of CCl 4 administration. Treatment with CCl 4 resulted in fibrosis evidenced by the staining of a-smooth muscle actin (a-SMA)-positive cells. CCl 4 induced an autophagic response measured as the presence of autophagic vesicles, protein 1 light chain 3 (LC3) staining, conversion of LC3-I to autophagosome- associated LC3-II, changes in expression of beclin-1, UV radiation resistance- associated gene (UVRAG), ubiquitin-like autophagy-related (Atg5), Atg12, Atg16L1, sequestosome 1 (p62/SQSTM1), and lysosome-associated membrane protein (LAMP)-2, and increased phosphorylation of the mammalian target of rapamycin (mTOR). There was an increase in the expression of the ER stress chaperones CCAAT/enhancer-binding protein homologous protein (CHOP), immunoglobulin-heavy-chain-binding protein (BiP/GRP78), and 94-kDa glucose-regulated protein (GRP94), and in the mRNA levels of pancreatic ER kinase (PERK), activating transcription factor 6 (ATF6), ATF4, inositol- requiring enzyme 1 (IRE1), and spliced X-box-binding protein-1 (XBP1). Phospho-IRE1, ATF6, and phospho-PERK protein concentration also increased significantly. Immunohistochemical staining of a-SMA indicated an abrogation of hepatic stellate cells activation by melatonin. Furthermore, treatment with the indole resulted in significant inhibition of the autophagic flux and the unfolded protein response. Findings from this study give new insight into molecular pathways accounting for the protective effect of melatonin in fibrogenesis. Beatriz San-Miguel 1 , Irene Crespo 1,2 , Diana I. S  anchez 1 , B  arbara Gonz  alez-Fern  andez 1 , Juan J. Ortiz de Urbina 1 , Mar  ıa J. Tu~ n on 1,2 and Javier Gonz  alez-Gallego 1,2 1 Institute of Biomedicine (IBIOMED), University of Le on, Le on, Spain; 2 Centro de Investigaci on Biom edica en Red de Enfermedades Hep aticas y Digestivas (CIBERehd), Le on, Spain Key words: autophagy, carbon tetrachloride, ER stress, fibrosis, liver, melatonin Address reprint requests to Mar  ıa J. Tu~ n on, PhD, Institute of Biomedicine, University of Le on, 24071-Le on, Spain. E-mail: mjtung@unileon.es Received March 5, 2015; Accepted May 5, 2015. Introduction Hepatic fibrosis is a reversible wound-healing response to chronic liver injury from a variety of causes, characterized by an excessive deposition of extracellular matrix (ECM) leading to liver dysfunction and irreversible cirrhosis, which results in high morbidity and mortality. The fibro- genic process is driven by transdifferentation of hepatic stellate cells (HSCs) to a-smooth muscle actin (SMA)-posi- tive myofibroblastic cells which synthesize excess ECM components [1]. Hepatic fibrosis may be reversed upon removal of the underlying etiological agent [2], which opens the possibility for the development of antifibrotic therapeutic approaches. However, effective clinical thera- pies are still lacking and there is a need for searching anti- fibrotic strategies which can prevent, halt, or reverse hepatic fibrosis. Melatonin is a versatile indolamine which has been reported to abrogate activation of HSCs induced by reac- tive oxygen species in vitro [3] and to prevent liver damage in mice and rats with fibrosis induced by bile duct ligation [4], dimethylnitrosamine [5], thioacetamide [6], or carbon tetrachloride (CCl 4 ) [7]. Using the CCl 4 -mediated model, it has been found that melatonin administration prevents liver histopathological changes, reduces hepatic hydroxy- proline content, inhibits oxidative stress and apoptosis or abrogates proinflammatory cytokine production, when administered to rats or mice [8–13]. We have recently shown that melatonin treatment impairs HSCs activation and results in a significant inhibition of the expression of profibrogenic factors in CCl 4 -treated mice [14]. Our data also indicated that melatonin lowers metalloproteinase (MMP)-9 activity, supporting previous results on melato- nin binding to the active site of the enzyme [14, 15]. Autophagy is a metabolic process which controls the clearance and recycling of intracellular constituents for the maintenance of cellular survival [16] and is known to par- ticipate in both sensing oxidative stress and removing oxidatively damaged proteins and organelles [17]. In autophagy, a doubled-membrane-bound vacuole, known as the autophagosome, engulfs fractions of the cytoplasm via the activity of the autophagy adaptors, such as seques- tosome 1 (p62/SQSTM1), which facilitates the autophagic degradation of ubiquitinated protein aggregates in 151 J. Pineal Res. 2015; 59:151–162 Doi:10.1111/jpi.12247 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Journal of Pineal Research Molecular, Biological, Physiological and Clinical Aspects of Melatonin