Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress

Detalhes bibliográficos
Autor(a) principal: Amorim, Ricardo Fernando Santos
Data de Publicação: 2022
Idioma: eng
Título da fonte: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
Texto Completo: https://hdl.handle.net/10316/100367
Resumo: Doutoramento em Biologia Experimental e Biomedicina apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra.
id RCAP_9d8deee49e123760e975d03f95947f7d
oai_identifier_str oai:estudogeral.uc.pt:10316/100367
network_acronym_str RCAP
network_name_str Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
repository_id_str https://opendoar.ac.uk/repository/7160
spelling Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stressDietary antioxidantsCaffeic acidMitochondriaMitochondria-targeted antioxidantsNrf2Antioxidant defensesNon-alcoholic fatty liver disease (NAFLD)Oxidative stressMitochondria dys(function)MitophagyMedical and Health SciencesBasic ScienceDoutoramento em Biologia Experimental e Biomedicina apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra.Non-alcoholic fatty liver disease (NAFLD) is a public health concern affecting 24% of the population worldwide. Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. NAFLD is a multifactorial disease and is considered the hepatic component of metabolic syndrome. Although mechanisms underlying disease pathophysiology are not fully clarified, mitochondrial dysfunction and oxidative stress (OxS) are potential key players. Mitochondria are key organelles involved in cellular survival, differentiation, and death induction. In this regard, mitochondrial morphology and/or function alterations are involved in stress-induced adaptive pathways, priming mitochondria for mitophagy or apoptosis induction. In this context and driven by the lack of effective pharmacological therapies, we propose a new approach using a mitochondria-targeted antioxidant (AntiOxCIN4) to prevent non-alcoholic fatty liver (NAFL) development. Previously studies shown that the mitochondriotropic antioxidant AntiOxCIN4 (100 μM; 48 h) presented significant cytoprotective effect without affecting the viability of human hepatoma-derived (HepG2) cells. Moreover, AntiOxCIN4 (12.5 μM; 72 h) caused a mild increase of reactive oxygen species (ROS) levels without toxicity to primary human skin fibroblasts (PHSF). As Nrf2 is a master regulator of the OxS response inducing antioxidant-encoding gene expression, we hypothesized that AntiOxCIN4 could increase the resistance of human hepatoma-derived HepG2 to oxidative by Nrf2-dependent mechanisms, in a process mediated by mitochondrial ROS (mtROS). In fact, in chapter 7, we showed that after an initial decrease in oxygen consumption paralleled by a moderate increase in superoxide anion levels, AntiOxCIN4 led to a time-dependent Nrf2 translocation to the nucleus. This was followed later by an increase in basal respiration (150 %) and extracellular acidification (120 %). AntiOxCIN4 treatment enhanced mitochondrial quality by triggering the clearance of defective organelles by autophagy and/or mitophagy, coupled with increased mitochondrial biogenesis. Anti-OxCIN4 also upregulated the cellular antioxidant defense system. AntiOxCIN4 seems to have the ability to maintain hepatocyte redox homeostasis, regulating the electrophilic/nucleophilic tone, and preserve cellular physiological functions. The obtained data in chapter 7 opened a new avenue to explore the effects of AntiOxCIN4 in the context of preserving hepatic mitochondrial function in disorders, such as NASH/NAFLD and type II diabetes. In agreement with that hypothesis, we next characterized the human HepG2 cells as an in vitro model for steatosis (chapter 8), in order to screen mitochondriotropic antioxidants lipid-lowering ability. Using an exploratory data analysis, we investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an increased expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. Unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function. Finally, to validate the beneficial effects of hydroxycinnamic-derived mitochondriotropic antioxidant AntiOxCIN4, C57BL/6J mice daily supplemented with 2.5 mg An-tiOxCIN4 were fed with a standard diet (SD) or Western diet (WD) (30% high-fat, 30% high-sucrose) for 16 weeks to induce simple steatosis in vivo. Additionally, we treated HepG2 cells with AntiOxCIN4 (100 μM, 48 h) before the exposure to bovine serum albumin (BSA) alone or free fatty acids (FFAs) mixture (250 µM, 24 h) to induce lipid accumulation in vitro (chapter 9). In WD-fed mice, AntiOxCIN4 supplementation de-creased body (by 43 %), liver weight (by 39 %) and plasma hepatocyte damage markers. The improvement in hepatic-related parameters was associated with a reduction of fat liver accumulation (600 %) and the remodeling of fatty acyl chain composition compared with the WD-fed group. Data on human HepG2 cells confirmed a lower lipid accumulation by the evident reduction of lipid droplets size and number in AntiOxCIN4-treated cells. AntiOxCIN4 supplementation induced mitochondrial metabolism remodeling by upregulating oxidative phosphorylation (OXPHOS) subunits, mediated by the PGC-1α-SIRT3. Accordingly, human HepG2 data corroborated that AntiOxCIN4 pre-treatment stimulated fatty acid oxidation-linked oxygen consumption rates and OXPHOS gene expression remodeling. AntiOxCIN4 also upregulated hepatic antioxidant defense system in WD-fed mice and counteracted cellular ROS exacerbation in FFA-treated human HepG2 cells. Finally, AntiOxCIN4 supplementation prevented lipid ac-cumulation-driven autophagic flux impairment, by increasing lysosomal proteolytic capacity as observed both in vivo and in vitro Overall, the results obtained in this dissertation added novel and relevant knowledge by showing that AntiOxCIN4 improved NAFL in vivo, via three main mechanisms: a) increasing mitochondrial function (fatty acid oxidation); b) regulating antioxidant capacity (enzymatic and non-enzymatic) and; c) preventing the impairment in autophagy. Together, the findings support the potential use of AntiOxCIN4 in the prevention/ treatment of NAFLD.2022-04-21doctoral thesisinfo:eu-repo/semantics/publishedVersionhttps://hdl.handle.net/10316/100367https://hdl.handle.net/10316/100367TID:101680163engAmorim, Ricardo Fernando Santosinfo:eu-repo/semantics/openAccessreponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiainstacron:RCAAP2022-06-14T20:31:37Zoai:estudogeral.uc.pt:10316/100367Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireinfo@rcaap.ptopendoar:https://opendoar.ac.uk/repository/71602025-05-29T05:49:36.235740Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiafalse
dc.title.none.fl_str_mv Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress
title Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress
spellingShingle Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress
Amorim, Ricardo Fernando Santos
Dietary antioxidants
Caffeic acid
Mitochondria
Mitochondria-targeted antioxidants
Nrf2
Antioxidant defenses
Non-alcoholic fatty liver disease (NAFLD)
Oxidative stress
Mitochondria dys(function)
Mitophagy
Medical and Health Sciences
Basic Science
title_short Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress
title_full Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress
title_fullStr Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress
title_full_unstemmed Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress
title_sort Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress
author Amorim, Ricardo Fernando Santos
author_facet Amorim, Ricardo Fernando Santos
author_role author
dc.contributor.author.fl_str_mv Amorim, Ricardo Fernando Santos
dc.subject.por.fl_str_mv Dietary antioxidants
Caffeic acid
Mitochondria
Mitochondria-targeted antioxidants
Nrf2
Antioxidant defenses
Non-alcoholic fatty liver disease (NAFLD)
Oxidative stress
Mitochondria dys(function)
Mitophagy
Medical and Health Sciences
Basic Science
topic Dietary antioxidants
Caffeic acid
Mitochondria
Mitochondria-targeted antioxidants
Nrf2
Antioxidant defenses
Non-alcoholic fatty liver disease (NAFLD)
Oxidative stress
Mitochondria dys(function)
Mitophagy
Medical and Health Sciences
Basic Science
description Doutoramento em Biologia Experimental e Biomedicina apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra.
publishDate 2022
dc.date.none.fl_str_mv 2022-04-21
dc.type.driver.fl_str_mv doctoral thesis
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://hdl.handle.net/10316/100367
https://hdl.handle.net/10316/100367
TID:101680163
url https://hdl.handle.net/10316/100367
identifier_str_mv TID:101680163
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.source.none.fl_str_mv reponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron:RCAAP
instname_str FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron_str RCAAP
institution RCAAP
reponame_str Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
collection Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
repository.name.fl_str_mv Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
repository.mail.fl_str_mv info@rcaap.pt
_version_ 1833602485613232128

Registros relacionados