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Aquaporins involvement in hepatic ischemia reperfusion injury
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A lesão de isquemia-reperfusão (IR) hepática é uma complicação inevitável durante a cirurgia hepática, causada pela interrupção do fluxo sanguíneo e subsequente inflamação durante a revascularização do fígado. Uma vez que esta condição patológica tem uma alta taxa de mortalidade associada, há um interesse crescente na investigação de métodos e estratégias para prevenir e atenuar a IR hepática.
A IR hepática é caracterizada por uma fase de isquemia, em que a falta de oxigénio e a depleção de ATP leva à interrupção da microcirculação e ao dano mitocondrial, e uma fase de reperfusão, distinguida pela ativação do sistema imunológico do fígado que envolve ativação de macrófagos e neutrófilos, produção de citocinas e quimiocinas, stress oxidativo e aumento da expressão de moléculas de adesão no endotélio hepático. Os mecanismos de resolução de inflamação no fígado são conhecidos uma vez que alguns mediadores anti-inflamatórios são capazes de induzir a cicatrização do tecido hepático e a proliferação de hepatócitos.
O sistema hepatobiliar é responsável por diversos processos metabólicos, incluindo formação e secreção biliar, desintoxicação e metabolismo do glicerol. Como a bile é predominantemente composta por água, a formação de bile canalicular é um processo de secreção osmótica que resulta da entrada de água em resposta a gradientes osmóticos criados pela secreção activa de solutos. Quando os hepatócitos são estimulados, o transporte de água ocorre significativamente pela via transcelular através da membrana plasmática.
Foi demonstrado que várias células do trato hepatobiliar expressam aquaporinas (AQPs), pequenas proteínas transmembranares conhecidas por auxiliar no transporte transmembranar de água, aumentando a permeabilidade à água e também permitindo o transporte de glicerol e outros pequenos solutos. Além disso, como durante a revascularização do fígado a permeabilidade da membrana é afectada por processos de inflamação, as AQPs situadas em várias células do fígado representam importantes reguladores do equilíbrio hemodinâmico durante a IR.
Considerando o papel que as AQPs podem desempenhar durante os processos de IR do fígado, o foco deste trabalho consistiu em avaliar o efeito da IR na expressão de AQPs. O ensaio in vitro com células HepG2 expostas a diferentes estímulos de IR permitiu distinguir o efeito da inflamação do estímulo de hipoxia na expressão de AQPs. Mediante estímulo de inflamação com LPS, as células induziram a expressão de AQPs a fim de restabelecer a homeostase e atenuar os processos biológicos instigados pela sobre-expressão de mediadores da inflamação. Opondo-se à resposta inflamatória, mediante estímulo de isquemia, as células submetidas a uma câmara de hipoxia suprimem a expressão de AQPs como um mecanismo de prevenção de influxo de água nas células e manutenção da homeostase da água durante o processo inflamatório inicial e prevenção de edema celular.
Além disso, a administração de formulações lipossomais de fármacos demonstrou ser eficaz na atenuação dos processos de inflamação causados pela exposição das células HepG2 a compostos tóxicos e disfunções relacionadas à inflamação nas células do fígado, já que ambos os fármacos testados (prednisolona e ibuprofeno) mostraram efeitos favoráveis na reversão da inflamação induzida por LPS, diminuindo significativamente a expressão de AQP3 e citocinas.
O ensaio in vivo do efeito de IR em células de fígado de rato permitiu delinear um perfil de expressão de AQPs ao longo dos diferentes tempos do processo de IR. As células suprimiram significativamente a expressão de AQPs após 2h de reperfusão, a fim de se opor aos mecanismos decorrentes do processo de isquemia e evitar o influxo de água na célula e edema celular nas primeiras horas de reperfusão, então induziram a expressão de AQPs após 6h de reperfusão a fim de restabelecer homeostase da água e, finalmente, após 24h de reperfusão as células aumentaram a expressão de AQPs. Como a expressão de AQPs e TNF-ɑ após 24h de reperfusão se assemelhava à condição de normóxia, este ensaio confirmou que após 24h de reperfusão as células passam por processos de resolução da inflamação e retornam à homeostase. Além disso, o tratamento com o composto natural quercetina foi ineficiente em atenuar os processos de inflamação induzidos por IR, pois os perfis de AQPs assemelham-se às amostras sem tratamento. Apesar disso, a administração intravenosa de quercetina lipossomal (formada por SPC) apresentou maior expressão de AQPs do que a administração intraperitoneal de quercetina lipossomal (formada por EPC).
Para uma melhor avaliação do papel das AQPs nos mecanismos de IR do fígado, devem ser realizados ensaios funcionais e, em última análise, devem ser investigadas estratégias para direcionar e regular as AQPs durante a IR do fígado com o objetivo de reduzir a alta taxa de mortalidade associada a essa condição.
Hepatic ischemia-reperfusion (IR) injury (IRI) is an unavoidable complication during liver surgery caused by the interruption of blood flow and subsequent inflammation during liver revascularization. Since this pathological condition has a high mortality rate associated, there is a growing interest in assessing methods and strategies to prevent and attenuate liver IRI. Liver IR is characterized by an ischemia phase, where the lack of oxygen supply and ATP depletion leads to microcirculation disruption and mitochondrial damage, and a reperfusion phase distinguished by activation of the liver immune system which involves activation of macrophages and neutrophils, production of cytokines and chemokines, generation of oxidative stress, and increased expression of adhesion molecules on the hepatic endothelium. Mechanisms of inflammation resolution have been reported as some anti-inflammatory mediators are capable of inducing tissue repair and hepatocytes proliferation. The hepatobiliary system is responsible for several metabolic processes including bile formation and secretion, detoxification and glycerol metabolism. As bile is predominantly composed of water, canalicular bile formation is an osmotic secretory process that results from the input of water in response to osmotic gradients created by active secretion of solutes. When hepatocytes are stimulated, water transport occurs significantly via transcellular pathway through the plasma membrane. Several cells from the hepatobiliary tract have been shown to express aquaporins (AQPs), which are small transmembrane proteins known to assist on the transcellular water transport by increasing water permeability and also allow the transport of glycerol and other small solutes. Additionally, since during the liver revascularization membrane permeability is affected by inflammation processes, AQPs situated in several liver cells pose as important regulators of hemodynamic equilibrium during IR. Considering the role that AQPs may play during liver IRI processes, the focus of this work consisted on assessing the IR effect on AQPs expression. The in vitro assay with HepG2 cells exposed to different IR stimulus allowed to distinguish the effect of inflammation and hypoxia stimulus on AQPs expression. Upon severe inflammation stimulus with LPS, cells induced AQPs expression in order to reestablish homeostasis and attenuate biological processes instigated by overexpression of inflammation mediators. Opposing the inflammatory response, upon severe ischemia stimulus, cells subjected to a hypoxia chamber suppress AQPs expression as a mechanism of preventing water influx into the cells and maintaining water homeostasis during the initial inflammatory process and preventing cell swelling. Moreover, administration of liposomal drugs formulations proved effective in attenuating inflammation processes caused by the HepG2 cells exposure to toxic compounds and inflammation-related dysfunction in liver cells, as both pharmaceutical drugs tested (prednisolone and ibuprofen) showed favorable effects in reversing LPS-induced inflammation by decreasing significantly AQP3 and cytokines expression. The in vivo assay of IR effect on rat liver cells allowed to delineate a profile of AQPs expression along the different timepoints of the IR process. Cells suppressed significantly the AQPs expression after 2h of reperfusion in order to oppose the mechanisms ensued in the ischemia process and prevent water influx into the cell and cell edema within the first hours of reperfusion, then induced AQPs expression after 6h of reperfusion in order to reestablish water homeostasis and finally after 24h of reperfusion cells increased AQPs expression. As AQPs and TNF-ɑ expression after 24h of reperfusion resembled the normoxia condition, this assay confirmed that at this timepoint the cells are undergoing processes of inflammation resolution and returning to homeostasis. Moreover, treatment with the flavonoid quercetin was inefficient in attenuating the IR-induced inflammation processes, as AQPs profiles resembled the samples without treatment. Regardless, intravenous administration of SPC liposomal quercetin presented higher AQPs expression than intraperitoneal administration of EPC liposomal quercetin. In order to further assess the role of AQPs in liver IR mechanisms, functional assays should be carried out and, ultimately, strategies to target and regulate AQPs during liver IR should be investigated aiming to reduce the high mortality rate associated with this condition.
Hepatic ischemia-reperfusion (IR) injury (IRI) is an unavoidable complication during liver surgery caused by the interruption of blood flow and subsequent inflammation during liver revascularization. Since this pathological condition has a high mortality rate associated, there is a growing interest in assessing methods and strategies to prevent and attenuate liver IRI. Liver IR is characterized by an ischemia phase, where the lack of oxygen supply and ATP depletion leads to microcirculation disruption and mitochondrial damage, and a reperfusion phase distinguished by activation of the liver immune system which involves activation of macrophages and neutrophils, production of cytokines and chemokines, generation of oxidative stress, and increased expression of adhesion molecules on the hepatic endothelium. Mechanisms of inflammation resolution have been reported as some anti-inflammatory mediators are capable of inducing tissue repair and hepatocytes proliferation. The hepatobiliary system is responsible for several metabolic processes including bile formation and secretion, detoxification and glycerol metabolism. As bile is predominantly composed of water, canalicular bile formation is an osmotic secretory process that results from the input of water in response to osmotic gradients created by active secretion of solutes. When hepatocytes are stimulated, water transport occurs significantly via transcellular pathway through the plasma membrane. Several cells from the hepatobiliary tract have been shown to express aquaporins (AQPs), which are small transmembrane proteins known to assist on the transcellular water transport by increasing water permeability and also allow the transport of glycerol and other small solutes. Additionally, since during the liver revascularization membrane permeability is affected by inflammation processes, AQPs situated in several liver cells pose as important regulators of hemodynamic equilibrium during IR. Considering the role that AQPs may play during liver IRI processes, the focus of this work consisted on assessing the IR effect on AQPs expression. The in vitro assay with HepG2 cells exposed to different IR stimulus allowed to distinguish the effect of inflammation and hypoxia stimulus on AQPs expression. Upon severe inflammation stimulus with LPS, cells induced AQPs expression in order to reestablish homeostasis and attenuate biological processes instigated by overexpression of inflammation mediators. Opposing the inflammatory response, upon severe ischemia stimulus, cells subjected to a hypoxia chamber suppress AQPs expression as a mechanism of preventing water influx into the cells and maintaining water homeostasis during the initial inflammatory process and preventing cell swelling. Moreover, administration of liposomal drugs formulations proved effective in attenuating inflammation processes caused by the HepG2 cells exposure to toxic compounds and inflammation-related dysfunction in liver cells, as both pharmaceutical drugs tested (prednisolone and ibuprofen) showed favorable effects in reversing LPS-induced inflammation by decreasing significantly AQP3 and cytokines expression. The in vivo assay of IR effect on rat liver cells allowed to delineate a profile of AQPs expression along the different timepoints of the IR process. Cells suppressed significantly the AQPs expression after 2h of reperfusion in order to oppose the mechanisms ensued in the ischemia process and prevent water influx into the cell and cell edema within the first hours of reperfusion, then induced AQPs expression after 6h of reperfusion in order to reestablish water homeostasis and finally after 24h of reperfusion cells increased AQPs expression. As AQPs and TNF-ɑ expression after 24h of reperfusion resembled the normoxia condition, this assay confirmed that at this timepoint the cells are undergoing processes of inflammation resolution and returning to homeostasis. Moreover, treatment with the flavonoid quercetin was inefficient in attenuating the IR-induced inflammation processes, as AQPs profiles resembled the samples without treatment. Regardless, intravenous administration of SPC liposomal quercetin presented higher AQPs expression than intraperitoneal administration of EPC liposomal quercetin. In order to further assess the role of AQPs in liver IR mechanisms, functional assays should be carried out and, ultimately, strategies to target and regulate AQPs during liver IR should be investigated aiming to reduce the high mortality rate associated with this condition.
Descrição
Tese de mestrado, Ciências Biofarmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia
Palavras-chave
Hepatic ischemia-reperfusion (IR) Aquaporins (AQPs) Ischemia stimulus Inflammation stimulus Liposomal drug formulations Cytokines expression Inflammation resolution Water homeostasis Teses de mestrado -2021