Exploração da Funcionalidade de Mitocôndrias Extracelulares e a sua Associação com Inflamção em Hepatócitos

Abstract

The discovery of mitochondria outside the cells and circulating in plasma has challenged the current knowledge about mitochondria and their role in cellular metabolism. This study proposes to verify if mitochondria are released by human hepatocytes (HepG2), to evaluate if an inflammatory stimulus (LPS) promotes their release, to compare the functionality of intracellular and released mitochondria and to identify possible cellular mechanisms involved in this process. The results demonstrated the existence of extracellular mitochondria, identified by the expression of TOM20 (western blot), capable of consuming O2 (OROBOROS Oxygraph-2k), although in uncoupled respiration. Interestingly, a preserved response to exogenous substrates of complex IV was detected in both control conditions and after the inflammatory stimulus (2.5 𝜇g/𝜇L LPS, 24h). LPS increased IL-6 expression, but did not induce an increase in mitochondrial release, although it altered the mitochondrial functionality of both intracellular and extracellular mitochondria. During this process, most cells remained viable (trypan blue, MTS and flow cytometry), although the expression of proteins involved in autophagy increased (LC3I and II and beclin 1). The expression of Mfn1, Mfn2 and OPA1 proteins did not allow to conclude about the implication of mitochondrial dynamic mechanisms, in the process, but the expression of mitophagy proteins such as Pink1 was detected in extracellular mitochondria. Our study confirmed and supported recent studies regarding the release of extracellular mitochondria and its appearance outside cells. Importantly, we could not prove that released mitochondria maintain their normal functionality, that seems to be lost, at least in part. We also were not able to confirm that inflammation promote their release. Thus, although our results clarify some points, several questions such as what promotes the release of mitochondria and their roles outside cells, need to be further explored.