Kalıtsal Metabolik Hastalıklarda Antioksidan Yanıt ve Otofajinin P62/Nrf2/Keap1 Yolağı Aracılığıyla Araştırılması

Abstract

Due to the enzyme, enzyme cofactor, carrier deficiency or disorder in the metabolic pathways, Inherited Metabolic Diseases (IMDs) cause the accumulation of the substrate and / or end product deficiency that are the single gene disorders, among the important causes of childhood morbidity and mortality. From a pathophysiological point of view, metabolic disorders are examined in three main groups: intoxication type, energy deficiency type and storage type.

Oxidative stress (OS) plays an important role in the pathophysiology of many diseases, including IMDs. In IMDs, ROS (reactive oxygen species) and RNS (reactive nitrogen species) contribute significantly to disease pathophysiology as well as accumulated toxic metabolites. It is known that OS causes mitochondrial dysfunction, the change of metabolites in cellular pathways, damage to molecules including proteins, lipids and DNA. Reductive stress is dangerous for ensuring cell homeostasis as OS that reduce cell growth responses, cause changes in the formation of disulfide bonds in proteins, decrease mitochondrial function and cellular metabolism.

The Nrf2/Keap1 pathway is considered the main regulator of cellular defense mechanisms against cellular stress that allows the cell to survive in stress conditions by regulating gene expressions of various cell protective protein networks involved in repairing or eliminating damaged macromolecules. Also, selective autophagy is linked via Nrf2/Keap1/p62. Before the cell is directed to death with increased OS, p62 functions as a protective alarm system, as a cargo receptor for autophagy.

The aim of this study is to investigate the relationship between OS, mitochondrial dysfunction and autophagy in IMDs. For this purpose, 6 healthy and 45 individuals with IMD (methylmalonic (MMA), propionic (PA), isovaleric (IVA) acidemia, mitochondrial diseases (MIT) and mucopolysaccharidosis IV (MPS IV) were examined. Six different experimental plans were created using peripheral blood including TAS (Total Antioxidant Status)/TOS (Total Oxidant Status) analysis, intracellular ATP, ROS and mitochondrial membrane potential (MMP) measurement and cell imaging, real-time polymerase chain reaction (RT-PCR) and Western blot analysis.

As a result of TAS/TOS analysis, the highest antioxidant response was found in the MMA group compared to the healthy group. In cell experiments, while high ROS values were determined in the MMA and IVA groups compared to healthy ones; low ROS values were detected in PA, MIT and MPS IV groups. In intracellular ATP amounts of alteration are compatible with ROS changes of IMD groups. Although there are no major changes in MMP in IMD groups compared to the healthy group, alteration of mitochondrial function have been detected. Cell imaging experiments demonstrate that each patient in the IMD groups should be evaluated separately. Induction of the Nrf2/Keap1/p62 pathway was detected in all IMD groups compared to the healthy group, and the increase in HMOX1 expression indicated that the main detoxification enzyme was HO-1.

The increase in LC3B-II and p62 protein expression detected in MIT and MPS IV groups indicates that there is a problem in autophagic flux; increased LC3B-II and decreased p62 in the MMA, PA and İVA groups indicate autophagic flux induction. Partial expression of Beclin1 observed in all IMDs is an adaptation that enables autophagy activation without directing the cells to the death pathways.