Analysis of oxidative modification of rat epididymal mitochondrial proteins under normobaric chronic hypoxia and modulation of nitric oxide (II) synthesis

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Abstract

Background. The molecular mechanism of NO effects in adaptation to hypoxia is of interest as a potential point of application in the therapy of fertility disorders.

Aim. To assess the degree of oxidative modification of epididymal mitochondrial proteins during hypoxia under conditions of experimentally modified NO synthesis.

Material and methods. Sexually mature rats were divided into four groups of 8 individuals: (1) control (animals without hypoxia); (2) chronic normobaric hypoxia, the animals were kept in a sealed chamber with oxygen reduced to 10% once a day for 14 days; (3) animals with hypoxia were injected with an inhibitor of the synthesis of nitric oxide (II) L-nitroarginine methyl ester at a dose of 25 mg/kg intraperitoneally once a day for 7 days; (4) animals with hypoxia were injected with the substrate for NO synthesis L-arginine at a dose of 500 mg/kg intraperitoneally once a day for 10 days. In the mitochondrial fraction of the head and tail of the epididymis, the activity of superoxide dismutase, the amount of NO metabolites, and the degree of oxidative modification of proteins were evaluated. Statistical analysis was performed using the Shapiro–Wilk, Mann–Whitney, and Spearman tests; differences were considered significant at p <0.05.

Results. Hypoxia led to an increase in the oxidative modification of proteins in the mitochondria of the head of the epididymis — 319.12 [240.98; 363.63] c.u./mg protein relative to control — 17.89 [15.31; 27.62] c.u./mg protein, p=0.0009. In the mitochondria of the tail of the epididymis, the oxidation of proteins under the studied conditions was less pronounced. The use of L-nitroarginine methyl ester, as well as L-arginine, led to a decrease in the level of oxidative modification of proteins in the head of the epididymis relative to the hypoxia model — 39.89 [29.25; 43.17] and 37.25 [34.91; 40.96] c.u./mg of protein, respectively, p=0.0009.

Conclusion. Mitochondrial proteins in the head of the epididymis are more susceptible to oxidative damage during hypoxia; a decrease in the level of NO metabolites under conditions of oxygen deficiency is associated with a decrease in the oxidative modification of mitochondrial proteins.

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About the authors

Yuliya A. Marsyanova

Ryazan State Medical University named after I.P. Pavlov

Author for correspondence.
Email: yuliyamarsyanova@yahoo.com
ORCID iD: 0000-0003-4948-4504
SPIN-code: 4075-3169

Assist., Depart. of Biological Chemistry with the Course of Clinical Laboratory Diagnostics

Russian Federation, Ryazan, Russia

Valentina I. Zvyagina

Ryazan State Medical University named after I.P. Pavlov

Email: vizvyagina@yandex.ru
ORCID iD: 0000-0003-2800-5789

Cand. Sci. (Biol.), Assoc. Prof., Depart. of Biological Chemistry with the Course of Clinical Laboratory Diagnostics

Russian Federation, Ryazan, Russia

Artem V. Petrov

Ryazan State Medical University named after I.P. Pavlov

Email: chasedflea089@icloud.com
ORCID iD: 0000-0002-0639-3891

student

Russian Federation, Ryazan, Russia

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2. Рис. 1. А — сравнение количества метаболитов оксида азота (II) (в нмоль/мг белка); В — активность супероксиддисмутазы (в у.е./мг белка); *p <0,05 по сравнению с группой контроля; #p <0,05 по сравнению с группой хронической нормобарической гипоксии (ХНГ); L-NAME — метиловый эфир L-нитроаргинина

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3. Рис. 2. Спектры поглощения 2,4-динитрофенилгидразин-производных аминокислот в белках; А — в митохондриях головки эпидидимиса; В — в митохондриях хвоста эпидидимиса; ось х — длина волны, ось у — условные единицы оптической плотности; ХНГ — хроническая нормобарическая гипоксия; L-NAME — метиловый эфир L-нитроаргинина

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4. Рис. 3. Сравнение изменения резервно-адаптационного потенциала; 100% — металл-зависимая окислительная модификация белков (ОМБ), моделируемая в лабораторных условиях, окрашенная часть шкалы — доля спонтанной окислительной модификации белков от металл-зависимой (%), неокрашенная часть шкалы — резервно-адаптационный потенциал (доля потенциально возможной окислительной модификации белков; %); *p <0,05 при сравнении с группой контроля; L-NAME — метиловый эфир L-нитроаргинина

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