Аннотация
Инсулин является одним из наиболее перспективных нейропротекторов. Существенным пробелом в понимании механизма его действия является отсутствие данных о том, способен ли он предотвращать аутофагическую гибель нейронов. Целью нашей работы была оценка вклада аутофагии и апоптоза в гибель нейронов коры мозга крыс в культуре при окислительном стрессе и изучение способности инсулина предотвращать эту гибель и ингибировать процессы аутофагии и апоптоза в нервных клетках. Изучено влияние перекиси водорода и инсулина на уровень двух основных маркеров аутофагии (LC3BII и SQSTM1/p62) и маркера апоптоза (расщепленная каспаза-3). Для оценки жизнеспособности нейронов использовали МТТ-тест, для измерения уровня маркерных белков применяли иммуноблоттинг. Найдено, что окислительный стресс вызывает активацию аутофагии и апоптоза в нейронах. Это проявляется в достоверном увеличении маркеров аутофагии LC3B-II и апоптоза (расщепленной каспазы-3) и в снижении уровня белка SQSTM1/p62. Содержание SQSTM1/p62, участвующего в образовании аутофагосом, снижается при активации аутофагии, т.к. белок подвергается деструкции в лизосомах. Перекись водорода вызывает аутофагическую и апоптотическую гибель нейронов, о чем свидетельствует значительное увеличение жизнеспособности нейронов в условиях окислительного стресса при действии ингибиторов аутофагии (3-метиладенина) и апоптоза (z-DEVD-FMK). В свою очередь инсулин, предотвращая гибель нейронов при окислительном стрессе, препятствует развитию аутофагии, вызывая снижение уровня липидированной формы LC3B-II и увеличение уровня SQSTM1/p62, и апоптоза, уменьшая уровень расщепленной каспазы-3. Защитное действие инсулина опосредуется активацией специфических сигнальных путей, связанных с рецепторами инсулина и IGF-1. Так, ингибитор рецепторов инсулина и IGF-1 BMS-754807 полностью блокирует нейропротекторный эффект инсулина. Таким образом, ярко выраженная активация аутофагии при окислительном стрессе является одной из причин гибели нейронов, а защита нейронов инсулином связана с подавлением не только апоптотической, но и аутофагической гибели этих клеток.
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