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ラット骨格筋における急性疲労運動による酸化ストレスのメカニズム
[Mechanism of oxidative stress in skeletal muscle of rats induced by acute exhaustive exercise].
PMID: 32476368 DOI: 10.12047/j.cjap.5841.2020.004.
抄録
目的:
ラットの骨格筋における酸化ストレス関連酵素の発現に及ぼす急性疲労運動の影響を観察する。
OBJECTIVE: To observe the effects of acute exhaustive exercise on the expressions of oxidative stress related enzymes in skeletal muscle of rats.
方法:
雄性SDラット40匹を4群に分け、各群10匹ずつ、対照群（C群）、疲弊運動群（E群）、運動＋PKC阻害剤投与群（EC群）、運動＋NOX阻害剤投与群（EA群）とした。3群の運動ラットを3日間トレッドミルランニングに慣れさせ（5m/min、1回/d、傾斜なし）、その後1日安静にした。ＥＣ群には運動の１日前と１時間前にＰＫＣ阻害剤クレリスリン（５ｍｇ／ｋｇ）を、ＥＡ群にはＮＡＤＰＨ酸化酵素阻害剤アポシニン（１０ｍｇ／ｋｇ）を同時に、Ｃ群とＥ群には同量の通常の生理食塩水を注射した。3群の運動ラットに1回のトレッドミル排せつ運動を行い、排せつ後の植物体を採取した。活性酸素種（ＲＯＳ）はＤＣＦ蛍光プローブで検出し、ＮＯＸ２、ＮＯＸ４、３-ＮＴはウエスタンブロットで、ＰＫＣ、ＮＯＸ２、ＮＯＸ４は免疫沈降法で分析した。
METHODS: Forty male SD rats were divided into 4 groups, 10 rats in each group, which were the control group (C group), exhausted exercise group (E group), exercise + PKC inhibitor group (EC group), exercise + NOX inhibitor group (EA group). Three groups of exercise rats were familiarized with treadmill running for 3 days (5 m/min, once/d, no incline), then rested for one day. EC group was injected with PKC inhibitor chelerythrine (5 mg / kg) one day before and one hour before exercise, EA group was injected with NADPH oxidase inhibitor apocynin (10 mg / kg) at the same time, group C and group E were injected with the same dose of normal saline. Three groups of exercise rats were subjected to a one-time treadmill exhaustion exercise, and the plantaris were taken after exhaustion. Reactive oxygen species (ROS) were detected by DCF fluorescent probe, NOX2, NOX4, 3-NT were analyzed by Western blot, and PKC, NOX2, NOX4 were analyzed by immunoprecipitation.
結果:
Ｃ群と比較して、Ｅ群では、ＲＯＳレベル、ＮＯＸ２およびＮＯＸ４タンパク質発現、ＰＫＣ-ＮＯＸ２およびＰＫＣ-ＮＯＸ４複合体レベル、３-ＮＴ産生が有意に増加し（Ｐ＜0.01、Ｐ＜0.05）、ＥＣ群とＥＡ群では、ＲＯＳレベルに有意差はなく（Ｐ＜0.05）、ＥＣ群では、ＮＯＸ４タンパク質発現が有意に増加した（Ｐ＜0.05）。E群と比較して、活性酸素レベル、NOX2およびNOX4タンパク質発現、PKC-NOX2およびPKC-NOX4複合体レベル、3-NT産生は有意に減少した(P＜0.01、P＜0.05)。
RESULTS: Compared with group C, ROS level, NOX2 and NOX4 protein expressions, PKC-NOX2 and PKC-NOX4 complex levels, and 3-NT production in group E were significantly increased (P＜0.01, P＜0.05), and ROS level was no significant difference in group EC and group EA (P＞0.05), and NOX4 protein expression in group EC was significantly increased (P ＜ 0.05). Compared with group E, ROS level, NOX2 and NOX4 protein expressions, PKC-NOX2 and PKC-NOX4 complex levels, 3-NT production were decreased significantly (P＜0.01, P＜0.05).
結論:
排他的運動は骨格筋におけるNOX2およびNOX4タンパク質の発現増加を誘導し、PKCはNOX2を調節することで活性酸素の産生を媒介していると考えられる。
CONCLUSION: Exhausted exercise induces increased expressions of NOX2 and NOX4 proteins in skeletal muscle, and PKC mediates the production of ROS by regulating NOX2.