毛蕊花苷对运动诱导的疲劳骨骼肌细胞肌浆网RyR1通道稳定性的调节与机制研究

The studies have shown that skeletal muscle sarcoplasmic reticulum RyR1 stability decreased, which made the excitation-contraction coupling disorders in skeletal muscle cells, is the important reason of skeletal muscle fatigue caused by exercise.The project team' previous studies showed that a main active ingredient of Pedicularis species-verbscoside could significantly retard skeletal muscle fatigue. This project aims to use the mice of exercise-induced skeletal muscle fatigue model and apply single-channel patch-clamp recording technique and laser scanning confocal microscope system etc.to research the effects of verbscoside on skeletal muscle sarcoplasmic reticulum RyR1 opening frequency, cytosolic calcium sparks, sarcoplasmic reticulum calcium intake back and calcium leakage etc.of mice of exercise-induced skeletal muscle fatigue ,which research regulations of verbascoside on the stability of sarcoplasmic reticulum RYR1 in skeletal muscle cells with fatigue caused by exercise.It also aims to apply the methods of in situ hybridization, immunoblotting, immunoprecipitation etc.to research the effects of verbscoside on nNOS/NO/ nitrosylation signaling pathway etc.of mice of exercise-induced skeletal muscle fatigue, which investigate mechanisms of verbscoside regulating sarcoplasmic reticulum RYR1 stability in skeletal muscle cells with fatigue caused by exercise. The study is to seek new mechanisms of verbscoside' anti-skeletal muscle fatigue and new prevention strategies for skeletal muscle fatigue caused by exercise.

研究表明骨骼肌细胞肌浆网RyR1通道稳定性下降，使骨骼肌兴奋-收缩偶联失调，是运动导致骨骼肌疲劳的重要机制。项目组的前期研究表明马先蒿属植物中主要活性成分-毛蕊花苷具有显著的抗骨骼肌疲劳作用。因此本项目拟运用小鼠运动性骨骼肌疲劳模型，应用膜片钳单通道记录技术、激光扫描共聚焦显微镜系统等方法，从骨骼肌细胞肌浆网RyR1通道开放率、胞浆钙火花、肌浆网钙回摄和钙渗漏等方面研究毛蕊花苷对运动诱导的疲劳骨骼肌细胞肌浆网RyR1通道稳定性的调节；应用原位杂交、免疫印迹和免疫沉淀等方法，从nNOS/NO/亚硝基化信号通路等方面探讨其调节运动诱导的疲劳骨骼肌细胞肌浆网RyR1通道稳定性的分子作用机制，以期探寻毛蕊花苷抗骨骼肌疲劳的新机制和骨骼肌疲劳防治的新策略。

研究表明骨骼肌细胞肌浆网RyR1通道稳定性下降，使骨骼肌兴奋-收缩偶联失调，是运动导致骨骼肌疲劳的重要机制。本项研究表明:疲劳运动后小鼠骨骼肌细胞肌浆网RyR1开放频率显著升高，肌浆网钙容量和钙火花事件明显减少。毛蕊花苷能通过降低运动小鼠骨骼肌细胞肌浆网RyR1的开放频率，提高运动小鼠骨骼肌细胞肌浆网钙容量和钙火花事件，从而具有较明显的保护小鼠骨骼肌损伤和提高小鼠运动能力作用。并呈一定的剂量依赖性，以高剂量的效果更明显。确定了毛蕊花苷能提高疲劳骨骼肌细胞肌浆网RyR1通道稳定性。并从nNOS/NO/亚硝基化信号通路等方面研究了毛蕊花苷调节运动诱导的疲劳骨骼肌细胞肌浆网RyR1通道稳定性的作用机制。结果表明：NO供体SNAP能降低小鼠的运动能力，能降低运动小鼠肌浆网钙容量和ΔF/F0值，提高运动小鼠骨骼肌组织NO和GSSG含量，降低GSH含量和GSH/GSSG比值，抑制运动小鼠骨骼肌组织细胞nNOS的蛋白表达，但对运动小鼠肌浆网 RyR1复合物中nNOS的蛋白表达作用相反，使RyR1复合物中Cys-NO蛋白表达水平明显升高，calstabin表达水平降低。nNOS抑制剂7-NI的作用则相反。毛蕊花苷能抵抗运动导致的小鼠骨骼肌肌酸激酶(CK)和钙蛋白酶-3（Calpain-3)活性的增高，提高运动小鼠肌浆网钙容量和ΔF/F0值，降低运动小鼠骨骼肌组织NO和GSSG含量，提高GSH含量和GSH/GSSG比值，抑制运动小鼠骨骼肌组织细胞和肌浆网 RyR1 复合物中nNOS的蛋白表达，降低运动小鼠骨骼肌细胞肌浆网 RyR1 复合物中Cys-NO蛋白表达水平，提高calstabin表达水平，并能抵抗NO供体SNAP的上述作用，与nNOS抑制剂7-NI有协同促进作用。显示毛蕊花苷可能通过nNOS/NO/亚硝基化信号通路提高运动诱导的疲劳骨骼肌细胞肌浆网RyR1通道的稳定性。为毛蕊花苷的进一步结构优化和开发提供了实验依据。