运动介导HSP70延缓增龄性骨骼肌萎缩的机理研究

The Role of Heat Shock Protein 70 in Exercise-attenuating Sarcopenia

目的:通过对自然衰老C57BL/6小鼠进行长期有氧运动干预,探讨增龄性骨骼肌萎缩发生发展过程中细胞凋亡相关蛋白的变化及运动诱导HSP70对其影响,揭示运动预防增龄性骨骼肌萎缩的作用机理。方法:27周龄雄性C57BL/6小鼠50只,经1周适应性喂养后,随机分为年轻对照组(YS组)、老年对照组(OS组)和老年运动组(OE组)。YS组(28周)小鼠经抓力测试后立即处死取材。OE组小鼠采用30周、每周3~4次、每次50分钟、75%VO2max有氧跑台运动训练(速度12米/分钟,坡度0°)。OS组与OE组小鼠喂养至60周龄后,检测抓力等指标后处死。随即分离各组小鼠腓肠肌,称重后检测小鼠骨骼肌萎缩指数(Sarcopenia index,SI),随后提取腓肠肌总蛋白。采用Western Blot检测小鼠腓肠肌组织HSP70,Cleaved Caspase-9蛋白表达水平;采用Co-IP法检测小鼠腓肠肌HSP70与Apaf-1蛋白结合量;采用TUNEL法检测小鼠腓肠肌细胞凋亡指数。结果:(1)SI变化:OS组小鼠腓肠肌SI较YS组显著减少23.09%,而OE组SI与OS组相比显著增加30.48%;(2)小鼠抓力:OS组小鼠抓力/体重比值比YS组显著减少42.74%,OE组比OS组显著增加21.51%;(3)凋亡指数检测:伴随年龄增加,OS组较YS组显著增加475.28%,而有氧运动之后,OE组小鼠凋亡指数较OS组显著减少46.41%;(4)Western Blot结果:OS组Cleaved Caspase-9表达水平较YS组显著增加45.40%,OE组较OS组显著减少33.40%;(5)腓肠肌HSP70与Apaf-1蛋白的结合量:OS组较YS组HSP70与Apaf-1结合量显著下降67.11%,运动后OE组较OS组显著增加306.31%。结论:(1)60周龄小鼠腓肠肌发生增龄性骨骼肌萎缩,30周有氧运动可显著延缓增龄性骨骼肌萎缩;(2)增龄性骨骼肌萎缩可能与骨骼肌凋亡细胞增多有关,30周有氧运动可显著抑制骨骼肌细胞凋亡;(3)30周有氧运动可增强HSP70与Apaf-1结合量,这可能是运动抑制骨骼肌细胞凋亡,延缓增龄性骨骼肌萎缩的机制之一。

Objectives To observe the changes of apoptosis＇related proteins during the development of sarcopenia and to explore the effect and mechanism of long-term aerobic exercises and heat shock pro- tein 70 （HSP70） on those changes for the natural aging C57BL/6 mice. Methods After 1 week of ac- climatization,50 27-week-old male C57BL/6 mice were randomly divided into a young sedentary con- trol （YS） group,an old sedentary control （OS） group and an old exercises （OE） group. After the grip-strength was measured the mice from group YS were sacrificed at the age of 28 weeks. The mice from group OE exercised on a flat treadmill at 12m/minute for 50 min 3 or 4 times a week for 30 weeks with the intensity of 75% VO2max. At the age of 60 weeks, the mice of group OS and OE were killed after their grip-strength was measured. The gastrocnemius muscle of each group were isolated and weighted, and the sarcopenia index （SI） was measured. Then the total protein of the gastrocnemius mus- cle was extracted. HSP70 and cleaved caspase-9 expression were examined.The binding rate of HSP70 and Apaf-1 was measured using Co-Immunoprecipitation. TUNEL staining was performed to detect the apoptosis of the gastrocnemius muscle. Results （1） Compared with group ~S, SI of group OS de- creased significantly by 23.09%, but significant increase was observed in group OE compared to group OS by 30.48%. （2） The grip strength per gram of body weight of group OS decreased significantly by 42.74% compared to group YS,while that of group OE increased significantly by 21.51% compared to group OS. （3） The apoptosis index increases by 475.28% with age. However, the index decreased by 46.41% in group OE over group OS. （4） Western blotting showed that the expression of cleaved cas- pase-9 increased significantly by 45.4% in group OS compared to group YS, while that of group OE decreased significantly by 33.4% compared to group OS. （5） The binding rate of HSP 70 with Apaf-1 of group OS decreased significantly by 67.11% compared to group YS, while that of group OE in- creased significanlty by 306.31% compared to group OS. Conclusions （1） Gastrocnemius atrophy occur- ring to mice of 60 weeks can be significanlty delayed through 30-week exercises training. （2） The in- creasing apoptosis may be involved in the development of age-related sarcopenia and 30-week exercis- es can significantly attenuate such apoptosis in rodent skeletal muscle. （3） Aerobic exercises increases HSP70/Apaf-1 interaction,which might play an important role in reducing age-related cellular and cell death in the skeletal muscle of rats.