细胞焦亡在长期抗阻训练对增...蛋白质代谢影响中的调节作用_龚丽景.pdf

中国运动医学杂志2022年12月第41卷第12期 Chin J Sports Med，Dec.2022，Vol.41，No.12细胞焦亡在长期抗阻训练对增龄大鼠胫骨前肌蛋白质代谢影响中的调节作用龚丽景1马芳源2杨璐瑶3唐舒宁4苏浩5付鹏宇61 北京体育大学运动与体质健康教育部重点实验室（北京 100084）2 南开大学生命科学学院3 浙江大学教育学院4 复旦大学公共卫生学院5 北京体育大学运动人体科学学院6 西北工业大学体育部摘要目的：探究长期抗阻训练通过调控细胞焦亡途径而影响骨骼肌蛋白质代谢的可能机制。方法：30只8月龄SD大鼠分为3组：基础值组（N组，n=10），干预前取材；安静对照组（C组，n=10），正常饮食32周，不运动；抗阻训练组（R组，n=10），进行30%负重（自身体重）、35坡度、跑速15 m/min的跑台训练，每次训练4组2个循环，隔天训练一次，共32周。干预后测试大鼠体重、瘦体重、胫骨前肌（TA）湿重；HE染色观察肌纤维形态并计算肌纤维横截面积；Western blot测试肌肉合成相关蛋白哺乳动物雷帕霉素靶蛋白（mTOR）和真核翻译起始因子4E结合蛋白1（4E-BP1），分解相关蛋白叉头状转录因子 O1（FoxO1）、肌肉环状指基因1（MuRF1）、肌肉萎缩盒F基因（Atrogin1）和泛素（Ub）的表达；细胞焦亡PCR芯片筛选TA中的焦亡差异表达基因，qPCR验证芯片结果准确性；WesternBlot测试细胞焦亡关键蛋白核因子-B（NF-B）、接头蛋白凋亡相关斑点样蛋白（ASC）、消皮素 D（GSDMD）和天冬氨酸蛋白水解酶 1（Caspase1/Casp1）的表达。结果：（1）C组大鼠体重显著高于N组，R组显著低于C组；C组瘦体重及其百分含量显著低于N组，R组显著高于C组（P0.05）。（2）C组肌纤维横截面积较N组显著降低，R组较C组显著增加（P0.05）。（3）C组磷酸化4E-BP1（p-4E-BP1）蛋白含量和p-4E-BP1/4E-BP1比值显著低于N组，R组p-4E-BP1蛋白含量显著高于C组（P0.05）；C组FoxO1和Ub蛋白含量显著高于N组，R组显著低于C组，C组p-FoxO1/FoxO1比值显著低于N组，R组显著高于C组（P0.05）。（4）焦亡PCR芯片结果显示，R组较C组下调的焦亡基因数目增加。对C/N组上调的差异基因Asc，C/N组上调且在R/C组下调的差异基因核苷酸结合寡聚化结构域样受体家族凋亡抑制蛋白6（Naip6）和R/C组下调的差异基因Casp1进行qPCR验证，与芯片结果一致。（5）C组NF-B和ASC蛋白含量显著高于N组，R组Caspase1蛋白含量显著低于N组（P0.05）。结论：长期抗阻训练通过降低骨骼肌蛋白质分解而缓解增龄性肌萎缩的发展，抑制细胞焦亡可能是其机制之一。关键词增龄；骨骼肌；蛋白质代谢；抗阻训练；细胞焦亡The Regulatory Effect of Pyroptosis on Protein Metabolism of Tibialis Anterior Muscles in Aging Ratsthrough Long-time Resistance TrainingGong Lijing1，Ma Fangyuan2，Yang Luyao3，Tang Shuning4，Su Hao，Fu Pengyu61 Key Laboratory of Sports and Physical Health of the Ministry of Education，Beijing Sport University，Beijing100084，China2 School of Life Sciences，Nankai University，Tianjin 300071，China3 College of Education，Zhejiang University，Hangzhou 310063，China4 School of Public Health，Fudan University，Shanghai 200032，China5 School of Kinesiology，Beijing Sport University，Beijing 100084，China6 Department of Physical Education，Northwestern Polytechnical University，Xi an 710072，ChinaCorresponding Author:Fu Pengyu，Email:收稿日期：2022.5.16基金项目：中央高校基本科研业务费专项资助课题（2021TD012）第1作者：龚丽景，Email：；通信作者：付鹏宇，Email： 9565DOI:10.16038/j.1000-6710.2022.12.014中国运动医学杂志2022年12月第41卷第12期 Chin J Sports Med，Dec.2022，Vol.41，No.12Abstract Objective To explore the possible mechanism of long-time resistance training affectingskeletal muscle protein metabolism by regulating pyroptosis.Methods Thirty Sprague-Dawley rats wererandomly divided into a baseline value group（group N），whose samples were collected before the intervention，a control group（group C）and a resistance training group（group R），each of 10.Group R underwent running with 30%weight-bearing on the slope of 35 and at the speed of 15 m/min，4 sets 2 cycles for each training，once every other day，for 32 weeks，while group C did not exercise.After the intervention，the body weight，lean body mass and tibialis anterior（TA）wet weight were measured and hematoxylin-eosin staining was conducted to observe the morphology of muscle fibers，andthe fiberscross-sectional area（FCSA）was calculated.The expression of muscle synthesis-related proteins mammalian target of rapamycin（mTOR）and eukaryotic translation initiation factor 4E-bingdingprotein 1（4E-BP1），decomposition-related proteins forkhead transcription factor O1（FoxO1），musclering finger 1（MuRF1），muscle atrophy F-box（MAFbx/Atrogin1），and ubiquitin（Ub）were tested byWestern blotting.The differentially expressed genes of pyroptosis of TA were screened by PCR array，and the accuracy of the array results were verified by qPCR.The expression of pyroptosis key proteinsnuclear factor kappa-B（NF-B），apoptosis speck-like protein containing a caspase recruitment domain（ASC），Gasdermin D（GSDMD）and Caspase1（Casp1）were tested by Western blotting.Results（1）The body weight of group C was significantly higher than group N，and that of group R was significantly lower than group C.Moreover，the lean body mass and its percentage of group C were significantly lower than group N，and those of group R were significantly higher than group C（P0.05）.（2）The FCSA of group C was significantly lower than group N，and that of group R was significantlyhigher than group C（P0.05）.（3）The protein content of phospho-4E-BP1（p-4E-BP1）and the ratioof p-4E-BP1/4E-BP1 of group C were significantly lower than group N，and those of group R weresignificantly higher than group C（P0.05）.The protein content of FoxO1 and Ub of group C were significantly higher than group N，while those of group R were significantly lower than group C.The ratio of p-FoxO1/FoxO1 in group C was significantly lower than N group，and that of group R was significantly higher than group C（P0.05）.（4）The results of pyroptosis PCR array showed that the number of down-regulated pyroptotic genes increased in group R compared with group C.The up-regulated differential gene Asc in group C/N，the differential gene NOD-like receptor family，apoptosis inhibitory protein 6（Naip6）up-regulated in group C/N and down-regulated in group R/C，and the down-regulated differential gene Casp1 in group R/C were verified by qPCR，which were consistent with the array results.（5）The