增龄性肌肉减少症小鼠肌肉组织抗氧化能力的特点

doi:10.13481/j.1671-587X.20220514

摘要/Abstract

摘要：

目的基于核因子E2相关因子2（Nrf2）依赖的抗氧化途径探讨增龄后肌肉组织抗氧化应激特点，阐明活性氧（ROS）生成和抗氧化间的动态平衡在增龄性肌肉减少症（肌少症）中的作用。方法 24只C57BL/6J（C57）雄性小鼠随机分为3月龄组（3 M组）、12月龄组（12 M组）和22月龄组（22 M组），取小鼠比目鱼肌组织，计算骨骼肌质量指数（SMI），HE染色观察各组小鼠肌肉组织病理形态表现，2'，7'-二氯荧光素二乙酸酯（DCFH-DA）荧光探针法检测各组小鼠肌肉组织中ROS水平，实时荧光定量PCR（RT-qPCR）法检测各组小鼠肌肉组织中过氧化物酶体增殖子活化受体γ共激活因子1α（PGC-1α）、Nrf2和抗氧化应激基因mRNA表达水平，Western blotting法检测各组小鼠肌肉组织中Nrf2蛋白表达水平。结果与3 M组比较，12 M组小鼠比目鱼肌肌纤维横截面积增大，部分肌纤维细胞核数量增多，部分肌纤维可见变性改变，SMI值降低，但差异均无统计学意义（P>0.05），肌肉组织中ROS水平升高（P<0.05），Nrf2 mRNA表达水平差异无统计学意义（P>0.05），PGC-1α mRNA表达水平明显升高（P<0.01），Nqol、Cat、Gclc、Sod1和Sod2 mRNA表达水平明显升高（P<0.01），Nrf2蛋白表达水平明显降低（P<0.01）；与12 M组比较，22 M组小鼠比目鱼肌白色肌纤维增多，形态不规则，肌纤维细胞核数量增多并出现核内移现象，肌细胞间隙增大，细胞数量减少，SMI值降低，但差异无统计学意义（P>0.05），肌肉组织中ROS水平明显升高（P<0.01），Nrf2 mRNA表达水平明显升高（P<0.01），Nqol、Cat、Sod1和Sod2 mRNA表达水平明显升高（P<0.05或P<0.01），而Gclc mRNA表达水平降低（P<0.05），Nrf2蛋白表达水平明显升高（P<0.01）。结论增龄性小鼠比目鱼肌表现为肌少症表型，以氧化代谢为主的比目鱼肌具有一定的抗氧化应激能力，与Nrf2依赖的抗氧化途径增强有密切关联。

关键词: 肌肉减少症, 骨骼肌, 核因子E2相关因子, 线粒体稳态

Abstract:

Objective To explore the characteristics of antioxidant stress of muscle tissue after aging based on the nuclear factor E2-related factor 2（Nrf2） dependent antioxidant pathway， and to clarify the effect of the dynamic balance between reactive oxygen species （ROS） production and antioxidation in the age-related sarcopenia. Methods Twenty four C57BL/6J （C57） male mice were randomly divided into 3-month-old group （3 M group）， 12-month-old group （12 M group） and 22-month-old group （22 M group）. The soleus muscle tissue of mice was taken， and the skeletal muscle mass index （SMI） was calculated. The pathomorphology of muscle tissue of the mice in various groups was observed by HE staining. The ROS levels of mice in various groups were detected by 2'，7'-dichlorodihydrofluorescein diacetate（DCFH-DA） fluorescence probe method， the mRNA expression levels of eroxisome proliferators-activated receptor-γ coactivator-1α （PGC-1α）， Nrf2 and antioxidant stress genes in muscle tissue of the mice in various groups were detected by real-time fluorescence quantitative（RT-qPCR） method，and the protein expression levels of Nrf2 in muscle tissue of the mice in various groups were detected by Western blotting method. Results Compared with 3 M group， the cross-sectional area of soleus muscle fibers of the mice in 12 M group was increased， the number of nuclei in some muscle fibers was increased， and some muscle fibers showed denaturation changes；the SMI value was decreased， but the difference was not statistically significant （P>0.05）；the level of ROS was increased （P<0.05），the expression level of Nrf2 mRNA had no significant difference（P>0.05）， the expression level of PGC-1α mRNA was significantly increased （P<0.01），the expression levels of Nqol， Cat， Gclc， Sod1 and Sod2 mRNA were significantly increased （P<0.01）， and the expression level of Nrf2 protein was significantly decreased （P<0.01）. Compared with 12 M group， the white muscle fibers of soleus muscle of the mice in 22 M group were increased， the morphology was irregular， the number of nuclei of muscle fibers was increased and the phenomenon of nuclear translocation occurred， the gap between muscle cells was increased， the number of cells was decreased， and the SMI value was decreased， but the difference was not statistically significant （P>0.05）； the level of ROS in muscle tissue was significantly increased （P<0.01）， the expression levels of Nrf2 mRNA was significantly increased （P<0.01）， the expression levels of Nqol， Cat， Sod1 and Sod2 mRNA were significantly increased （P<0.05 or P<0.01）， while the expression level of Gclc mRNA was decreased （P<0.05）， and the expression level of Nrf2 protein was significantly increased （P<0.01）. Conclusion The soleus muscle of aging mice shows sarcopenia phenotype. The soleus muscle with oxidative metabolism as the main has certain characteristics of antioxidant stress ability， which is closely related to the enhancement of Nrf2 dependent antioxidant pathway.

Key words: Sarcopenia, Skeletal muscle, Nuclear factor E2-related factor, Mitochondrial homeostasis

中图分类号:

R739.5

郭丹,温旭,吴丹妮,王秦玉,刘珊珊,季铎淳,王力可,王丹,董营,于春艳. 增龄性肌肉减少症小鼠肌肉组织抗氧化能力的特点[J]. 吉林大学学报(医学版), 2022, 48(5): 1209-1215.

Dan GUO,Xu WEN,Danni WU,Qinyu WANG,Shanshan LIU,Duochun JI,Like WANG,Dan WANG,Ying DONG,Chunyan YU. Characteristics of antioxidant capacity of muscle tissue in aging sarcopenia mice[J]. Journal of Jilin University(Medicine Edition), 2022, 48(5): 1209-1215.

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Gene	Primer sequence
Nqo1	F:5'-TTCTCTGGCCGATTCAGAGT-3'
	R:5'-GGCTGCTTGGAGCAAAATAG-3'
Cat	F:5'-ACATGGTCTGGGACTTCTGG-3'
	R:5'-CAAGTTTTTGATGCCCTGGT-3'
Gclc	F:5'-CAGTCAAGGACCGGCACAAG-3'
	R:5'-CAAGAACATCGCCTCCATTCAG-3'
Sod1	F:5'-CCAGTGCAGGACCTCATTTT-3'
	R:5'-TTGTTTCTCATGGACCACCA-3'
Sod2	F:5'-CCGAGGAGAAGTACCACGAG-3'
	R:5'-GCTTGATAGCCTCCAGCAAC-3'
Nrf2	F:5'-TTCTTTCAGCAGCATCCTCTCCAC-3'
	R:5'-ACAGCCTTCAATAGTCCCGTCCAG-3'
PGC-1α	F:5'-TATGGAGTGACATAGAGTGTGCT-3' R:5'-CCACTTCAATCCACCCAGAAAG-3'
GADPH	F:5'-TTGTGATGGGTGTGAACCACGAGA-3'
	R:5'-CATGAGCCCTTCCACAATGCCAAA-3'

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