肠道稳态破坏对后肢制动诱导的小鼠废用性肌萎缩的影响初探

Preliminary exploration of effects of gut homeostasis disruption on disuse muscle atrophy induced by hindlimb immobilization in mice

中文关键词: 肌萎缩  抗生素  肠道稳态  肠—肌轴  炎症

英文关键词: muscular atrophy  antibiotic  gut homeostasis  gut-muscle axis  inflammation

中文摘要:

      目的 通过后肢石膏固定构建小鼠废用性肌萎缩模型,联合四联广谱抗生素干预,从肠—肌轴视角探讨肠道稳态破坏对肌肉萎缩的影响.方法 采用6～8 周龄雄性 C57BL/6J 小鼠随机分为对照组、固定组和固定+抗生素组,每组各8 只.对照组不做处理,固定组采用石膏固定小鼠双后肢持续制动 2 周,固定+抗生素组小鼠在固定组基础上采用四联抗生素包括氨苄青霉素、硫酸新霉素、甲硝唑及万古霉素进行饮水干预.检测体重、摄食量、抓力、跑台耐力和肌肉湿重;苏木精-伊红染色观察肌纤维形态并计算肌纤维横截面积;蛋白质印迹检测肌肉蛋白合成与降解标志物,包括成肌蛋白(MYOG)和肌肉萎缩相关 F-box 蛋白(Atrogin-1);免疫组织化学染色检测肠道紧密连接蛋白(闭锁小带蛋白-1和闭合蛋白);酶联免疫吸附试验测定血清白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α);通过 16S rRNA 测序分析肠道菌群结构.结果 后肢制动可导致显著废用性肌萎缩,较对照组,固定组小鼠后肢肌肉相对质量下降[股四头肌(4.61±0.34)mg/g比(6.37±0.38)mg/g、胫骨前肌(1.97±0.18)mg/g 比(2.59±0.15)mg/g、腓肠肌(4.56±0.36)mg/g 比(6.09±0.30)mg/g、比目鱼肌(0.61±0.06)mg/g 比(1.06±0.09)mg/g,P<0.001],相对抓力[(2.96±0.59)F/g 比(4.20±0.63)F/g,P<0.001]及跑步距离[(438.10±69.53)m 比(570.20±49.81)m,P<0.001]减少,肌纤维形态萎缩且横截面积缩小[(1 084.64±64.32)μm2 比(1 674.86±77.94)μm2,P<0.001],MYOG表达降低(0.59±0.04 比1.00±0.09,P<0.001).而抗生素处理进一步加重肌萎缩表型,并在固定基础上放大肠道屏障结构受损,固定+抗生素组较固定组闭合蛋白(0.12±0.01 比0.17±0.01,P<0.05)表达降低,IL-6 水平[(28.63±4.88)pg/mL 比(18.59±6.38)pg/mL,P<0.01]与TNF-α 水平[(6.77±0.93)pg/mL 比(4.04±0.84)pg/mL,P<0.001]升高.16S rRNA 测序表明,抗生素导致肠道菌群多样性显著下降,群落结构显著分离.结论 肠道稳态破坏加重后肢制动诱导的小鼠废用性肌萎缩.

英文摘要:

      Objective To investigate the effects of gut homeostasis disruption on disuse muscle atrophy from the perspective of the gut-muscle axis by means of a hindlimb immobilization mouse model and the intervention with a quadruple broad-spectrum antibiotics.Methods Male C57BL/6J mice aged 6--8 weeks were randomly divided to three groups:control group,immobilization(IM)group,and immobilization+antibiotics(IM+AB)group,with 8 mice in each group.The control group did not receive any treatment.The IM group underwent continuously bilateral hindlimb immobilization with plaster for 2 weeks.On the basis of the IM group,the IM+AB group were given a quadruple antibiotic intervention through drinking water,including ampicillin,neomycin sulfate,metronidazole and vancomycin.The body weight,food intake,grip strength,treadmill endurance and muscle wet weight were measured.Hematoxylin-eosin staining was used to observe muscle fiber morphology and the cross-sectional areas were calculated.Western blotting was performed to detect protein synthesis and degradation markers,including myogenin(MYOG)and muscle atrophy F-box protein(Atrogin-1).Immunohistochemistry was used to detect intestinal tight junction proteins(zonula occludens-1 and occludin).The enzyme-linked immunosorbent assay was used to measure the levels of serum interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α).16S rRNA sequencing was applied to analyze gut microbiota structure.Results Hindlimb immobilization induced significant disuse muscle atrophy.Compared with the control group,the IM group showed reduced relative muscle mass in the hindlimbs[quadriceps:(4.61±0.34)mg/g vs.(6.37±0.38)mg/g;tibialis anterior:(1.97±0.18)mg/g vs.(2.59±0.15)mg/g;gastrocnemius:(4.56±0.36)mg/g vs.(6.09±0.30)mg/g;soleus:(0.61±0.06)mg/g vs.(1.06±0.09)mg/g,P<0.001],decreased relative grip strength[(2.96±0.59)F/g vs.(4.20±0.63)F/g,P<0.001],and shorter running distance[(438.10±69.53)m vs.(570.20±49.81)m,P<0.001].Additionally,muscle fibers atrophied with reduced cross-sectional area[(1 084.64±64.32)μm2 vs.(1 674.86±77.94)μm2,P<0.001],along with lower MYOG expression(0.59±0.04 vs.1.00±0.09,P<0.001).Antibiotic treatment further exacerbated the muscle atrophy phenotype and aggravated the damage to the intestinal barrier structure on the basis of the IM group.Compared with the IM group,the IM+AB group displayed lower occludin expression(0.12±0.01 vs.0.17±0.01,P<0.05),increased levels of serum IL-6[(28.63±4.88)pg/mL vs.(18.59±6.38)pg/mL,P<0.01],and TNF-α[(6.77±0.93)pg/mL vs.(4.04±0.84)pg/mL,P<0.001].16S rRNA sequencing indicated that antibiotics led to a significant decline in the diversity of the intestinal microbiota and a significant separation of the community structure.Conclusion Disruption of gut homeostasis exacerbates immobilization-induced disuse muscle atrophy in mice.