短期间歇和急性低氧对大鼠快慢肌萎缩的影响及其作用机制

Effects of short-term intermittent and acute hypoxia on fast-and slowtwitch muscle atrophy in rats and its mechanism

目的:探讨不同低氧干预模式对大鼠快肌和慢肌萎缩的影响差异及可能的分子机制。方法:24只10周龄雄性SD大鼠分为常氧对照组(C组)、短期间歇低氧组(IH组,于12.4%O_(2)暴露8 h/d,持续4周)和急性低氧组(AH组,连续3 d暴露于12.4%O_(2)),每组8只。干预后测定抓力,胫骨前肌(TA,快肌)和比目鱼肌(SOL,慢肌)湿重,以及肌纤维横截面积(FCSA)。Western blot法检测嘌呤霉素(Puro)和泛素(Ub)的含量,计算蛋白质累积量(Puro/Ub)。使用RNA测序法筛选两种肌肉中IH/C组和AH/C组的差异表达基因,分析差异基因所富集的生物过程和通路,RT-qPCR验证关键差异基因表达。结果:干预后,AH组相对抓力显著低于C组,AH组TA和SOL相对湿重均高于IH组(P<0.05),IH组和AH组TA和SOL的FCSA均显著低于C组(P<0.01)。IH组TA的蛋白质累积量显著高于C组,而AH组显著低于IH组(P<0.01);IH组SOL的蛋白质累积量显著低于C组,而AH组显著高于IH组(P<0.01)。IH组/C组TA的差异基因以上调为主,AH/C组SOL的差异基因亦以上调为主。TA中,IH组/C组上调基因的功能主要为抗氧化和糖代谢等,PPAR通路参与调节;AH/C组上调基因的功能主要为快慢肌间的转化、氧化应激、炎症和细胞死亡等。SOL中,IH/C组和AH/C组上调基因功能主要为细胞凋亡、免疫反应和氧化应激等,TNF、TGF-β和JAK-STAT通路参与调节。关键差异基因表达验证结果与测序结果一致。结论:短期间歇低氧可能通过提高大鼠快肌(TA)的抗氧化能力而抵抗萎缩;急性低氧可能通过增加氧化应激、炎症反应和细胞凋亡过程促进快肌(TA)和慢肌(SOL)的萎缩。

AIM:To investigate the effects of different hypoxia intervention modes on fast-and slow-twitch muscle atrophy in rats and the possible molecular mechanism.METHODS:Twenty-four 10-week-old male SD rats were divided into normoxic control group(C group),short-term intermittent hypoxia group(IH group,exposed to 12. 4% oxygen for 8 h/d for 4 weeks)and acute hypoxia group(AH group,living in hypoxic room with 12. 4% oxygen for 3 d),with 8 rats in each group. After intervention,the holding power,wet weight of tibialis anterior muscle(TA,fast-twitch muscle)and soleus muscle(SOL,slow-twitch muscle),and muscle fiber cross-sectional area(FCSA)were tested. The content of puromycin(Puro)and ubiquitin(Ub)was measured by Western blot,and the protein accumulation(Puro/Ub)was calculated. Differentially expressed genes in IH/C group and AH/C group were screened by RNA sequencing. The enriched biological processes and pathways of these genes were analyzed. The expression of key differential genes was verified by RTqPCR.RESULTS:After intervention,the relative holding power in AH group was significantly lower than that in C group(P<0. 01). The relative wet weight of TA and SOL in AH group was higher than that in IH group(P<0. 05). The FCSA of TA and SOL in IH group and AH group were significantly lower than those in C group(P<0. 01). The protein accumulation of TA in IH group was significantly higher than that in C group,while that in AH group was significantly lower than that in IH group(P<0. 01). The protein accumulation of SOL in IH group was significantly lower than that in C group,while that in AH group was significantly higher than that in IH group(P<0. 01). The differential genes of TA in IH/C group were mainly up-regulated,and the differential genes of SOL in AH/C group were mainly up-regulated. In TA,the up-regulated genes in IH/C group were mainly enriched in antioxidant and glucose metabolism,and PPAR signaling pathway was involved. The up-regulated genes in AH/C group were mainly enriched in the transformation between fast-and slow-twitch muscle,oxidative stress,inflammation and cell death. In SOL,the up-regulated genes in IH/C group and AH/C group were mainly enriched in apoptosis,immune response and oxidative stress,and the TNF,TGF-β and JAK-STAT pathways were involved. The key differentially expressed genes verified by RT-qPCR were consistent with the results of sequencing.CONCLUSION:Short-term intermittent hypoxia exposure may resist atrophy by increasing the antioxidant capacity of fast-twitch muscle(TA)in rats,while acute hypoxia may promote the atrophy of fast-twitch muscle(TA)and slow-twitch muscle(SOL)by increasing the capacity of oxidative stress,inflammatory response and apoptosis in rats.