FoxO转录因子与NF-κB通路在冬眠达乌尔黄鼠不同类型骨骼肌中的差异性调控

发布时间：2018-03-18 12:14

本文选题：冬眠　切入点：废用性肌萎缩　出处：《西北大学》2016年博士论文　论文类型：学位论文

【摘要】：Atrogin-1(又称MAFbx/FBXO32)与MuRF-1(又称Trim63)是哺乳动物细胞内重要的肌萎缩因子。Atrogin-1与MuRF-1基因表达在动物骨骼肌废用期间受到上游FoxO转录因子(包括FoxO1与FoxO3a)的调控而显著上调,而MuRF-1基因表达还受到NF-κB通路的调控。此外,之前对的研究还发现肌肉萎缩的程度受到了肌肉类型的影响。目前冬眠动物因为长期冬眠不活动而未导致大多数骨骼肌发生明显肌萎缩而受到关注。但是FoxO转录因子与NF-κB通路是如何参与到冬眠动物骨骼肌atrogin-1与MuRF-1基因表达的调控,与冬眠动物的抗肌萎缩机制之间的关系还未可知,而冬眠动物骨骼肌中FoxO转录因子与NF-κB通路对atrogin-1与MuRF-1的调控是否也受到了肌肉类型的影响也还不清楚。本次研究通过对比FoxO转录因子与NF-κB通路在达乌尔黄鼠(Spermophilus dauricus)不同类型骨骼肌(慢缩比目鱼肌,混合型腓肠肌与快缩趾长伸肌)中表达的变化,探讨黄鼠抗废用性肌萎缩的潜在机制。通过检测FoxOs,总蛋白相对表达(total FoxOs)及磷酸化FoxOs (p-FoxOs)的相对蛋白表达,计算两者的比值即p-FoxOs/total FoxOs代表了FoxOs在细胞内的磷酸化水平,该比值的大小与FoxOs促进atrogin-1与MuRF-1基因表达的强度正好相反。研究结果发现冬眠中黄鼠通过抑制比目鱼肌中FoxOs;总蛋白表达与磷酸化作用,维持了FoxOs的磷酸化水平最终抑制了atrogin-1基因表达的上调；腓肠肌内磷酸化作用的增加抑制了FoxOs磷酸化水平的降低从而抑制了atrogin-1基因表达的上调；趾长伸肌中FoxO3a磷酸化水平未发生明显改变,而FoxO1总蛋白表达的显著减少也并未导致FoxO1的磷酸化水平的降低,两者均有利于atrogin-1基因表达上调的抑制。由此可见黄鼠在长期冬眠不活动中,不同类型的骨骼肌中存在不同的机制可以维持FoxO对atrogin-1的转录活性,最终抑制骨骼肌蛋白合成的增加。此外,在短暂的阵间觉醒期间,比目鱼肌与腓肠肌中FoxO转录因子与atrogin-1基因表达的变化相一致。出眠时黄鼠通过增加腓肠肌与趾长伸肌中FoxO的磷酸化水平而抑制了atrogin-1基因表达的上调。通过检测NF-κB途径(IKKβ/p50途径)在黄鼠腓肠肌中基因与蛋白表达,结合之前实验室的研究结果发现冬眠中时黄鼠通过维持比目鱼肌中FoxO转录因子磷酸化水平与NF-κB通路而抑制了MuRF-1基因表达的上调；腓肠肌中NF-κB信号的活化可能促进了MuRF-1基因表达的上调,从而促进了腓肠肌肌肉蛋白的降解；趾长伸肌中NF-κB通路被活化,但并未促进MuRF-1基因表达的增加。由此可见,在长期的冬眠不活动中,黄鼠腓肠肌与趾长伸肌中FoxO转录因子与NF-κB通路单独作用于MuRF-1基因表达的调控,而在比目鱼肌中则是FoxO转录因子与NF-κB通路协同作用于MuRF-1基因表达的调控。此外,相对于比目鱼肌,黄鼠冬眠期间腓肠肌与趾长伸肌对atrogin-1与MuRF-1基因表达的调控机制更为复杂。Atrogin-1和MuRF-1的表达具有组织特异性,只在骨骼肌与心肌中表达,而心肌在动物冬眠随伴随着的低氧低代谢条件下可以维持收缩功能,这与心肌重量的维持密切相关的。本次研究发现整个冬眠期间黄鼠心肌中的atrogin-1和MuRF-1的基因表达维持在低水平,有利于心肌重量的维持。同骨骼肌(腓肠肌与趾长伸肌)类似,冬眠期间黄鼠心肌中的NF-κB信号被活化,但不同的是心肌细胞内p50蛋白表达的增加并未促进MuRF-1基因表达的上调,说明冬眠动物心肌中NF-κB途径对MuRF-1基因表达的调控较弱。
[Abstract]:Atrogin-1 (also called MAFbx/FBXO32) and MuRF-1 (also called Trim63) is important in mammalian cells, muscle atrophy factor.Atrogin-1 and MuRF-1 gene expression in skeletal muscle of animal waste by the upstream transcription factor during FoxO (including FoxO1 and FoxO3a) control was significantly increased, while the expression of MuRF-1 gene is regulated by NF- B pathway. In addition, before the study also found that the degree of muscle atrophy of affected muscle type. The hibernating animal hibernation is not because of the long-term activities resulted in the most obvious skeletal muscle atrophy and attention. But the FoxO and NF- transcription factor kappa B pathway is how to participate in the regulation of Atrogin-1 and MuRF-1 gene expression in skeletal muscle of hibernating animal the relationship between the hibernating animal dystrophin mechanism is not known, but the hibernating animal skeletal muscle FoxO and NF- transcription factor kappa B pathway on Atrogin-1 and MuRF-1 The regulation is also affected by the type of muscle is still unclear. This study through the comparison of FoxO and NF- transcription factor kappa B pathway in the ground squirrel (Spermophilus dauricus) of different types of skeletal muscle (slow twitch soleus, gastrocnemius and mixed fast twitch extensor digitorum longus) in the form of change, the potential mechanism to investigate the squirrel anti muscle atrophy. Through the detection of FoxOs, total protein expression (total FoxOs) and phosphorylated FoxOs (p-FoxOs) expression of relative protein, calculating the ratio between the p-FoxOs/total FoxOs on behalf of FoxOs in intracellular phosphorylation, and the ratio of the size of FoxOs promotes the expression of Atrogin-1 and MuRF-1 gene the strength of the opposite. The results found that hibernation by inhibiting FoxOs squirrel soleus muscle; total protein expression and phosphoric acid, to maintain the level of FoxOs phosphorylation can inhibit Atrogin-1 gene expression The effect of increase; reduce in the gastrocnemius muscle of phosphate can inhibit the phosphorylation of FoxOs can inhibit the expression of Atrogin-1 gene up-regulated; EDL FoxO3a phosphorylation levels did not change significantly, while the expression of FoxO1 protein was significantly reduced also did not lead to the phosphorylation level of FoxO1 decreased, both for Atrogin-1 gene expression is suppressed. Thus in the long hibernation is in ground squirrel, different mechanisms exist for different types of skeletal muscle can maintain the transcription activity of FoxO to Atrogin-1, increase the final inhibition of protein synthesis in skeletal muscle. In addition, during the awakening in the short array, consistent expression of FoxO transcription factor Atrogin-1 gene soleus and gastrocnemius muscle. The sleep ground squirrel by increasing the FoxO of gastrocnemius muscle and extensor digitorum longus in phosphorylation and inhibition of Atrogin-1 gene expression The raised. By detection of NF- kappa B pathway (IKK beta /p50 pathway) in ground squirrels in gastrocnemius muscle of gene and protein expression, combined with the research results found in laboratory before hibernation when Citellus by maintaining the soleus muscle transcription factor FoxO phosphorylation and NF- B pathway and inhibition of MuRF-1 gene expression; NF- K B signal in gastrocnemius muscle activation may contribute to the regulation of MuRF-1 gene expression, so as to promote the degradation of gastrocnemius muscle protein; extensor digitorum longus NF- kappa B pathway is activated, but did not increase the expression of MuRF-1 gene. Thus, in the long hibernation is activity, regulation of Citellus gastrocnemius muscle and extensor digitorum longus FoxO and NF- transcription factor kappa B pathway alone on the expression of MuRF-1 gene in soleus muscle is the regulation of the FoxO transcription factor kappa B pathway and NF- synergistic effects on MuRF-1 gene expression. In addition, compared with the halibut Fish muscle, tissue specific regulation mechanism of ground squirrel during hibernation of gastrocnemius muscle and extensor digitorum longus on the expression of Atrogin-1 and MuRF-1 gene expression of.Atrogin-1 and MuRF-1 is more complex, only expressed in skeletal muscle and cardiac muscle, and myocardium in animal hibernation with hypoxia with low metabolic conditions can be maintained and the systolic function. Maintain the myocardial weight closely related. This study found that the expression of myocardial hibernation in ground squirrels Atrogin-1 and MuRF-1 gene maintained at a low level, which is conducive to the maintenance of myocardial weight. With skeletal muscle (gastrocnemius muscle and extensor digitorum longus muscle) is similar to that of NF- kappa B signal is activated in squirrel myocardial hibernation but the difference is the increased expression of P50 protein in myocardial cells did not promote the regulation of MuRF-1 gene expression, indicating weak regulation of hibernating animal myocardial NF- kappa B pathway on the expression of MuRF-1 gene.

【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q445

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