尾加压素Ⅱ对Irisin的负性调节及在慢性肾衰竭骨骼肌萎缩中的作用及机制

It is high prevalence of protein energy waste in chronic renal failure and its manifestation is skeletal muscle atrophty. In our previous study, we found that plasma urotensin Ⅱ(UⅡ) levels were higher whereas Irisin levels were lower than normal controls. UII levels were negativley correlated with Irisin, and UII level was negatively correlated with the muscle mass while Irisin level was positively correlated with muscle mass. We hypothesized that UⅡ may regulate negatively Irisin by inhibiting the release of irisin from skeletal muscle and inducing skeletal muscle atrophagy. This study will verify UⅡ can induce skeletal atrophy in chronic renal failure through 5/6 nephrectomy animal experiments, skeletal cell culture in vitro and UⅡ receptor knockout mice with 5/6 nephrectomy animal model. The mechanisms are to inhibit irisin secretion, to enhance skeletal muscle autophagy through mTOR-AMPK pathway, to activate ubiquitin-proteasome system, to inhibit FOXO phosphoration, to activate Atrogin-1 and MuRF-1 signal pathway, to inhibit proliferation of skeletal satellite cell. Through our study, we verify UⅡ can induce skeletal atrophy through inhibiting release of irisin and other mechanisms which these provide new treatment target sites for skekelatal muscle atrophy in chronic renal failure.

慢性肾衰竭患者普遍存在蛋白质能量消耗，表现为骨骼肌萎缩。我们前期研究发现血液透析患者血浆尾加压素Ⅱ（Urotensin Ⅱ，UⅡ）水平增高而Irisin水平下降, UⅡ和Irisin水平负相关;UⅡ与身体成分肌肉量负相关,Irisin与肌肉量正相关。我们提出假想：UⅡ是一种新的导致骨骼肌萎缩的致病因子,对Irisin有负性调节作用，通过多种机制来介导慢性肾衰竭骨骼肌萎缩。本研究通过5/6肾切除小鼠动物模型，体外小鼠骨骼肌细胞培养，.UⅡ受体基因敲除叠加5/6肾切除模型，验证慢性肾衰竭时UⅡ能够介导骨骼肌萎缩，抑制骨骼肌细胞释放Irisin,并直接诱发骨骼肌细胞自噬活性增强经mTOR-AMPK途径,激活泛素蛋白酶系统,抑制叉头转录因子磷酸化,活化肌萎缩蛋白（Atrogin-1）和肌环指蛋白1(MuRF-1),并抑制骨骼肌卫星增殖分化。通过本研究，为慢性肾衰竭骨骼肌萎缩提供新的干预靶点。

慢性肾衰竭（CRF）患者普遍存在蛋白质能量消耗，表现为骨骼肌萎缩，发病机制未明，缺乏有效干预措施。本项目首先研究血液透析患者蛋白质能量消耗与尾加压素II (UII)和Irisin的相关性; UII和Irisin在慢性肾衰竭患者骨骼肌组织中的表达及与骨骼肌萎缩及自噬水平的关系; 通过UII受体敲除叠加5/6肾切除动物模型小鼠，以及小鼠骨骼肌C2C12细胞暴露不同浓度UII及Irisin，UII受体敲减再加入UII进行体外细胞实验，研究UII对Irisin的负性调节作用及在慢性肾衰竭骨骼肌萎缩中的作用。研究结果（1）血液透析患者血清irisin和UII水平较正常对照组显著下降，irisin和UII呈负相关，合并蛋白质能量消耗的血透患者血清irisin水平比没有蛋白质能量消耗的患者更低。（2）CRF患者存在骨骼肌萎缩及自噬过度活化现象，UII和骨骼肌组织自噬过度活化及肌萎缩正相关，而Irisin前体FNDC5表达与自噬水平及肌萎缩呈负相关，提供了临床上UII可能负性调节骨骼肌组织Irisin前体FNDC5水平的证据。（3）C57BL/6小鼠5/6肾切除（WT CRF）双后肢腓肠肌平均湿重较假手术组（NC组）显著降低；骨骼肌HE染色结果显示，WT CRF组小鼠骨骼肌肌纤维间隙增大，且其横截面积较NC组明显下降。当UII作用被阻断后（UII受体基因敲除，再叠加5/6肾切除即UTKO CRF），与WT CRF 小鼠相比，小鼠骨骼肌横截面积增大，双后肢湿重显著增加。小鼠骨骼肌C2C12 细胞暴露于浓度10-7M UII，48小时及以上，与正常磷酸盐缓冲液比较，肌管显著萎缩。体内动物实验及体外细胞实验均证实UII能直接介导骨骼肌萎缩，抑制Irisin的表达，而Irisin可以拮抗UII介导的肌管萎缩。（4）UII介导慢性肾衰竭骨骼肌萎缩的机制是促进骨骼肌细胞自噬。（5）Irisin拮抗UII介导的肌管萎缩与上调FOXO3A磷酸化，抑制肌肉特异性E3泛素连接酶（MuRF1和MAFbx）有关。（6）WT CRF小鼠骨骼肌卫星细胞标志物Pax7表达较多，但MyoD表达量很少，表明在应激状态下，肌卫星细胞的自我更新能力虽然依然存在，但其成肌分化潜能被抑制，UII可抑制小鼠骨骼肌卫星细胞分化。本研究结果表明：干预UII的作用、或者补充Irisin，可能是CRF患者骨骼肌萎缩新的治疗方向。

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