CCN1(富含半胱氨酸61) 促进老年人肌少症和尿毒症肌少症的机制研究

The extent of muscle loss in older people serves as a significant predictor of mortality. Our preliminary experiment provided evidence that the CCN1 (Cyr61) induces muscle satellite cell (MSC) senescence, which contributes to aging sarcopenia. We have also observed that CCN1 induces adipogenesis in fibro/adipogenic progenitors (FAP) in culture. Our central hypothesis is that an increase in senescence and adipogenesis, combined with a decrease in myogenesis, contributes to aging sacopenia and uraemic sarcopenia, and that these processes are induced by increased CCN1. We plan to test our central hypothesis with the following specific methods: ① We will measure senescence pathway markers and myogenesis biomarkers in animals with muscle hypertrophy (exercise), atrophy (denervation) and injury (CTX) produced in muscle specific CCN1 knock out mice and 5/6 nephrectomy mice. ② We will track the process of muscle senescence, adipogenesis and myogenesis using CCN1 knock out, 5/6 nephrectomy and age-matched wild type control mice through their life span from 6 to 30 months. ③ We will isolate FAP and MSC from muscles of GFP mice, then transplant these cells into CCN1 knock out mice and the mice overexpressing CCN1 in muscle and 5/6 nephrectomy mice, and track the progression of adipogenesis in the FAP cells and senescence in the integrin alpha-7 positive MSC. ④ We will determine if Wnt-3a is involved in muscle loss, possibly through CCN1. We are going to monitor the Wnt-3a expression and its relationship with CCN1 in the life span of CCN1 knock out mice and 5/6 nephrectomy mice. ⑤ We need to identify which integrins are critical for CCN1-mediated senescence or adipogenesis. One synthetic peptide (T1: GQKCIVQTTSWSQCSKS) will compete with CCN1 for integrin binding and should reduce the senescence response. The other is a similar peptide but with 2 amino acids substituted (T1-mut; GQKCIVQTTSAAQCSKS) making it unable to compete for integrin binding. It is a negative control for the competition studies. FAPs will be cultured in medium with or without CCN1 and their progression to adipocytes determined by oil-Red O staining. ⑥ We will test serum Wnt-3a and CCN1 in sarcopenic with elderly patients and uraemic sarcopenia patient. .Our proposed project is innovative on several levels. First, to our knowledge, this is the first study to identify whether CCN1 contributes to sarcopenia by promoting muscle cell senescence and adipogenesis. Second, we have bred a new muscle specific CCN1 knock out mouse specifically to monitor the muscle progenitor cell growth and differentiation to determine the role of CCN1 in muscle senescence and adipogenesis. Third, the idea that CCN1 impacts different populations of progenitor cells, inducing senescence in satellite cells (muscle progenitors) and inducing adipogenesis in FAP cells (mesenchymal progenitors), is a novel concept. Fourth, integrins have been identified in muscle for some time, however, they have not been previously been connected to muscle senescence or adipogenesis. The characterization of integrin involvement in muscle processes connected to aging sarcopenia and uraemic sarcopenia is novel. Although there are many studies of sarcopenia, the concept that prevention of senescence can impact and attenuate sarcopenia is new. Lastly, revealing these connections may allow us to use the inhibition of CCN1 as an innovative therapeutic strategy for treating sarcopenia.

老年人肌少症是一种新的老年病，我国发病率男性12.3%、女性4.8%。尿毒症肌少症未引起重视。肌少症会增加死亡率，发病机理不清、亚洲与欧洲的诊断标准不统一、疗效差。我们预实验发现CCN1(富含半胱氨酸61)会引起肌少症；推测：①CCN1诱导肌卫星细胞（MSC）衰老、诱导纤维/脂肪细胞的祖细胞（FAP）生成脂肪细胞；② 抑制CCN1、阻止肌肉衰老能改善老年人肌少症；③整合素是CCN1的受体，CCN1与不同的整合素结合，起不同的作用。整合素在肌肉中的功能未知。这三个观点未见报道。通过CCN1敲除和CCN1过表达、5/6肾切除小鼠模型、MSC和FAP细胞培养、老年人肌少症和尿毒症肌少症患者来研究①CCN1对骨骼肌衰老、脂肪细胞和肌细胞的作用；② CCN1和Wnt信号通路上激动剂Wnt-3a的关系；③CCN1与整合素结合位点。创新性强、有探索价值和临床意义。

全球老年人肌少症的发病率正在上升。尿毒症肌少症未引起重视。肌少症会增加死亡率，其发病机理不清、诊断标准不统一、疗效差。我们通过小鼠实验（CCN1骨骼肌条件性敲除小鼠、5/6肾切除小鼠、去神经小鼠、肌注心脏毒素CTX小鼠）、细胞培养（小鼠骨骼肌细胞株C2C12、纤维/脂肪细胞的祖细胞FAP和肌卫星细胞SC）、老年人肌少症患者和尿毒症肌少症患者来研究CCN1(富含半胱氨酸61) 促进老年人肌少症和尿毒症肌少症的发病机制。结果发现：⑴老年人肌少症和尿毒症肌少症患者均伴有低BMI、低骨骼肌质量指数和低握力；CCN1在老年人肌少症和尿毒症肌少症患者的血清中表达增加。⑵发现CCN1通过二条信号通路来促进老年人肌少症和尿毒症肌少症的发生，①CCN1通过促进P53、P21、RB、p16 衰老相关的蛋白表达增加，促进骨骼肌衰老；②CCN1通过Akt/mTOR 通路，抑制骨骼肌蛋白合成和增加蛋白分解，促进骨骼肌萎缩。⑶首次通过“CCN1骨骼肌条件性敲除小鼠”干预CCN1的表达，能够改善小鼠骨骼肌萎缩，为阐明CCN1是老年人肌少症和尿毒症肌少症的治疗靶点提供了直接的实验依据。⑷首次报道了纤维/脂肪细胞的祖细胞（FAP）是5/6肾切除小鼠肌肉中脂肪细胞的主要来源，5/6肾切除（造模20周）小鼠的血清中CCN1升高，CCN1通过结合整合素α2β1受体，调节FAP细胞向脂肪细胞分化，增加肌肉中的脂肪细胞。为阐明尿毒症肌少性肥胖的发病机制提供重要实验依据。⑸尿毒症微环境通过启动血清中无翅型MMTV整合位点家族成员3a（Wnt3a），上调CCN1表达，Wnt3a/CCN1这一途径可能是尿毒症肌少症的关键的发病环节之一。DKK-1（Wnt3a抑制剂）可能成为尿毒症肌少症的潜在药物。为临床防治尿毒症肌少症提供新的策略。结论：CCN1在老年人肌少症和尿毒症肌少症的发病机制中起着重要的促进作用。本研究成果创新性强、有科学意义和临床价值。