FOXO1-CPT1信号通路在肉碱防治肿瘤恶液质骨骼肌减少中的机制研究

Skeletal muscle atrophy is a defining feature of cancer cachexia and affects physiologic function, chemotherapy response, surgical outcomes, and survival. Skeletal muscle contains at least 95% of the total body L-carnitine (LC). LC supplementation ameliorates cancer cachexia in mice and cancer patients. However, the effects of LC on muscle atrophy in cancer cachexia and the precise molecular mechanism(s) are not well understood. Carnitine palmitoyltransferase 1 B (CPT1B) is located on the outer mitochondrial membrane of muscle cells and is essential for transporting long chain fatty acids into the mitochondria for oxidation. CPT1B and PDK4 also through PDH play gatekeeping roles in fatty acid oxidation (FAO) and glucose oxidation. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is known to regulate genes involved in skeletal muscle function, include CPT1B and PDK4. Thus, CPT1 is an idea experimental and pharmacological target of studies aimed at elucidating the relationship between fat oxidation and muscle atrophy. Forkhead box class O family member proteins 1 (FOXO1) are key regulators of protein breakdown, as they modulate the activity of several actors in the ubiquitin proteasome, Atrogen-1, Murf-1 and ZNF216. FOXO1 also binds and inhibits PGC-1α leading to a decrease in type I fiber expression. Our preliminary data showed that LC decreased TNF-α treatment induced atrophic responses in C2C12 mytubes, and LC also reduced cachexia-induced muscle atrophy and protein levels of atrogin-1 and MuRF1 in tumor-bearing mice. The objective of the present investigation is to elucidate if LC administration is able to ameliorate muscle wasting in tumor-bearing cachexia mice and chemotherapy patients. We also try to ascertain the possible molecular mechanisms, which FOXO1-CPT1 signaling networks involved with CPT1B overexpression mice and FOXO1 knockout mice . Together, all of these findings will provide a basis for the future development of LC for cancer cachexia muscle atrophy therapy.

骨骼肌减少是肿瘤恶液质核心表现。骨骼肌中肉碱(LC)占机体总量95%,恶液质患者肌肉LC下降。研究提示LC可缓解恶液质的疲倦等症状,但少见改善肌肉减少的研究。LC通过肉碱棕榈酰转移酶1B(CPT1B)促进脂肪酸氧化供能,利于肌肉合成,CPT1B受PGC-1α调控;叉头转录因子FOXO1促进肌蛋白过度降解,并受PGC-1α调控而抑制脂肪酸供能。推测FOXO1-CPT1B共同调节恶液质肌细胞蛋白平衡及脂代谢。预实验发现LC削弱TNF-α对肌细胞C2C12肌管形成的抑制,降低肌细胞及荷瘤小鼠骨骼肌FOXO1,下游泛素化酶Atrogen-1及Murf-1表达,促进CPT1B活性。本研究通过C2C12细胞分化模型,荷瘤恶液质小鼠,CPT1B过表达小鼠, FOXO1基因敲除小鼠模型及患者LC干预临床研究,探讨LC干预与FOXO1-CPT1B相关的分子机制,为LC防治肿瘤恶液肌肉减少提供理论及临床依据。

肿瘤患者随病情进展，常出现不可逆的食欲下降、体重下降、营养状况恶化，直至最后死亡，这就是肿瘤恶液质。研究显示，恶液质患者骨骼肌肉碱缺乏，且肉碱缺乏与恶液质进展有关，动物实验及临床研究提示，肉碱干预能有效缓解肿瘤恶液质症状。.本研究的结果证明，肉碱下调骨骼肌中肌细胞降解的关键酶MuRF-1和MAFbx表达。进一步利用siRNA-Akt证实，肉碱下调MAFbx是经由Akt/FOXO3a通路，且影响骨骼肌的降解。同时证明肉碱下调Akt上游Mystatin的表达，且通过分子对接证实肉碱可能与Mystatin存在直接结合位点。除上述机制，本研究也证实肉碱改善恶液质与调控糖脂代谢相关关键酶- PGC-1α、CD36、FOXO1和PDK4相关。. 除分子机制以外，细胞的超微结构在恶液质中的发生发展中也具有重要的作用。线粒体是细胞中ATP主要来源的场所。本研究证实恶液质可引起线粒体肿胀，肉碱缓解这一现象。T管与肌浆网构成肌细胞中特有的三联体，其主要存储细胞中的Ca离子，及给线粒体输送葡萄糖进行能量代谢。本研究证实，恶液质可以引起T肿胀，肉碱可以逆转这一效应。. 除对肌细胞的影响以外，本研究也从根源上证实肉碱对恶液质的作用。在恶液质中，肿瘤细胞主要通过分泌炎症因子影响肌肉状态，导致恶液质的发生。本研究证实，肉碱干预18天后可以抑制血液中IL-6和IL-1的含量，其中以IL-6最为明显。但肉碱干预7天，并不能改善血液中IL-6,IL-1和TNF-α的含量。.本研究主要证明：1)肉碱经由Akt/FOXO3a通路抑制MuRF-1和Atrogin-1表达，同时以Akt非依赖的方式上调蛋白合成关键酶p70S6K；2)肉碱通过调节糖脂代谢关键酶；3)肉碱改善恶液质对超微结构线粒体及T管的损伤；4) 肉碱抑制血液中IL-6和IL-1的表达，并不影响TNF-α的表达。