β2-AR激动剂福莫特罗调控BCKDC改善恶病质骨骼肌萎缩的作用机制研究

Cachexia is a multifactorial metabolic syndrome characterized by ongoing loss of body weight and lean body mass. It is common in chronic wasting disease, such as cancer. However, the mechanism is still controversial. It is known that the preserve of skeletal muscle mass is helpful for the prognosis and outcome of cancer cahceixa. The intervention of skeletal muscle atrophy is the main target for the treatment of cachexia. Our previous study showed that β2-adrenoceptor (β2-AR) agonist fomrmoterol could prevent the wasting of skeletal muscle and alleviate the loss of body weight. Moreover, the preliminary experiments showed formoterol treatment could regulate the metabolism of branched chain amino acid, thus resulting in the accumulation of α-ketoisocaproate, which was the substrate of enzyme BCKDC. It is known that branched chain amino acid are essential for the synthesis of protein and they can promote the synthesis of muscle protein. So we proposed the hypothesis that BCKDC is a potential target for the treatment of muscle waste in cachexia, and β2-AR agonist formoterol can regulate the activity of BCKDC and alleviate the syndrome of cancer cachexia. To state the metabolic mechanism of branched chain amino acid, stable isotope 13C was employed to trace the dynamic changes of leucine metabolism. The activity of BCKDC was measured using enzyme kinetics method. What is more, to evaluate the effect of BCKDC in the muscle wasting of cachexia in vitro and in vivo, the activity of BCKDC was suppressed by the method of gene disruptions or addition of key inhibitors. The present research will not only reveal the novel metabolic mechanism of muscle atrophy in cancer cachexia, but also provide a new approach for the potential drug treatment of muscle wasting.

恶病质是恶性肿瘤等疾病伴发的以进行性体重减轻为特征的多因素综合征，研究表明改善骨骼肌萎缩有利于其预后。β2肾上腺能受体（β2-AR）激动剂能促进骨骼肌蛋白合成，我们研究也证明福莫特罗能改善模型动物骨骼肌萎缩和体重减轻，但机制不明；体外预实验发现其能调控支链氨基酸代谢并导致支链α酮酸脱氢酶复合物（BCKDC）上游代谢产物α-酮异己酸的积累。BCKDC是亮氨酸代谢的限速酶，能调控骨骼肌蛋白质代谢。因此我们提出假说：BCKDC是干预骨骼肌萎缩的潜在靶点，福莫特罗可能通过调控BCKDC改善骨骼肌萎缩和恶病质预后。本课题拟采用稳定同位素示踪技术动态分析亮氨酸代谢改变，以酶动力学方法测定BCKDC活性，结合分子抑制剂和基因转染等手段研究BCKDC对骨骼肌萎缩的意义，通过体内外实验探索福莫特罗的作用机制和代谢靶点。本研究一方面可揭示骨骼肌萎缩的关键限制性因素；另一方面有助于为恶病质的防治研究提供新思路。

恶病质是恶性肿瘤等疾病伴发的蛋白质分解代谢增加，合成代谢不足，导致严重体重下降和肌肉丢失为特征的多因素综合征。但传统促进骨骼肌蛋白合成的药物由于广泛的药理活性和容易产生心肌肥大副作用而不能用于临床。支链氨基酸不仅是肌蛋白的重要构成成分，而且对蛋白质合成具有重要意义。我们分析肌组织中支链氨基酸浓度变化：难治性恶病质阶段支链氨基酸浓度下降，而恶病质早期和典型恶病质期，支链氨基酸浓度升高。稳定同位素示踪发现肌萎缩伴随特异性的α-酮异己酸信号峰强度减弱。α-酮异己酸是BCKDC的上游代谢物，提示肌萎缩表现为BCKDC功能增强。进一步实验发现：①BCKDE1A的干扰会抑制C2C12成肌细胞分化为多核肌小管；②以腺病毒干扰BCKDK表达时，多核肌小管MyHC、MyoD和Myogeinin的表达明显减少；③在CT26模型小鼠的腓肠肌组织局部注射干扰BCKDK表达的腺病毒14天后，腓肠肌质量减轻，腓肠肌局部肌纤维萎缩。这些研究结果提示BCKDK可通过调控BCKDC活性而负向影响骨骼肌纤维蛋白合成。BCKDK是丝氨酸激酶，我们研究BCKDK对恶病质骨骼肌萎缩的影响发现：采用BT2抑制BCKDK时，BCKDE1A磷酸化程度升高，肌蛋白构成性成分Myogeinin 和MyHC表达下降。福莫特罗改善恶病质骨骼肌萎缩是因为增强BCKDK而抑制BCKDE1A表达，促进骨骼肌蛋白合成，抑制蛋白降解。这些研究提示福莫特罗通过BCKDK/BCKDC抑制支链氨基酸分解代谢促进骨骼肌蛋白合成。