结直肠癌诱导骨骼肌自噬和有氧糖酵解致脾虚肌肉失养的机制研究

Colorectal cancer (CRC) induced malnutrition and even cachexia are important death causes. Spleen-deficiency is an important pathogenesis and a major clinic syndrome of CRC with malnutrition. CRC cells secrete reactive oxygen species (ROS) and high-mobility group box 1 (HMGB1) to drive autophagy and aerobic glycolysis of muscle cells. The “parasitic” metabolic coupling converts the muscle into a “factory” for the local production of recycled and high-energy nutrients (such as L-lactate) - to fuel oxidative mitochondrial metabolism of CRC cells. The reverse “Warburg effects” conform to the essense of Spleen-deficiency theory in traditional Chinese medicine. Our previous work showed that HMGB1 induces autophagy and aerobic glycolysis through interaction between PGAM5, RIPK2 and PKM1. We thus propose a hypothesis that CRC usurps glatamate, ketone and lactate from skeletal muscle as an energy repository through inducing skeletal muscles autophagy. To elucidate the hypothesis, the relationship between energy metabolism reprogramming and the internal essence of Spleen-deficiency in CRC patients would be analyzed. RAGE-/- and PGAM5-/- would be used to analyze the mechanism of skeletal muscles autophagy induced by CRC. Shenling Baizhu San would be used as a ounterevidence to explain the correlation between Spleen-deficiency and skeletal muscle autophagy. Finally, overexpression and siRNA would be used to elucidate the regulatory mechanism in cells. Novel connotation between skeletal muscle autophagy and Spleen -deficiency would be infused to the theory “Spleen governing muscles”.

结直肠癌(CRC)释放肿瘤源型因子产生慢性炎症和营养不良，契合于中医脾虚证内涵。CRC分泌的HMGB1和ROS通过RAGE诱导肌肉自噬和有氧糖酵解，产生的酮体、乳酸、谷氨酰胺等代谢物供肿瘤细胞进行氧化磷酸化，其消耗肌肉、因癌致虚的反向瓦博格效应与“脾虚肌肉失养”的论述一致。我们发现，HMGB1通过PGAM5与RIPK2、PKM1的相互作用诱导肌肉细胞自噬和有氧糖酵解。基于此，我们提出假说：CRC通过RAGE及PGAM5通路诱导骨骼肌自噬和有氧糖酵解，致脾虚肌肉失养；CRC利用产生的谷氨酰胺等代谢物进行氧化磷酸化并加重脾虚。拟首先分析临床CRC脾虚病人能量代谢重编程与RAGE和PGAM5关系；再应用RAGE和PGAM5基因敲除小鼠阐明肿瘤诱导肌肉自噬和有氧糖酵解的机制，以参苓白术散反佐骨骼肌自噬与脾虚证关系；提炼CRC诱导骨骼肌自噬致脾虚肌肉失养的机制，有望提供脾主身之肌肉经典论述的新认识。

背景.结直肠癌肿瘤微环境是结直肠癌（CRC）发生和复发的必要物质基础，脾虚是该微环境形成的关键病机。CRC分泌炎症因子通过PGAM5诱导骨骼肌自噬致脾虚肌肉失养。 .主要研究内容、结果及数据. 本项目从临床、动物模型和细胞调控机制等三个层面研究了MDSC参与CRC免疫抑制肿瘤微环境形成的作用及其调控机制。发现参苓白术散加减显著减少血MDSC比例，改善CRC病人免疫抑制微环境。同时在细胞水平上发现肿瘤分泌HMGB1因子通过PGAM5通路诱导肌肉自噬，对脾虚肌肉失养有了新的认识。.1．发现参苓白术散加减方可抑制结直肠肿瘤干细胞的增殖，该抑制作用主要归因于本方可下调TGF-beta表达，使Smad信号通路失活，从而抑制肿瘤细胞EMT的发生。2. 利用结肠癌原位移植瘤模型在转基因PGAM5-/-小鼠上观察小鼠体重及肌肉量的变化，发现PGAM5-/-抑制肿瘤引起的肌肉减少，动物实验腓肠肌标本WB，荷瘤小鼠表达更多自噬相关蛋白LC3、P62，PGAM5-/-后表达减少。体外细胞实验，用HMGB1因子刺激小鼠成肌细胞C2C12，MDC法染色显示自噬，提示HMGB1能剂量依赖性和时间依赖性的诱导C2C12发生自噬。3. 自噬并非都是不利的，健脾化湿中药白术有效成分白术内酯I（AT1）具有抗肿瘤和抗炎活性。在APCmin/+小鼠中激活自噬减少腺瘤的发生，并确定D-dopachrome（D-DT）是AT1的潜在靶标。4. 在非酒精性脂肪性肝炎（NASH）中溶酶体酸化，自噬流抑制。中药虎杖苷可以通过上调TFEB从而修复自噬-溶酶体的损伤来发挥其在NASH中的保护作用。.科学意义.提出脾虚是CRC肌肉消耗导致恶病质的主要病机，肌肉过度自噬导致脾虚肌肉失养又加重CRC病情进展，健脾扶正调控肌肉自噬能有效改善CRC肌肉失养。发表论文共13篇，其中SCI论文9篇，累计影响因子32。中文核心期刊文章4篇。