机械损伤导致Ca2+内流经CaMKKβ/AMPK/mTOR通路调控脊髓神经元自噬的作用及机制研究

Traumatic spinal cord injury (TSCI), which could result in dysfunction of sensation, motion, or defecation for patients, even be life-threatening, has long been an intractable health problem worldwide. Autophagy, as a crucial mechanism to maintain cell homeostasis, participates in the secondary injury following TSCI. Besides, AMPK/mTOR is an important signal transduction pathway in autophagy. Our previous study has showed that after TSCI, autophagy related proteins, Beclin-1 and LC3 II increased in neurons, however, the specific mechanism how mechanical injury regulates autophagy has not been fully elucidated. It is well known that the concentration of Ca2+ elevates in the cytoplasm after injury, and CaMKKβ is an important Ca2+ dependent kinase, which could activate AMPK. Accordingly, we put forward the hypothesis that the mechanical injury leads to Ca2+ influx and intracellular Ca2+ elevation, the latter of which regulates autophagy via CaMKKβ/AMPK/mTOR signaling pathway, and then further influences neural function. The project aims to clarify the mechanism how mechanical injury regulates neuron autophagy via Ca2+ influx, in vitro and in vivo, by means of establishing mechanical injury model of spinal cord neurons and rat spinal cord clamping model, thus providing new ideas and direction for clinical treatment of TSCI.

创伤性脊髓损伤，可导致患者感觉、运动、大小便功能障碍，甚至危及生命，其治疗一直是世界性难题。自噬，作为维持细胞内环境稳态的重要机制，参与了脊髓外伤后的继发性损伤过程，AMPK/mTOR是调控自噬的重要信号通路。我们的前期研究表明，在脊髓损伤中，神经元的自噬相关蛋白 Beclin-1和LC3 II表达增加，但机械损伤调控自噬的具体机制仍未完全阐明。已知细胞损伤后其胞浆内的Ca2+浓度升高，CaMKKβ是依赖Ca2+的重要激酶，可以激活AMPK。据此，我们提出假说，机械损伤导致Ca2+内流，胞内Ca2+升高，后者经CaMKKβ/AMPK/mTOR信号通路调控自噬，进而影响神经功能。本项目拟通过构建脊髓神经元机械损伤模型、大鼠脊髓钳夹模型，从体外和体内水平阐明机械损伤通过Ca2+内流对神经元自噬的调控作用及具体机制，为临床上脊髓损伤后的治疗提供新的思路和方向。

自噬，作为维持细胞内环境稳态的重要机制，参与了脊髓外伤后的继发性损伤过程。本课题探究机械性脊髓损伤中，Ca2+调控神经元自噬的作用及相关机制。采用PC12细胞替代原代脊髓神经元，建立神经元机械损伤（Mechanical Injury， MI）模型，Western blot检测自噬和细胞存活情况，检测自噬相关蛋白LC3-II和Beclin-1表达情况，免疫组化检测自噬相关蛋白LC3-II表达，Tunel检测细胞凋亡情况；测定神经元胞内 Ca2+浓度，采用离子霉素和BAPTA调节胞内 Ca2+浓度，检测自噬相关蛋白表达和细胞凋亡情况；采用STO-609拮抗CaMKKβ，检测CaMKKβ/AMPK/mTOR 信号通路各蛋白表达情况，检测自噬相关蛋白表达和细胞凋亡情况。建立大鼠胸段脊髓机械损伤模型，BBB法评估造模后下肢神经功能。Western blot 检测自噬情况，检测自噬相关蛋白LC3-II和Beclin-1表达情况，免疫组化检测自噬相关蛋白LC3-II表达，Tunel检测细胞凋亡情况；测定神经元胞内 Ca2+浓度，采用离子霉素和BAPTA调节胞内 Ca2+浓度，检测自噬相关蛋白表达和细胞凋亡情况采用STO-609拮抗CaMKKβ，检测蛋白表达及测自噬和凋亡情况。结果：成功建立神经元钳夹损伤（Mechanical Injury， MI）模型。Western blot检测损伤后自噬相关蛋白LC3-II和Beclin-1表达升高，具有时序性；凋亡相关蛋白casepase-3表达升高。胞内Ca2+升高，免疫组化示LC3-II表达增加，Tunel示凋亡增加。离子霉素促进胞内Ca2+升高，促进自噬，而BAPTA作用相反。STO-609拮抗CaMKKβ，促进AMPK磷酸化，抑制mTOR磷酸化，抑制自噬和凋亡。成功建立大鼠脊髓机械损伤模型，BBB评分0-1分。Western blot结果与细胞实验类似。结论：体外和体内实验证明，机械性脊髓损伤中，Ca2+通过CaMKKβ/AMPK/mTOR 信号通路，促进神经元自噬和凋亡。