益肾养髓方基于Akt/mTOR通路调控细胞自噬对脊髓慢性损伤的保护机制研究

In early clinical researches，it has been proved that yishenyangsui decoction（YSYSD） can effectively improve the limb motor function of patients with cervical spondylotic myelopathy, and basic experiments showed that it had a protective effect on neurons in the rats model of chronic spinal cord injury(CSCI). Literature and network pharmacological studies indicate that down-regulation of Akt protein can inhibit the expression of mTOR protein, improve the level of autophagy, and reduce the damage of neurons. YSYSD can regulate Akt protein expression in the Akt/mTOR pathway. Hypothesis: YSYSD affects the expression of key proteins in the Akt/mTOR pathway via regulating Akt protein, regulates the level of autophagy and plays a neuroprotective role. In this study, a rat model of chronic spinal cord injury will be established to observe the autophagy of neurons via electron microscopy and test the expression of p-akt, Akt, p-mtor, mTOR and other proteins by tissue transparency and western blot etc. We will establish a injuried spinal cord neuron cell model to further verify the regulatory effect of YSYSD on the expression of key factors in Akt/mTOR pathway. Innovation: applying the CUBIC method to transparentize tissue and observing the tissue with the help of fluorescence tracing technology break through the limitation of traditional sections technology which is difficult to determine the interorganizational relationship. This study is a prospective exploration of Chinese herb in promoting functional recovery of CSCI from the perspective of Akt/mTOR pathway regulating autophagy.

前期临床证实益肾养髓方可有效改善脊髓型颈椎病患者肢体运动功能，基础研究发现其对慢性脊髓损伤大鼠模型神经元有保护作用。文献及网络药理学研究发现：下调Akt蛋白可抑制mTOR蛋白表达，提高自噬水平，减少神经元细胞损伤；益肾养髓方可调节Akt/mTOR通路中Akt蛋白表达。假说：益肾养髓方通过调控Akt蛋白影响Akt/mTOR通路关键蛋白表达，调节细胞自噬水平，发挥神经保护作用。本研究将建立慢性脊髓损伤大鼠模型，通过电镜、组织透明、蛋白印迹等方法，观测神经元自噬及p-Akt、Akt、p-mTOR、mTOR等蛋白表达；建立脊髓神经元损伤细胞模型，采用抑制剂验证该复方对通路中关键因子表达的调控作用。创新点：采用CUBIC方法进行组织透明，结合荧光示踪技术进行观察，突破传统切片难以明确组织间关系的局限。从Akt/mTOR通路调控细胞自噬角度开展研究，是中药促进慢性脊髓损伤功能恢复研究的前瞻性探索。

前期临床研究证实益肾养髓方可有效改善脊髓型颈椎病(CSM)患者肢体运动功能，且经过基础研究发现其对慢性脊髓损伤大鼠模型神经元有保护作用。本研究通过在体建立(CSM)动物模型与神经细胞糖氧剥夺损伤（OGD）模型，并分别予益肾养髓方中药水煎剂与益肾养髓方含药血清进行干预。结果发现CSM后以LC3B、P62蛋白表达同步增加为表现的自噬小体降解受阻可能是导致细胞损伤的重要机制，益肾养髓方干预可以显著改善CSM大鼠的BBB评分与斜板试验评分，促进大鼠运动功能与受损脊髓形态的恢复，减少P62、Bax、caspase3的表达，降低细胞凋亡率。同时进一步发现益肾养髓方干预可以降低AKT/mTOR蛋白的表达水平。明确了自噬流受阻是大鼠脊髓慢性压迫损伤后导致细胞凋亡的主要病理机制之一，促进自噬水平可以减轻脊髓慢性损伤后的细胞凋亡，而抑制自噬可能会引发神经元细胞的凋亡；初步明确了益肾养髓方促进脊髓慢性损伤恢复的主要作用机制是通过促进自噬流的恢复，从而减轻神经元细胞的凋亡；明确了益肾养髓方可能是通过抑制Akt/mTOR通路从而发挥促进自噬、抑制神经细胞凋亡的作用，丰富了中药复方治疗中枢性神经病变的科学内涵，为治疗CSM药物开发提供科学依据。