基于cAMP/PKA/RhoA信号通路探讨益气活血法促进BMSCs移植修复脊髓损伤的机制

Axonal retrograde degeneration may result in death and atrophy of soma after spinal cord injury(SCI), so rescuing soma and keeping its physiologic function is the prime therapeutic target for axonal restoration and regeneration. Bone marrow mesenchymal stem cells(BMSCs) transplantation is a promising experimental treatment for SCI, but the low activity limits their survival, proliferation and differentiation. SCI has been regarded as the "syndrome of blood stasis due to qi deficiency" in traditional Chinese medicine. Our previous stuty reported that Buyang Huanwu Decoction(BYHWD) prevented axotomy-induced rubrospinal neuronal death and atrophy, enhanced axonal regeneration, promoted functional recovery of forelimb in a rat model of SCI. In the present study, the soma which is far away from the site of injury will be the research target. Detection methods include behavioristics, neural tract tracing, immunohistochemistry, western blot, etc. Firstly, whether BYHWD combined with BMSCs transplantation will be cooperative remarkably for SCI therapy will be explored. Then, on the basis of cooperation, we continue to clarify whether BYHWD promotes BMSCs transplantation to restore SCI by up-regulating cAMP and then activating cAMP/PKA/RhoA signaling pathway. The results will reveal the molecular mechanism of "supplementing QI and activating blood circulation method" and offer novel treatments for SCI with traditional Chinese medicinal herbs.

脊髓损伤（SCI）后轴突可通过继发逆行性溃变，导致神经元胞体发生死亡与萎缩，因此挽救神经元胞体，维持其正常生理功能，是轴突修复与再生的前提。BMSCs移植是治疗SCI的理想选择，但BMSCs活力低，在体内不容易存活、增殖和分化。SCI归属中医"气虚血瘀"证，我们的前期研究发现益气活血中药复方补阳还五汤（BYHWD）能提高大鼠红核脊髓束（RST）横断后红核神经元的存活率，抑制萎缩，促进轴突再生及部分功能修复。本项目拟选择远离损伤部位的神经元胞体作为研究靶点，采用行为学分析、神经束路示踪、免疫组化、蛋白质印迹等方法，探索BYHWD联合BMSCs移植修复SCI能否产生协同效应。在确定具有协同增效的基础上，可望合理解释BYHWD是否通过上调cAMP、激活cAMP/PKA/RhoA信号通路来促进BMSCs移植修复SCI，从而实现其"益气活血"作用的分子机制，为中医药治疗SCI提供新的思路和方法。

脊髓损伤（SCI）归属中医“气虚血瘀证”范畴，SCI后轴突可通过继发逆行性溃变，导致神经元胞体发生死亡与萎缩，因此挽救神经元胞体，维持其正常生理功能，是轴突修复与再生的前提。骨髓间充质干细胞（BMSCs）移植是治疗SCI的理想选择，但BMSCs活力低，在体内不容易存活、增殖和分化。本研究选择SCI局部以及远离损伤部位的神经元胞体作为研究靶点，采用中医宏观表征观察、行为学分析、神经束路示踪、免疫组化、PCR、蛋白质印迹等方法，探索益气活血中药复方补阳还五汤（BYHWD）联合BMSCs移植修复SCI能否产生协同效应。在确定具有协同增效的基础上，可望合理解释BYHWD是否通过上调cAMP、激活cAMP/PKA/RhoA信号通路来促进BMSCs移植修复SCI，从而实现其“益气活血”作用的分子机制。. 结果发现：① 游泳力竭法联合RST横断术能顺利制备SCI气虚血瘀证大鼠模型；② BYHWD能促进移植的BMSCs在脊髓局部存活、增殖并向神经干细胞方向分化。③ BYHWD联合BMSCs移植对保护SCI气虚血瘀证脊髓局部轴突、改善前肢运动功能可产生协同增效作用，其作用机制可能与上调脊髓局部微环境cAMP浓度，激活cAMP/RhoA信号通路，上调NGF、BDNF有关。④ BYHWD联合BMSCs移植对保护SCI气虚血瘀证逆行性溃变的红核神经元胞体可产生协同增效作用，其作用机制可能与上调红核胞体内cAMP浓度，激活cAMP/PKA/RhoA信号通路，上调NGF、BDNF有关。这既为SCI 的中医药治疗提供实验依据和科学参考，又为SCI 的临床治疗提供了新的思路。