A型肉毒毒素干预交感神经通路治疗雷诺综合征的机制研究

Raynaud's syndrome is a kind of blood vessel disorder with terminal small arterial excessive spasm and stenosis usually induced by cold or emotion. Several clinical studies suggest that Botulinum toxin type A (BoNT/A) had significant effectiveness in treatment of Raynaud 's syndrome, but the exact mechanism is unknow. BoNT/A was firstly used in treatment of patient with Raynaud's syndrome at home, we found that blood flow in hand was significantly faster than before, ischemic symptoms improved and ulcer healing.Our preliminary experiments show that BoNT/A can significantly antagonize the vasoconstriction of the small artery induced by electrical stimulation,and the immediate intensity was equal to the effect of α -adrenergic receptor antagonist. The result indicate that BoNT/A may antagonize the vasoconstriction mainly by the inhibition of the release of norepinephrine.We plan to use PLAP transgenic mice Raynaud's syndrome model and cultured sympathetic neurons as experimental materials to study mechanism and effect of BoNT/A on the sympathetic neurons in the small arteries and norepinephrine neurotransmitter release,to clarify the prosess of the changes in morphological and functional recovery of the sympathetic schedule after chemical blockade, and to reveal the evolvement rule of secondary vascular endothelial and smooth muscle of small arteries, providing a theoretical basis about BoNT/A in the treatment of small vessel disease caused by sympathetic dysfunction.

雷诺综合征是由于寒冷或情绪刺激诱发的肢端小动脉过度痉挛、管腔狭窄性疾病。国外多项临床研究提示A型肉毒毒素（BoNT/A）治疗雷诺综合征疗效显著，但具体机制尚不清楚。本课题组在国内率先应用BoNT/A治疗雷诺综合征，发现患者手部小动脉血流明显增快，缺血症状改善、溃疡愈合。我们前期实验证明BoNT/A能够显著拮抗电刺激诱发的小动脉收缩效应，其即刻作用强度与肾上腺素α受体拮抗剂相当，表明BoNT/A可能主要通过抑制血管交感神经去甲肾上腺素释放从而拮抗血管异常收缩。本课题以PLAP转基因小鼠的雷诺综合征模型及培养的交感神经元为实验材料，研究BoNT/A对小动脉交感神经元去甲肾上腺素递质释放的影响及机制，阐明化学性阻滞后交感神经形态及功能恢复的时程变化，并揭示小动脉血管内皮细胞和血管平滑肌的继发性演变规律，为BoNT/A治疗交感神经功能异常引起的小血管疾病提供理论依据。

雷诺综合征是由于寒冷或情绪刺激诱发的肢端小动脉过度痉挛、管腔狭窄性疾病。国外多项临床研究提示A型肉毒毒素（BoNT/A）治疗雷诺综合征疗效显著，但具体机制尚不清楚。本课题组在国内率先应用BoNT/A治疗雷诺综合征，发现患者手部小动脉血流明显增快，缺血症状改善、溃疡愈合。我们前期实验证BoNT/A能够显著拮抗电刺激诱发的小动脉收缩效应，其即刻作用强度与肾上腺素α受体拮抗剂相当，表明BoNT/A可能主要通过抑制血管交感神经去甲肾上腺素释放从而拮抗血管异常收缩。本课题通过大鼠提睾肌动脉电刺激模型，发现BoNT/A、哌唑嗪、BQ123均可显著抑制电刺激诱发的微动脉收缩反应，育亨宾无明显抑制效应。BoNT/A引起的抑制效应可被内皮素受体拮抗剂增强，而不被α1-肾上腺素受体拮抗剂、α2-肾上腺素受体拮抗剂增强，提示BoNT/A可能主要通过拮抗α1肾上腺素能受体介导的交感神经通路抑制血管收缩，对血管内皮细胞分泌内皮素无明显影响。应用大鼠颈上交感神经元原代培养，用BoNT/A处理颈上交感神经节（SCGNS）30分钟后，FM1-43荧光强度下降速度减慢，且与BoNT/A剂量有关。此外，在高剂量BoNT/A（25μ/ml）干预后24小时，高效液相色谱法测定的上清液中的NE释放量显著降低。BoNT/A（50μ/ml）干预24小时后，用Western blotting可检测到裂解的SNAP-25。此外，在交感神经细胞上可检测到类似于胆碱能神经元的受体SV2c、GM1和FGFR3，可特异性与BoNT/A的重链结合。研究结果提示BoNT/A通过裂解SNAP-25，抑制囊泡循环，从而抑制去甲肾上腺素（NA）的释放。本课题初步阐明了交感神经通路治疗雷诺综合征的作用机制，为BoNT/A治疗交感神经功能异常引起的小血管疾病提供理论依据。