硫化氢对心跳骤停复苏后血脑屏障的影响及PKC-TJ通路在其中作用的研究

Adjusting and maintenance of the integrity of blood brain barrier (BBB) is one of the most difficulty problem to improve the neurological function after cardiac arrest and resuscitaion.Recent researchs demonstrated that as the third gaseous signaling molecule, hydrogen sulfide (H2S) played an important role in reducing and ameliorating the damagement of neurological function. However, there is rare report about the effect of H2S on BBB and the underlying mechanisms. In our previous study, we found that the damagement of BBB after cardiac arrest is closely related to the brain edema, neuron apoptosis and neurological function deficit. Inhaling of exogenous 80ppm H2S reduced the permeability of FITC-dextran,the damagement of tight junction (TJ), the fracture of basement membrane and improvement the neurological function. In this program, we will explore the effects of H2S on BBB using the cardiac arrest and resustitation model in rats.The expression of PKC protein family and the TJ, the changes of histology and the neurological function will be studied. Moreover, we will testify whether the H2S-PKC-TJ signal pathway is one of the mechanisms using this in vivo studies. These findings will give us a new idea and a new target point for portection of BBB, which will establish a theoretical base for the clinical application of H2S.

调控和维持血脑屏障（BBB）完整性是改善患者心跳骤停复苏后神经功能转归的棘手难题。H2S是近期被发现的第三类气体信号分子，研究证实H2S在减轻神经功能损伤和改善神经功能转归方面具有重要的调节作用，但H2S对BBB的影响和内在机制尚不清楚，国内外研究较少。我们前期的研究显示，心肺复苏后BBB的破坏与复苏后脑水肿含量的增加、神经元的凋亡和神经功能的缺陷密切相关。80ppmH2S能减轻BBB损伤后FITC-dextran的渗漏，减少电镜下紧密连接（tight junction, TJ）的破坏和基底膜的断裂，并改善神经功能转归。本项目在此基础上利用大鼠心跳骤停复苏模型，从PKC蛋白家族及TJ表达、组织学及功能学等方面探讨H2S对心肺复苏后BBB的影响及H2S-PKC-TJ信号通路在其中的作用及调控机制。以期寻找一种能改善心肺复苏后患者神经功能转归的新途径，进而为H2S的临床应用提供重要的理论基础。

本研究采用免疫组织化学、功能学、分子生物学和电镜等手段和技术通过大鼠心跳骤停复苏模型，研究发现：（1）40ppm和80ppm外源性H2S吸入能减轻心搏骤停复苏后神经功能的损伤，其机制与显著抑制心跳骤停复苏后脑组织皮质与海马血脑屏障（BBB）的破坏，减轻皮质及海马组织脑水肿相关。（2）H2S减少心跳骤停复苏后基质金属蛋白酶-9（MMP-9）的高表达，抑制VEGF的表达，增加Ang-1的表达，H2S可能通过影响VEGF-Ang1-MMP9通路改善心肺复苏后BBB的功能。（3）H2S抑制PKC-α, β I, β II and δ的激活，增加PKC-ε的激活，增加紧密连接蛋白Claudin-5, Occludin and ZO-1的表达，维持Claudin-5, Occludin and ZO-1的结构。H2S通过调控PKC-TJ通路减少心跳骤停复苏后BBB的破坏，减轻脑水肿的发生，改善心跳骤停复苏后大鼠的存活率。.上述研究结果证实了吸入外源性低浓度H2S能改善心跳骤停复苏后神经功能的转归，其机制可能与维持BBB的稳定，减轻脑水肿发生相关。抑制MMP-9在心跳骤停复苏后的高表达，抑制VEGF的表达，增加Ang-1的表达，抑制PKC-α, β I, β II and δ的激活，增加PKC-ε的激活，增加紧密连接蛋白Claudin-5, Occludin and ZO-1的表达，维持Claudin-5, Occludin and ZO-1的结构可能是H2S维持BBB稳定的分子信号通路，这些为为改善心跳骤停复苏后患者神经功能的转归提供新的思路，为外源性H2S 的实际临床应用提供了重要的参考依据。