亚低温影响心肺复苏后补体介导脑皮质神经细胞自噬与凋亡“分子对话”的机制研究

At present, brain ischemic-reperfusion (I/R) injury remains a major cause of very low rate of survival to discharge in patients resuscitated from a sudden cardiac arrest. Therapeutic mild hypothermia is the only effective treatment that has been confirmed clinically to improve neurological outcomes. However, how to maximize the therapeutic effects of mild hypothermia and the mechanisms of its cerebral protection are not yet fully understood. Increasing studies showed that I/R-induced autophagy is a double-edged sword for ischemic cerebral injury; autophagy and apoptosis share the same upstream regulatory pathways and therefore crosstalk occurs between them. As a result of this crosstalk, excessive autophagy may facilitate cell apoptosis and thus exacerbate cerebral injury. Our previous study observed an activation of complement in pig brain tissue after cardiopulmonary resuscitation, whereas this activation was inhibited by mild hypothermia. Based on the above findings, we hypothesize that the complement activation in pig brain tissue after cardiopulmonary resuscitation mediates the crosstalk between autophagy and apoptosis of nerve cells from brain cortex, and this pathological process is inhibited by mild hypothermia. In the present study, we will determine the effects of mild hypothermia on complement activation- induced crosstalk between autophagy and apoptosis of nerve cells from brain cortex and its regulatory pathways at neurological function, histopathological and cellular and molecular levels by blocking the actions of C3a, C5a and C5b-9 and intervening regulatory pathways of autophagy in a pig model of cardiac arrest and a human neuron model of oxygen glucose deprivation/reoxygenation. This study will further probe into the mechanisms of brain I/R injury after cardiopulmonary resuscitation and the mechanisms of cerebral protection by mild hypothermia, and thus provide new experimental and theoretical basises for optimizing therapeutic mild hypothermia in clinic.

目前心肺复苏后患者脑缺血-再灌注（I/R）损伤仍然是存活率低的主要原因之一。亚低温是唯一被临床证实能改善神经功能预后的措施，而如何使亚低温疗效最佳化及脑保护机制均未完全确定。研究表明，脑I/R诱导的神经细胞自噬对脑损伤是一把“双刃剑”；自噬和凋亡有相同的上游调控通路，存在“分子对话”，过度自噬可促进凋亡，加重脑损伤。我们前期研究表明心肺复苏后脑组织存在补体激活且可被亚低温抑制。我们设想心肺复苏后补体激活可介导脑皮质神经细胞自噬与凋亡“分子对话”且亚低温抑制该病理过程。故，本课题采用猪心跳骤停模型和人神经元氧糖剥夺/复氧模型，分别阻断补体C3a、C5a和C5b-9作用并干预自噬调控通路，从神经功能、组织病理和细胞分子水平检测亚低温对补体激活介导的神经细胞自噬与凋亡“分子对话”及其调控通路的影响，进一步探讨心肺复苏后脑I/R损伤和亚低温脑保护机制，为临床上优化亚低温治疗提供新的实验和理论依据。

目前心肺复苏后患者脑缺血-再灌注（I/R）损伤仍然是存活率低的主要原因之一。亚低温是唯一被临床证实能改善神经功能预后的措施，而如何使亚低温疗效最佳化及脑保护机制均未完全确定。研究表明，脑I/R诱导的神经细胞自噬对脑损伤是一把“双刃剑”；自噬和凋亡有相同的上游调控通路，存在“分子对话”，过度自噬可促进凋亡，加重脑损伤。我们前期研究表明心肺复苏后脑组织存在补体激活且可被亚低温抑制。然而，心肺复苏后补体能否介导脑皮质神经细胞的自噬与凋亡和两者之间的“分子对话”及亚低温是否影响该机制均不清楚。本课题研究发现自主循环恢复后24 h猪大脑皮层神经细胞存在自噬和凋亡及补体系统激活，而亚低温可抑制神经细胞的自噬和凋亡及补体系统激活；在体外培养的神经元上通过阻断补体C3a、C5a和膜攻击复合物C5b-9的形成，阻断或激动自噬信号通路或给予亚低温处理，结果发现氧糖剥夺/复氧后神经元自噬和凋亡之间的存在“分子对话”，且均与补体激活相关，而亚低温可抑制补体诱导的神经元自噬和凋亡。该研究从神经功能、组织病理和细胞分子水平上探讨了亚低温对补体介导的神经细胞自噬与凋亡“分子对话”及其调控通路的影响，进一步明确了心肺复苏后脑缺血/再灌注损伤和亚低温脑保护机制，为临床上优化亚低温治疗提供了新的实验和理论依据。