脑缺血对免疫系统乙酰胆碱酯酶及Caspase-3表达的影响

The effects of rat brain ischemia on the expression of Acetyl-cholinesterase and Caspase-3 of the immune system

目的:探讨脑缺血对免疫系统的影响以及神经免疫损伤过程中乙酰胆碱酯酶与Caspase-3表达的作用。方法:ELISA检测不同缺血时间模型大鼠血清TNF-α、IL-2、IL-12、IL-4、IL-10含量,脑组织、胸腺、脾脏AChE含量;采用Western blot方法检测脑组织、胸腺、脾脏Caspase-3活化片段P32、P17的表达;免疫组化标示AChE在神经细胞的表达状况;分析了AChE与细胞凋亡、TNF-α、IL-2、IL-12、IL-4、IL-10、NK细胞活性变化之间的关系。结果:缺血侧脑组织AChE随缺血时间的延长逐渐增高,在缺血12小时时间点达高峰,随后逐渐降低;胸腺、脾脏AChE在缺血1小时增高,与缺血侧脑组织趋势基本一致;在缺血2小时之前TNF-α、IL-2、IL-12亦与缺血侧脑AChE的变化一致,血清IL-4、IL-10无明显变化。自缺血3小时之后NK活性、TNF-α、IL-2、IL-12与缺血侧脑AChE的变化完全相反,而IL-10与缺血侧脑AChE的变化基本一致,IL-4含量在各缺血时间点变化不明显,仅在缺血12小时时间点略有升高。P17/(P32+P17)比值与AChE有关,且随缺血时间的延长逐渐增高。结论:脑缺血导致AChE持续增高通过活化Casepase-3,诱导缺血脑组织、胸腺、脾脏细胞凋亡,进而导致Th1/Th2失衡影响免疫功能。

Objective： To explore the effects of brain ischemia on the immune system and the expression of Acetyl-cholinesterase （ACHE） and Caspase-3 after neuro-immunological injury.Methods： The serum IL-4,IL- 10, TNF-α, IL-2 and IL- 12 concentrations and ACHE level in brain,thymus and spleen of the rat brain ischemia at different time spots were detected by ELISA. The active fragments of Caspase-3 P17 and P32 in brain, thymus and spleen were tested by Westem blot. The expression of ACHE in nerve cells was examined immunohistochemistry. The relationship between amount of ACHE and concentrations of the cytokines or NK activity as well as cell apoptosis was analyzed. Results： The concentration of ACHE in brain increased gradually after ischemia, the level reached peak after 12 hours, and then decreased afterward. The ACHE level in thymus and spleen increased after 1 h in accordance with the ischemic brain. The NK activity, serum TNF-α, IL-2 and IL-12 levels also increased at 0.5 h, 1 h and 2 h after iscbemia,but the trend reversed after 3 hours. Serum IL-4 and IL- 10 levels did not change significandy at 0.5 h, 1 h and 2 h after ischemia, but serum IL- 10 raised after 3 hours to 48 hours. Serum IL-4 level slightly increased only appearing at ischemia 12 h point. The ratio of P17/（ P17 ＋ P32） became higher with iscbemic time. Conclusion： Sustained rising of ACHE level due to brain ischemia induces the apoptosis of cells in the brain, thymus and spleen via activation caspase-3, leading to Th1/Th2 imbalance and immune dysfunctions.