不同脑微血管内皮细胞条件培养液抗缺血及再灌神经元损伤的比较

Conditioned mediums of different rat cerebral microvascular endothelial cells against damage of ischemia and ischemia/reperfusion neurons

目的:通过乳酸脱氢酶(lactate dehydrogenase,LDH)的检测,观察解毒通络药物干预后的脑微血管内皮细胞条件培养液(conditioned medium of cerebral microvascular endothelial cells,CMECs-CM)特征及其抗缺血及再灌皮质神经元损伤的效应,探讨脑微血管内皮细胞(cerebral microvascular endothelial cells,CMECs)调控神经元功能的机制。方法:通络救脑注射液(Tongluo Jiunao Injection,TLJNI)作用于正常、缺血和缺血再灌三种培养状态的大鼠CMECs,分别收集有药物干预及无干预的三对无血清内皮细胞条件培养液,并测定其LDH值。同步原代培养大鼠脑皮质神经元,亦分为正常、缺血、缺血再灌三组,用低(10%)、中(50%)、高(100%)三种浓度的各类CMECs-CM分别作用上述三种培养状态的神经元,测定其LDH漏出率。结果:比较TLJNI干预后的CMECs-CM与无干预的CMECs-CM中LDH漏出值,CMECs缺血组与缺血再灌组经药物干预后均明显降低(P<0.01)。对于缺血神经元,缺血内皮细胞条件培养液(conditioned medium of ischemic CMECs,Is-CM)高浓度(100%)和TLJNI干预后的缺血内皮细胞条件培养液(conditioned medium of ischemic CMECs with drug treatment,IsT-CM)高浓度(100%)作用后表现为增加LDH漏出率(P<0.01),而低浓度10%的IsT-CM则降低LDH漏出率(P<0.05);对于缺血再灌神经元,与对照组比较,各类CMECs-CM作用后均能降低神经元LDH漏出率(P<0.05或P<0.01)。比较每种条件液相邻浓度间的效应差异,均以10%或50%的CM降低损伤为佳,正常内皮细胞条件培养液(conditioned medium of normal CMECs,N-CM)及缺血再灌内皮细胞条件培养液(conditioned medium of ischemic/reperfusional CMECs,Rp-CM)组间差异有统计学意义(P<0.05或P<0.01);对于正常神经元,各类CMECs-CM作用后均增加了神经元LDH漏出率(P<0.05或P<0.01)。比较每种条件液相邻浓度间的效应差异,N-CM组间差异有统计学意义(P<0.05或P<0.01)。结论:TLJNI能够防御CMECs缺血及缺血再灌的损伤,可以通过内皮细胞介导发挥抗缺血及缺血再灌神经元损伤的效应。提示CMECs-CM及TLJNI干预后的CMECs-CM均存在着抗缺血再灌神经元损伤的活性物质。

Objective： Using the method of lactate dehydrogenase （LDH） assay, to observe the activities of rat cerebral microvascular endothelial cells （CMECs） intervened by Tongluo Jiunao Injection （TLJNI）, a traditional Chinese compound drug removing toxin to dredge brain collaterals, and then further study the effects of different kinds of conditioned mediums （CMECs-CM） of cerebral microvascular endothelial cells on ischemia and ischemia/reperfusion cerebral cortex cells, and to probe into the drug pharmacological mechanisms of CMECs in modulating the neurons.Methods： Three kinds of CMECs （normal, ischemic and ischemic/reperfusional） were all treated by TLJNI previously, and then the three pairs of CMECs-CM without serum were collected respectively for LDH assay. Rat cerebral cortex neurons were also primarily cultured and then divided into similar three groups （normal, ischemic and ischemic/reperfusional）. The neuron responses caused by CMECs-CM at different concentrations were observed by using LDH transudation rate assay.Results： The LDH release values of ischemic and ischemic/reperfusional CMECs with TLJNI treatment were obviously reduced （P〈0.01） compared with the same kinds of CMECs untreated. For ischemic neurons, both conditioned medium of ischemic CMECs （Is-CM） and conditioned medium of ischemic CMECs with drug treatment （IsT-CM） in high concentration of 100% increased the LDH transudation rate （P〈0.01）, while in low concentration of 10%, IsT-CM reduced the transudation rate （P〈0.05）. For ischemia/reperfusion neurons, all kinds of CMECs-CM reduced the transudation rate respectively （P〈0.05 or P〈0.01）. As far as each group concentration was concerned, 10% or 50% showed relatively stronger effects, and both conditioned medium of normal CMECs （N-CM） group and conditioned medium of ischemic/reperfusional CMECs （Rp-CM） group had statistical significance （P〈0.05 or P〈0.01）. For normal neurons, all kinds of CMECs-CM increased the transudation rate respectively （P〈0.05 or P〈0.01）. As far as each group concentration was concerned, only conditioned medium of normal CMECs （N-CM）had statistical significance （P〈0.05 or P〈0.01）. Conclusion： The study shows that TLJNI is capable of preventing the damage of CMECs from both ischemia and ischemia/reperfusion states. Chinese drug can restrain the brain ischemia and ischemia/reperfusion damage by the media that CMECs modulate the neurons, demonstrating the pharmacological mechanisms of TLJNI. This work also indicates that there exist some active substances against ischemia/reperfusion injury secreted from CMECs-CM with TLJNI treatment.