不同内皮细胞条件培养液对皮层神经元线粒体功能的影响及通络救脑注射液的保护作用

Influence of Different Endothelial Cells Conditioned Medium on the Function of Mitochondria of Cortical Neurons and the Protective Effect of Tongluo Jiunao Injection

目的探讨脑微血管内皮细胞条件培养液对损伤神经元线粒体功能的影响,以及通络救脑注射液对此过程的干预作用。方法制备(1)正常内皮细胞条件培养液(N-CM):正常培养的内皮细胞不作任何处理;(2)正常用药内皮细胞条件培养液(NT-CM):通络救脑注射液按1μl/ml浓度作用10h;(3)缺氧损伤内皮细胞条件培养液(I-CM);(4)缺氧损伤用药内皮细胞条件培养液(IT-CM):于损伤前4h,加入通络救脑注射液1μl/ml;分别作用于糖氧剥夺损伤的神经元模型,通过测定神经元线粒体活性、线粒体膜电位(MMP)及细胞色素C(CytC),分析各个条件培养液对损伤神经元线粒体功能的影响。结果(1)与模型组比较,N-CM组与NT-CM组(P<0.05)均可阻抑损伤神经元线粒体活性的下降,尤其是NT-CM保护作用更为明显。(2)与正常组比较,模型组MMP明显下降;I-CM使受损神经元的线粒体功能进一步下降;I-CM组可进一步降低受损神经元MMP,IT-CM可逆转神经元MMP下降,明显改善这种损伤状态。(3)N-CM组与NT-CM组均可降低损伤神经元的CytC释放,分别与模型组、I-CM组比较,差异均有显著性(P<0.05)。结论脑微血管内皮细胞的旁分泌功能对损伤神经元的存活有重要的调节作用,该作用的实现可能与维持神经元线粒体功能有关,通络救脑注射液可促进该作用的发挥。

Objective To study the influence of conditioned medium of rat brain microvascular endothelial cells on mitochondrial function of cortical neurons and the protective effect of Tongluo Jiunao Injection （TJI） on it. Methods Four kinds of conditioned endothelial cell （EC） cultured medium were prepared, i.e. the N-CM medium prepared by EC cultured in the normal conditioned medium without any treatment; the NT-CM prepared by EC cultured in N-CM and treated with TJI 1 μl/ml for 10 h; the I-CM prepared by EC cultured in the non-glucose kreb medium under hypoxia condition; and the IT-CM by EC pre-treated with TJI 1 μl/ml for 4 h and cultured as that of I-CM. The levels of neuronic mitochondrial activity, membrane potential （MMP） and cytochrome C （Cyt C） were determined before and after the glucose-oxygen deprived model neurons of brain cortex being cultured with different kinds of conditioned EC cultured medium for assessing the effects of these media on mitochondria of injured neuron. Results As compared with those of the normal neuron, the mitochondrial activity and MMP of all injured neurons decreased and Cyt C level increased significantly. But comparison of these indexes among neurons cultured with different conditioned EC culture media showed that the greatest extent abnormality revealed in the N-CM cultured neurons, which even greater than that in the model neuron; while that was less in the N-CM cutured neuron than in model neuron; as for those cultured in the NT-CM and IT-CM, i.e. the TJI treated cuture medium, the abnormal changes were reduced significantly when compared with those cultured in medium untreated with TJI （N-CM and I-CM）, respectively （all P〈0.05）. Conclusion The paracrine secretion of the brain microvascular endothelial cells has evident regulatory effect on survival of the injured neurons, which might possibly be related to its protective effect on neuron mitochondrial function, and TJI could enhance the protective effect.