葛根素抗“脑神经血管单元”体外模型内神经元氧糖剥夺损伤的分子机制研究

Heat stress, which has brought great damage to breeding industry at present, may be with a main pathological process of cerebral ischemia. Our previous studies have found that puerarin had significant protection on apoptosis of neurons induced by oxygen glucose deprivation. In this present study, the neurovascular unit (NVU) of SD rat is co-cultured with neurons, cerebral microvascular endothelial cells and astrocytes and contrasted by separately cultured neurons, and then treated with puerarin when the cells are injuried by oxygen glucose deprivation, and the inhibitors of MAPKs are used to pretreated the cells as comparison. The functions of NVU are assessed by 4 hours permeability, transepithelial electrical resistance(TEER), western bolt of specific proteins in component cells and transmission electron microscope analysis of tight junction between cerebral microvascular endothelial cells and astrocytes. Apoptosis rate and concentration of calcium ion in neurons are detected by flow cytometry, activity of neurons are tested by MTT method, content of glutamic acid and oxidative stress indicators such as content of NO, ROS and MDA and activity of iNOS and SOD are tested by respective kit, mRNA rxpression of HSP70, ERK, JNK and p38 are detected by fluorescence quantitative polymerase chain eaction, protein expression of HSP70, ERK1/2, JNK, p38, p-ERK1/2, p-JNK and p-p38 are detected by western bolt, so as to expound the anti-apoptosis molecular mechanism of puerarin on oxygen glucose deprivation induced neurons in NVU, and discuss the theoretical basis of puerarin for alleviating heat stress in breeding industry.

养殖业热应激危害严重，脑缺血是其重要的病理过程。本项目在前期发现葛根素抗氧糖剥夺诱导神经元凋亡活性的基础上，以大鼠神经元、脑微血管内皮细胞、星形胶质细胞共培养构建“脑神经血管单元”体外模型（NVU），以单独培养神经元为对照，氧糖剥夺诱导神经元凋亡的同时以葛根素干预，并以MAPK通路特异性抑制剂预处理细胞作对比，通过4h渗透率、跨内皮电阻值、WB法分析细胞特异性蛋白表达量、透射电镜检测胞间紧密连接进行NVU功能评估，流式细胞仪检测神经元凋亡率及胞内钙离子浓度，MTT法检测细胞活性，试剂盒法分析细胞内Glu含量及氧化应激指标，FQ-PCR分析HSP70、ERK1/2、JNK、p38 mRNA表达量，WB分析HSP70、ERK1/2、JNK、p38、p-ERK1/2、p-JNK、p-p38蛋白表达量，以阐释葛根素抗氧糖剥夺诱导NVU内神经元凋亡的分子机制，为其用于抗养殖业热应激展开理论探索。

本研究构建 “脑神经血管单元”(NVU)体外模型，氧糖剥夺（OGD）构建NVU损伤模型，同时以葛根素干预，评估葛根素的神经保护效果及可能机制。. 结果：. （1）NVU模型中三种细胞生长良好，胞间连接紧密，4h渗透率及跨内皮电阻值表明NVU具有显著的屏障功能。氧糖剥夺损伤可显著降低模型的屏障功能，并显著上调Caspase 3蛋白表达，提示损伤可引起神经细胞凋亡，而NVU共培养能显著降低损伤后GAP-43、Claudin-5、AQP-4的表达。. （2）葛根素可改善OGD损伤中的星型胶质细胞形态，显著提高OGD损伤星型胶质细胞的存活率（CCK-8法），降低星型胶质细胞凋亡率（流式细胞术），减少星型胶质细胞凋亡数量（荧光双标法）并抑制caspase-3蛋白的表达（WB法）。. （3）与正常对照组相比，缺氧6 h模型组mRNA表达量上调的基因为404个，下调的基因为454个。其中HIF-1、MAPK信号通路上调，PI3k/Akt信号通路下调。与缺氧6 h模型组相比，500 μg•mL-1梓葛冻干粉针组mRNA表达量上调的基因为31个，下调的基因为40个。. （4）与正常对照组相比，缺氧6 h/复氧12 h组mRNA表达量上调的基因为515个，下调的基因为634个。其中JAK/STAT信号通路上调，PI3k/Akt信号通路下调。与缺氧6 h/复氧12 h模型组相比，500 μg•mL-1梓葛冻干粉针组mRNA表达量上调的基因为36个，下调的基因为88个。其中PI3k/Akt信号通路上调，JAK/STAT信号通路下调，逆转了损伤模型的基因表达变异。. （5）PI3K、Akt、JAK2、STAT3 mRNA表达量qPCR检测结果与转录组结果相似。. 上述结果提示葛根素对神经细胞OGD损伤保护作用机制可能与其调控JAK/STAT及PI3K/Akt信号通路有关。