广西医学

目的分析益肺宣肺降浊方对星形胶质细胞-神经元氧糖剥夺（OGD）损伤的缓解作用，并基于胆碱乙酰转移酶(ChAT)、α7烟碱型乙酰胆碱受体（α7-nAChR）及炎症因子探讨其可能的作用机制。方法（1）制备对照血清及益肺宣肺降浊方含药血清。（2）分离培养新生大鼠海马星形胶质细胞和神经元。将细胞分为对照组、OGD组、OGD+YFXF组、OGD+YFXF+山莨菪碱组。在对照组中，常规培养原代海马神经元和星形胶质细胞；在OGD组、OGD+YFXF组、OGD+YFXF+山莨菪碱组中，建立星形胶质细胞-神经元OGD模型，并分别使用含5%对照血清的高糖DMEM、含5%益肺宣肺降浊方含药血清的高糖DMEM、含5%益肺宣肺降浊方含药血清及100 μg/mL山莨菪碱的高糖DMEM进行培养。（3）干预24 h后，观察各组细胞形态，检测星形胶质细胞和神经元的标志物表达情况，星形胶质细胞活性氧簇含量、ChAT活性、α7-nAChR和ChAT的mRNA和蛋白表达量，以及上清液乳酸脱氢酶（LDH）、白细胞介素（IL）-1β、肿瘤坏死因子α(TNF-α)和IL-6水平。结果与对照组相比，OGD组星形胶质细胞及神经元标志物荧光强度减弱，神经元形态异常，星形胶质细胞的活性氧簇含量及上清液LDH、IL-1β、TNFα、IL-6水平增加，星形胶质细胞的ChAT活性降低且α7-nAChR、ChAT的mRNA和蛋白表达量下调（P＜0.05）。与OGD组相比，OGD+YFXF组星形胶质细胞及神经元标志物荧光强度增强，神经元形态有所改善，星形胶质细胞的活性氧簇含量及上清液LDH、IL-1β、TNF-α、IL-6水平降低，星形胶质细胞的ChAT活性增强且α7-nAChR、ChAT的mRNA和蛋白表达量上调（P＜0.05）。与OGD+YFXF组相比，OGD+YFXF+山莨菪碱组的星形胶质细胞和神经元的标志物荧光强度减弱，神经元形态异常，星形胶质细胞的活性氧簇含量及上清液LDH、TNF-α水平增加，星形胶质细胞的α7-nAChR mRNA和蛋白表达量降低（P＜0.05）。结论益肺宣肺降浊方可能通过上调海马星形胶质细胞中α7-nAChR的表达、增强ChAT活性、抑制炎症反应，来缓解星形胶质细胞-神经元OGD损伤，这或许是该方改善缺血再灌注损伤介导的血管性痴呆的作用机制。

ObjectiveTo analyze the effect of Yifei Xuanfei Jiangzhuo Prescription for alleviating astrocytes-neuronal oxygen glucose deprivation (OGD) damage, and to explore its possible mechanism based on choline acetyltransferase (ChAT), α7-nicotinic acetylcholine receptor (α7-nAChR), and inflammatory factors. Methods(1) Control serum and drug-contained serum of Yifei Xuanfei Jiangzhuo Prescription were prepared. (2) Hippocampal astrocytes and neurons were isolated and cultured from neonatal rats. Cells were assigned to control group, OGD group, OGD+YFXF group, or OGD+YFXF+anisodamine group. In the control group, primary hippocampal neurons and astrocytes were routinely cultured; furthermore, in the OGD, OGD+YFXF, and OGD+YFXF+anisodamine groups, astrocytes-neuronal OGD model was established, and high glucose DMEM of 5% control serum, high glucose DMEM of 5% Yifei Xuanfei Jiangzhuo Prescription with drug-contained serum, and high glucose DMEM of 5% Yifei Xuanfei Jiangzhuo Prescription with drug-contained serum and 100 μg/mL anisodamine were used to culture, respectively. (3) After 24 hours of intervention, cell morphology was observed in various groups. The expressions of astrocytes and neurons markers, and mRNA and protein expressions of content of reactive oxygen species, ChAT activity, α7-nAChR, and ChAT in astrocytes, as well as supernate lactate dehydrogenase (LDH), interleukin (IL)-1β, tumor necrosis factor α (TNF-α), and IL-6 levels were detected.ResultsCompared with the control group, the OGD group yielded weakened fluorescence intensity of astrocytes and neurons markers, and presented as abnormal neuronal morphology, increased content of reactive oxygen species of astrocytes, elevated supernate LDH, IL-1β, TNF-α, and IL-6 levels, decreased ChAT activity of astrocytes, and down-regulated mRNA and protein expressions of α7-nAChR and ChAT of astrocytes (P＜0.05). Compared with the OGD group, the OGD+YFXF group exhibited enhanced fluorescence intensity of astrocytes and neurons markers, and presented as improved neuronal morphology, decreased content of reactive oxygen species of astrocytes and supernate LDH, IL-1β, TNF-α, and IL-6 levels, enhanced ChAT activity of astrocytes, and up-regulated mRNA and protein expressions of α7-nAChR and ChAT of astrocytes (P＜0.05). Compared with the OGD+YFXF group, the OGD+YFXF+anisodamine group interpreted weakened fluorescence intensity of astrocytes and neurons markers, and presented as abnormal neuronal morphology, increased content of reactive oxygen species of astrocytes and supernate LDH and TNF-α levels, and down-regulated mRNA and protein expressions of α7-nAChR of astrocytes (P＜0.05).ConclusionYifei Xuanfei Jiangzhuo Prescription may alleviate astrocytes-neuronal OGD damage through up-regulating α7-nAChR expression, enhancing ChAT activity, inhibiting inflammatory responses in hippocampal astrocytes, and it may be the mechanism of this prescription for ameliorating vascular dementia mediated by ischemia reperfusion injury.