补阳还五汤类方对氧糖剥夺再复糖复氧海马神经元细胞的体外保护作用及其机制研究

Effects and Mechanism of Buyang Huanwu Decoction and the like on Oxygen Deprivation and Reoxygenation of Hippocampal Neurons in Vitro

目的探讨补阳还五汤类方(补阳还五汤、脑心通胶囊、养阴通脑颗粒)对乳鼠氧糖剥夺/复糖复氧(OGD/R)海马神经元细胞的体外保护作用及其机制。方法原代培养海马神经元细胞,建立海马神经元OGD/R模型,CCK-8法确定补阳还五汤类方含药血清于海马神经元细胞的作用浓度。实验设对照组、模型组、补阳还五汤含药血清组(10%)、脑心通胶囊含药血清组(10%)、养阴通脑颗粒含药血清组(10%)、补阳还五汤含药血清+TAK-242组(10%,1μmol·L^-1)、脑心通胶囊含药血清+TAK-242组(10%,1μmol·L^-1)、养阴通脑颗粒含药血清+TAK-242组(10%,1μmol·L^-1)共8组。倒置相差显微镜下观察海马神经元生长形态,试剂盒检测肿瘤坏死因子α(TNF-α)和白介素1β(IL-1β)的含量,Hoechst 33358染色荧光显微镜观察神经元细胞的凋亡情况,蛋白免疫印迹(Western blot)检测TLR4/NF-κB信号通路上关键蛋白的表达情况。结果补阳还五汤类方均可以降低海马神经元细胞TNF-α和IL-1β的释放量,显著降低细胞凋亡数,抑制TLR4/NF-κB信号通路中TLR4和NF-κB蛋白的表达,对OGD/R海马神经元细胞的炎症反应起到了保护作用。TLR4特异性抑制剂TAK-242的加入,阻断了该信号通路的传导,降低了补阳还五汤类方的保护作用。结论补阳还五汤类方对OGD/R致海马神经元细胞发生的炎症反应具有保护作用,其作用机制可能与TLR4/NF-κB信号通路有关,且脑心通胶囊的抗炎保护作用相对明显。

OBJECTIVE To investigate the effects and mechanism of Buyang Huanwu Decoction and the like on primary cultured neonate hippocampal neurons induced by oxygen deprivation and reoxygenation.METHODS The primary cultured hippocampal neurons cell model was established,oxygen sugar deprivation/reoxygenation(OGD/R)model was established in vitro.CCK-8 was used to determine the concentration of Buyang Huanwu Decoction and the like drug-containing serum to protect hippocampal neurons cell.The experimental groups were divided into control group,model group,Buyang Huanwu Decoction drug-containing serum group(10%),Naoxintong Capsule drug-containing serum group(10%),Yangyin Tongnao Granules drug-containing serum group(10%),Buyang Huanwu Decoction drug-containing serum+TAK-242 group(10%,1μmol·L^-1),Naoxintong Capsule drug-containing serum+TAK-242 group(10%,1μmol·L^-1),Yangyin Tongnao Granules drug-containing serum+TAK-242 group(10%,1μmol·L^-1).The morphology of hippocampal neuron was observed under inverted microscope.The expression of tumor necrosis factor-α(TNF-α)and interleukin 1(IL-1β)in the cell supernatant was detected by ELISA kit.The neuronal cell apoptosis was observed by Hoechst 33358 staining fluorescence microscope and the expression of TLR4/NF-κB signaling pathway proteins was detected by Western blot.RESULTS The results showed that hgher purity neuronal cells can be obtained by primary culture.Compared with model group,Buyang Huanwu Decoction and the like all can reduce the release of TNF-αand IL-1βin hippocampal neurons,significantly reduced the number of apoptosis.At the same time,the three traditional Chinese medicine compounds all can inhibit the expression of TLR4 and NF-κB protein in TLR4/NF-κB signaling pathway,and protect the inflammatory response of oxygenated sugar deprivation and reoxygenated hippocampal neurons.The addition of TLR4-specific inhibitor TAK-242 blocked the conduction of this signaling pathway and reduced the protective effect of Buyang Huanwu Decoction and the like.CONCLUSION Buyang Huanwu Decoction and the like have protective effects on OGD/R-induced inflammatory response in hippocampal neurons,its mechanism may be related to TLR4/NF-κB signaling pathway,and the anti-inflammatory protective effect of Naoxintong Capsule is relatively obvious.