【摘要】 目的 探讨脑通合剂含药血清对脂多糖诱导的人小胶质细胞（HMC3）介导的神经元炎症反应的影响，及脑通合剂治疗血管性痴呆（VD）潜在的作用机制。方法 制备脑通合剂含药血清，利用CCK-8法筛选脂多糖的最佳诱导浓度及含药血清的最佳干预浓度。脂多糖刺激HMC3细胞，收集其上清液并与人神经母细胞瘤细胞（SH-SY5Y）共培养，建立血管性痴呆体外炎症模型。将HMC3和SH-SY5Y细胞随机分为6组：正常组、模型组、空白血清组、含药血清组、空白血清+抑制剂TAK-242组及含药血清+抑制剂TAK-242组。各组HMC3细胞经相应处理后，将HMC3细胞上清液加入对应组别的SH-SY5Y细胞中。ELISA法检测脑通合剂含药血清对脂多糖诱导的HMC3细胞介导的SH-SY5Y细胞上清液中IL-1β、IL-6和TNF-α的表达；Western Blot法检测SH-SY5Y细胞内Toll样受体4（TLR4）、丝裂原活化蛋白激酶p38（p38MAPK）和核因子κB（NF-κB）的蛋白表达水平；CCK-8法检测含药血清对脂多糖诱导的HMC3细胞介导的SH-SY5Y细胞活力的影响。结果 脂多糖诱导HMC3细胞的最佳浓度为1.0μg·mL^-1（P<0.01）。空白血清和含药血清对HMC3细胞的最佳干预浓度均为10%（P<0.05,P<0.01）。ELISA结果显示，与SH-SY5Y细胞正常组比较，模型组IL-1β、IL-6、TNF-α含量增加（P<0.01）；与模型组比较，空白血清组与含药血清组炎症因子IL-1β、IL-6、TNF-α含量降低（P<0.01），且含药血清组IL-1β、IL-6、TNF-α含量显著低于空白血清组（P<0.01）。同时，空白血清+TAK-242组和含药血清+TAK-242组的IL-1β、IL-6、TNF-α含量分别低于空白血清组和含药血清组（P<0.01）。Western Blot结果表明，与正常组比较，模型组TLR4表达升高（P<0.01）,p-p38MAPK/p38MAPK和p-p65NF-κB/p65NF-κB比值升高（P<0.01）；与模型组比较，空白血清组和含药血清组TLR4表达降低、p-p38MAPK/p38MAPK和p-p65NF-κB/p65NF-κB比值降低（P<0.01），且含药血清组表达低于空白血清组（P<0.01），同时空白血清组和含药血清组的p-p65NF-κB/p65NF-κB分别高于空白血清+TAK-242组和含药血清+TAK-242组（P<0.01）。CCK-8结果表明，脂多糖可以显著降低HMC3细胞介导的SH-SY5Y细胞存活率（P<0.01），而含药血清能够提高其细胞存活率（P<0.01），且加入TAK-242可进一步增强细胞存活率（P<0.01）。结论 脑通合剂含药血清可抑制TLR4/p38MAPK/NF-κB信号通路，减轻小胶质细胞介导的神经元炎症反应，从而发挥对血管性痴呆的神经保护作用。更多还原

【Abstract】 Objective To investigate the effects of Naotong Mixture（NTM）-medicated serum on lipopolysaccharide（LPS）-induced inflammatory responses in human microglia clone 3（HMC3）-mediated neuronal cells and explore its potential mechanism in treating vascular dementia（VD）. Methods NTM-medicated serum was prepared, and the optimal concentrations of LPS induction and serum intervention were determined using CCK-8 assay. An in vitro VD inflammatory model was established by stimulating HMC3 cells with LPS and co-culturing their supernatants with human neuroblastoma cells（SH-SY5Y）. Cells were divided into six groups:control,model,blank serum,NTM-medicated serum,blank serum + TAK-242,and NTM-medicated serum + TAK-242. After treatment,HMC3 supernatants were transferred to corresponding SH-SY5Y cultures. ELISA measured IL-1β, IL-6, and TNF-α levels in SH-SY5Y supernatants;Western Blot analyzed the protein expression of TLR4,p38 MAPK,and p65 NF-κB;CCK-8 assessed SH-SY5Y viability. Results The optimal concentration of LPS for inducing HMC3 cells was 1.0 μg·mL^-1（P<0.01）. The optimal intervention concentrations of blank serum and medicated serum for HMC3 cells were both 10%（P<0.05,P<0.01）. ELISA results showed that compared with the normal group of SH-SY5Y cells, the levels of IL-1β,IL-6,and TNF-α increased in the model group（P<0.01）; compared with the model group, the levels of inflammatory factors IL-1β,IL-6,and TNF-α decreased in the blank serum group and medicated serum group（P<0.01）,and the levels of IL-1β,IL-6,and TNF-α in the medicated serum group were significantly lower than those in the blank serum group（P<0.01）. Meanwhile,the levels of IL-1β,IL-6,and TNF-α in the blank serum + TAK-242 group and medicated serum + TAK-242 group were lower than those in the blank serum group and medicated serum group,respectively（P<0.01）. Western Blot results showed that compared with the normal group, the expression of TLR4 increased in the model group（P<0.01）, and the ratios of p-p38 MAPK/p38 MAPK and p-p65 NF-κB/p65 NF-κB increased（P<0.01）; compared with the model group, the expression of TLR4 decreased and the ratios of pp38 MAPK/p38 MAPK and p-p65 NF-κB/p65 NF-κB decreased in the blank serum group and medicated serum group（P<0.01）,and the expression in the medicated serum group was lower than that in the blank serum group（P<0.01）. Meanwhile,p-p65 NF-κB/p65 NF-κB in the blank serum group and mediciated serum group were higher than that in the blank serum + TAK-242 group and medicated serum + TAK-242 group,respectively（P<0.01）. CCK-8 results showed that LPS significantly reduced the survival rate of SH-SY5Y cells mediated by HMC3 cells（P<0.01）,while medicated serum could improve the cell survival rate（P<0.01）,and adding TAK-242 could further enhance the cell survival rate（P<0.01）. Conclusion NTM-medicated serum attenuates microglia-mediated neuroinflammation by inhibiting the TLR4/p38 MAPK/NF-κB pathway,suggesting neuroprotective potential for VD.更多还原