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。
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,p38MAPK,and p65 NF-κB;CCK-8 assessed SH-SY5Y viability.Results The optimal concentration of LPS for inducing HMC3 cells was 1.0μg·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-p38MAPK/p38MAPK and p-p65NF-κB/p65NF-κB increased(P<0.01);compared with the model group,the expression of TLR4 decreased and the ratios of pp38MAPK/p38MAPK and p-p65NF-κB/p65NF-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-p65NF-κB/p65NF-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/p38MAPK/NF-κB pathway,suggesting neuroprotective potential for VD.
作者
王德秀
李婧涵
巩玉瑾
姚慧莹
李欣宇
刘若凡
王艺翔
温振川
陈泽涛
WANG Dexiu;LI Jinghan;GONG Yujin;YAO Huiying;LI Xinyu;LIU Ruofan;WANG Yixiang;WEN Zhenchuan;CHEN Zetao(School of Basic Medical Sciences,Shandong Second Medical University,Weifang 261053 Shandong,China;School of Clinical Medicine,Shandong Second Medical University,Weifang 261053 Shandong,China;Department of Geriatric Medicine,Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250011 Shandong,China)
出处
《中药新药与临床药理》
北大核心
2025年第10期1710-1719,共10页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
国家大学生创新创业计划项目(202410438014,202410438002)
山东省自然科学基金青年项目(ZR2022QF123)
山东省医药卫生科技项目(保健项目)(2023BJ000051)
山东第二医科大学校级大学生创新创业计划项目(X2024008)。
