银杏二萜内酯葡胺注射液通过调控炎症和凋亡信号通路减轻OGD/R诱导的细胞损伤

doi:10.5246/jcps.2020.07.043

摘要/Abstract

摘要：

银杏二萜内酯葡胺注射液(DGMI)是一种银杏叶提取物注射液, 目前用于缺血性中风恢复期的治疗。在本研究中, 我们旨在证实DGMI是否可以抑制炎症反应和细胞凋亡, 并探讨这些作用的潜在机制。将细胞暴露于氧-糖剥夺/复氧(OGD/R)后, 通过MTS和LDH测定法测量细胞活力和乳酸脱氢酶(LDH)释放量。使用Annexin V-FITC/PI双重染色分析试剂盒通过流式细胞仪检测了DGMI的抗凋亡作用。通过特定的Bio-Plex Pro^TM剂盒对包括TNF-α、IL-1β、IL-6和IL-10在内的促炎性细胞因子进行定量检测。此外, 分别通过蛋白质印迹分析和免疫荧光染色测定TLR2/4, NF-κB p65, MAPK通路激活和凋亡相关蛋白的活性以及NF-κB p65的细胞定位。结果显示, 50 μg/mL的DGMI在OGD/R诱导的BV2小胶质细胞中显著提高了细胞活力并降低了IL-1β, IL-6, IL-10和TNF-α的分泌。通过LDH测定和Annexin V-FITC/PI染色也证实了这些作用。同时, DGMI不仅抑制TLR2, TLR4, MyD88, p-TAK1, p-IkBk, p-IKKK和Bak的蛋白表达, 且降低了OGD/R诱导的BV2小胶质细胞中C-caspase-3/caspase-3, Bax/Bcl-2和C-PARP-1/PARP-1的比率。此外, DGMI还部分抑制了OGD/R增强的p-JNK1/2和p-p38 MAPK表达以及NF-κB p65的核移位。本研究表明, OGD/R激活的BV2小胶质细胞中触发了炎症反应, 导致促炎细胞因子的释放和细胞凋亡。DGMI通过调节TLR/MyD88/NF-κB信号通路以及下调p-JNK1/2和p-p38 MAPK激活来抑制炎症反应和细胞凋亡。

关键词: 银杏内酯, BV2神经小胶质细胞, TLR, 脑缺血, NF-κB, MAPK, 炎症

Abstract:

Diterpene ginkgolides meglumine injection (DGMI), a kind of Ginkgo biloba special extract injection, is now used for the treatment of ischemic stroke in convalescence. In the present study, we aimed to conﬁrm whether DGMI could suppress inflammatoryresponses and apoptosis and explore the potential mechanisms underlying these effects. Cell viability and lactate dehydrogenase (LDH) release were measured by MTS and LDH assays after the cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R). The extent of anti-apoptotic effect of DGMI was detected by flow cytometry using Annexin V-FITC/PI double staining assay kit. Pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IL-10, were quantified by a specific Bio-Plex Pro^TM Reagent Kit. Additionally, activities of TLR2/4, NF-κB p65, MAPK pathway and apoptosis-related proteins as well as cellular localization of NF-κB p65 were determined by Western blotting analysis and immunofluorescence staining, respectively. DGMI at 50 μg/mL significantly increased the cell viability and decreased the secretion of IL-1β, IL-6, IL-10 and TNF-α in OGD/R-induced BV2 microglia cells. These effects were also confirmed by LDH assay and Annexin V-FITC/PI staining. Meanwhile, DGMI not only inhibited the protein expressions of TLR2, TLR4, MyD88, p-TAK1, p-IkBα, p-IKKβ and Bak, but also decreased the cleaved caspase-3/caspase-3, Bax/Bcl-2 and cleaved PARP-1/PARP-1 ratio in OGD/R-induced BV2 microglia cells. Furthermore, OGD/R-enhanced p-JNK1/2 and p-p38 MAPK expressions and nuclear translocation of NF-κB p65 were also partially inhibited by DGMI. The present study showed that inflammatory responses were triggered in BV2 microglia cells activated by OGD/R, leading tothe release of pro-inflammatory cytokines and apoptosis. DGMI suppressed the inﬂammatory response and apoptosis by regulating the TLR/MyD88/NF-κB signaling pathways and down-regulation of p-JNK1/2 and p-p38 MAPK activation.

Key words: Ginkgolides, BV2 microglia cells, TLR, Cerebral ischemia, NF-κB, MAPK, Inflammation

中图分类号:

R966

Supporting:

洪倩, 王增慧, 杨阳, 高璐, 闫兆. 银杏二萜内酯葡胺注射液通过调控炎症和凋亡信号通路减轻OGD/R诱导的细胞损伤[J]. 中国药学(英文版), 2020, 29(7): 455-469.

Qian Hong, Zenghui Wang, Yang Yang, Lu Gao, Zhao Yan. DGMI alleviates OGD/R-induced cell injury by regulating inflammatory and apoptosis signaling pathways[J]. Journal of Chinese Pharmaceutical Sciences, 2020, 29(7): 455-469.

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