title: Ginkgolides and bilobalide protect BV2 microglia cells against OGD/reoxygenation injury by inhibiting TLR2/4 signaling pathways.
abstract: Ginkgolide and bilobalide are major trilactone constituent of Ginkgo biloba leaves and have been shown to exert powerful neuroprotective properties. The aims of this study were to observe the inhibitory effects of ginkgolide and bilobalide on the activation of microglial cells induced by oxygen-glucose deprivation and reoxygenation (OGD/R) and the specific mechanisms by which these effects are mediated. For detecting whether ginkgolide and bilobalide increased cell viability in a dose-dependent manner, BV2 cells were subjected to oxygen-glucose deprivation for 4 h followed by 3 h reoxygenation with various concentrations of drugs (6.25, 12.5, 25, 50, and 100 μg/ml). The extent of apoptosis effect of OGD/R with or without ginkgolide and bilobalide treatment were also measured by Annexin V-FITC/PI staining. Similarly, the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and IL-10 were detected using a specific Bio-Plex Pro™ Reagent Kit. The effects of ginkgolide and bilobalide on protein levels of TLR2/4, MyD88, p-TAK1, p-IKKβ, p-IkBα, NF-κB p65, Bcl-2, Bax, Bak, RIP3, cleaved-Caspase-3, cleaved PARP-1 and cellular localization of NF-κB p65 were evaluated by Western blot and double-labeled immunofluorescence staining, respectively. OGD/R significantly decreased the cell viability and increased the release of IL-1β, IL-6, IL-8, IL-10, TNF-α in BV2 microglia cells; these effects were suppressed by ginkgolide and bilobalide. Meanwhile, ginkgolide and bilobalide also attenuated the OGD/R-induced increases in TLR2, TLR4, MyD88, Bak, RIP3 levels and reversed cleaved caspase-3/caspase-3, Bax/Bcl-2 and cleaved PARP-1/PARP-1 ratio. Furthermore, ginkgolide and bilobalide also downregulated p-TAK1, p-IkBα, and p-IKKβ and inhibited the OGD/R-induced transfer of NF-κB p65 from cytoplasm to nucleus in BV2 microglia cells. The results showed that ginkgolide and bilobalide can inhibit OGD/R-induced production of inflammatory factors in BV2 microglia cells by regulating the TLRs/MyD88/NF-κB signaling pathways and attenuating inflammatory response. The possible mechanism of anti-inflammatory and neuroprotective effects of ginkgolides results from the synergistic reaction among each monomer constituents.
authors: State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Modern TCM Research Institute, Lianyungang, 222001, China. jianmin8920@163.com.
authors: Jian-Ming
authors: JM
authors: Zhou
authors: State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Modern TCM Research Institute, Lianyungang, 222001, China.
authors: Sha-Sha
authors: SS
authors: Gu
authors: State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Modern TCM Research Institute, Lianyungang, 222001, China.
authors: Wang Hong
authors: WH
authors: Mei
authors: State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Modern TCM Research Institute, Lianyungang, 222001, China.
authors: Jun
authors: J
authors: Zhou
authors: State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Modern TCM Research Institute, Lianyungang, 222001, China.
authors: Zhen Zhong
authors: ZZ
authors: Wang
authors: State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Modern TCM Research Institute, Lianyungang, 222001, China.
authors: Wei
authors: W
authors: Xiao
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015687:Cell Hypoxia; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology*; D016207:Cytokines / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005663:Furans / Q000494:pharmacology*; D046934:Ginkgolides / Q000494:pharmacology*; D005947:Glucose / Q000494:pharmacology; D051379:Mice; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism
chemical_list: D003517:Cyclopentanes; D016207:Cytokines; D005663:Furans; D046934:Ginkgolides; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D018696:Neuroprotective Agents; C498144:Rela protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA; D005947:Glucose; C073710:bilobalide
keywords: BV2 microglia cells; Bilobalide; Cerebral ischemia; Ginkgolides; Inflammatory; NF-κB; TLRs