Xiang Li1, Liangliang Huang2, Ge Liu3, Wenxiang Fan4, Binbin Li5, Rui Liu6, Ziyu Wang7, Qiru Fan8, Wei Xiao9, Yunman Li10, Weirong Fang11. 1. State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: 13022519260@163.com. 2. State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: 541873948@qq.com. 3. State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: 2193146353@qq.com. 4. Department of Pharmacy, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, 210009, China. Electronic address: 349164503@qq.com. 5. State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: 1149183859@qq.com. 6. State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: liurui09cpu@163.com. 7. State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: cpuwangziyu@qq.com. 8. Faculty of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: fanqiru@gmail.com. 9. State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, 222001, China. Electronic address: xw_kanion@163.com. 10. State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: yunmanlicpu@hotmail.com. 11. State Key Laboratory of Natural Medicines, School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: weirongfang@cpu.edu.cn.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. (Ginkgoaceae) is a traditional Chinese medicine known to treating stroke and other cardio-cerebrovascular diseases for thousands of years in China. Ginkgo diterpene lactones (GDL) attracted much attention because of their neuroprotective properties. AIM OF THE STUDY: To uncover the effects of GDL, which consist of ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), on ischemic stroke, as well as the underlying molecular mechanisms. MATERIALS AND METHODS: We used middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) models mimicking the process of ischemia/reperfusion in vivo and in vitro, respectively. Anticoagulant effects of GDL were investigated on platelet activating factor (PAF), arachidonic acid (AA) and adenosine diphosphate (ADP)-induced platelet aggregation both in vivo and in vitro. We also evaluated the effects of GDL on lipopolysaccharide (LPS)-induced inflammatory response in primary cultured rats' astrocytes. Infarct size, neurological deficit score, and brain edema were measured at 72 h after MCAO. Immunohistochemistry was utilized to analyze neurons necrosis and astrocytes activation. Expression of pro-inflammatory cytokines, including tumor necrotic factor-α (TNF-α) and interleukin-1β (IL-1β) were detected using enzyme-linked immunosorbent assay (ELISA) and real time PCR. The levels of toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) were assessed by real time PCR or Western blot. RESULTS: Compared with MCAO/R rats, GDL significantly reduced infarct size and brain edema, improved neurological deficit score. Meanwhile, GDL suppressed platelet aggregation, astrocytes activation, pro-inflammatory cytokines releasing, TLR4 mRNA expression and transfer of NF-κB from cytoplasm to nucleus. Furthermore, GDL alleviated OGD/R injury and LPS-induced inflammatory response in primary astrocytes, characterized by promoting cell viability, decreasing lactate dehydrogenase (LDH) activity, and inhibiting IL-1β and TNF-α releasing. CONCLUSIONS: In summary, GDL attenuate cerebral ischemic injury, inhibit platelet aggregation and astrocytes activation. The anti-inflammatory activity might be associated with the downregulation of TLR4/NF-κB signal pathway. Our present findings provide an innovative insight into the novel treatment of GDL in ischemic stroke therapy.
ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. (Ginkgoaceae) is a traditional Chinese medicine known to treating stroke and other cardio-cerebrovascular diseases for thousands of years in China. Ginkgoditerpene lactones (GDL) attracted much attention because of their neuroprotective properties. AIM OF THE STUDY: To uncover the effects of GDL, which consist of ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), on ischemic stroke, as well as the underlying molecular mechanisms. MATERIALS AND METHODS: We used middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) models mimicking the process of ischemia/reperfusion in vivo and in vitro, respectively. Anticoagulant effects of GDL were investigated on platelet activating factor (PAF), arachidonic acid (AA) and adenosine diphosphate (ADP)-induced platelet aggregation both in vivo and in vitro. We also evaluated the effects of GDL on lipopolysaccharide (LPS)-induced inflammatory response in primary cultured rats' astrocytes. Infarct size, neurological deficit score, and brain edema were measured at 72 h after MCAO. Immunohistochemistry was utilized to analyze neurons necrosis and astrocytes activation. Expression of pro-inflammatory cytokines, including tumor necrotic factor-α (TNF-α) and interleukin-1β (IL-1β) were detected using enzyme-linked immunosorbent assay (ELISA) and real time PCR. The levels of toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) were assessed by real time PCR or Western blot. RESULTS: Compared with MCAO/R rats, GDL significantly reduced infarct size and brain edema, improved neurological deficit score. Meanwhile, GDL suppressed platelet aggregation, astrocytes activation, pro-inflammatory cytokines releasing, TLR4 mRNA expression and transfer of NF-κB from cytoplasm to nucleus. Furthermore, GDL alleviated OGD/R injury and LPS-induced inflammatory response in primary astrocytes, characterized by promoting cell viability, decreasing lactate dehydrogenase (LDH) activity, and inhibiting IL-1β and TNF-α releasing. CONCLUSIONS: In summary, GDL attenuate cerebral ischemic injury, inhibit platelet aggregation and astrocytes activation. The anti-inflammatory activity might be associated with the downregulation of TLR4/NF-κB signal pathway. Our present findings provide an innovative insight into the novel treatment of GDL in ischemic stroke therapy.