Ginkgo diterpene lactones inhibit cerebral ischemia/reperfusion induced inflammatory response in astrocytes via TLR4/NF-κB pathway in rats

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 72h after MCAO. Immunohistochemistry was utilized to analyze neurons necrosis and astrocytes activation. Expression of proinflammatory cytokines, including tumor necrotic factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) were detected using enzyme-linked immunosorbent assay (ELISA) and real time PCR. The levels of toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-kappa B) 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-kappa B 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-1 beta and TNF-alpha 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-kappa B signal pathway. Our present findings provide an innovative insight into the novel treatment of GDL in ischemic stroke therapy.