Ginkgolide B

Bay k-8644, an activator of L-type voltage-gated calcium channels, induces self-injurious behaviors in mice. Although previous studies using animal models have suggested the possible implications of neuroinflammation in self-injurious behaviors, this has not yet been elucidated in the context of Bay k-8644-induced self-injurious behaviors. In this study, Bay k-8644 (50 µg, i.c.v.)-induced self-injurious behaviors were accompanied by increased expression of endothelin (ET)-1, platelet-activating factor (PAF) receptors, and Iba-1 in the striatum. Pretreatment with the ET receptor antagonist bosentan (10 mg/kg, i.p.), the PAF receptor antagonist ginkgolide B (10 mg/kg, i.p.), or the microglial activation inhibitor minocycline (40 mg/kg/day for 5 days, i.p.) significantly inhibited Bay k-8644-induced self-injurious behaviors and microglial activation in the striatum. Interestingly, bosentan also suppressed Bay k-8644-induced PAF receptor expression, indicating that ET-1 may act as an upstream modulator of the PAF signaling under these experimental conditions. Bay k-8644-induced ET-1 expression and consequent pro-inflammatory changes were reversed by the protein kinase C (PKC) inhibitor NPC-15,437 and the Ca²⁺/calmodulin-dependent kinase II (CaMKII) inhibitor KN-93. Moreover, Bay k-8644-induced self-injurious behaviors and microglial activation were significantly potentiated by exogenous ET-1 administration (10 pmol, i.c.v.) or by weak neuroinflammation in the striatum induced by systemic injection of low-dose lipopolysaccharide (LPS; 1 mg/kg, i.p.). Our results suggest that neuroinflammatory changes associated with ET-1/PAF signaling in the striatum contribute to Bay k-8644-induced self-injurious behaviors.

Excessive osteoclast activity could cause skeletal diseases including osteoporosis. Additionally, autophagy plays an initial role in osteoclast differentiation and function. Ginkgolide B (GB), a key compound in Ginkgo biloba, improves bone mass and suppresses mature osteoclast formation in vitro. This study examines the role of GB role in regulating osteoclast formation via autophagy. Using murine bone marrow-derived macrophages in vitro, we explored GB's effects on autophagy and osteoclast formation. We also assessed bone loss prevention in an ovariectomized (OVX) mouse model using GB combined with an autophagy inhibitor. Tartrate-resistant acid phosphatase staining was used to observe osteoclast formation. Autophagy-related proteins and intracellular microtubule associated protein 1 light chain 3 beta puncta were observed using western blotting and immunofluorescence. The impact of GB on OVX mice was evaluated using micro-computed tomography and hematoxylin and eosin staining. GB directly promoted autophagy in osteoclast precursors (OCPs), but inhibited osteoclast differentiation by reducing receptor activator of nuclear factor kappa B ligand (RANKL)-induced autophagy. GB inhibits the phosphorylation of RANKL downstream signaling pathways, such as Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). Anisomycin (ANI), a JNK activator, reversed GB's inhibitory effects on RANKL-induced autophagy and osteoclastogenesis. Inhibiting autophagy using 3-Methyladenine (3-MA) or small interfering RNA significantly suppressed osteoclast differentiation both in vitro and in vivo. Our findings suggested that GB inhibits osteoclast formation by decreasing JNK phosphorylation and autophagy under RANKL stimulation. Interestingly, GB also directly promotes autophagy in OCPs. Thus, GB markedly reduces osteoclast differentiation and prevents bone loss, with its anti-osteoclastogenesis effect being enhanced by 3-MA. Accordingly, inhibiting GB-induced direct autophagy could further increase its therapeutic effect on bone disease resulting from excessive osteoclast activity.