Objective To explore the mechanism of Diterpene Ginkgolides Meglumine Injection (银杏二萜内酯葡胺注射液, DGMI) in treatment of acute ischemic stroke (IS) based on platelet generation and activation. Methods Sham group, model group, ibrutinib (10 mg/kg) group, DGMI low-, medium- and high-dose (6.25, 12.50, 25.00 mg/kg) groups were established. A transient middle cerebral artery occlusion (tMCAO) model was prepared in C57BL/6 mice using suture method. Medication was administered continuously for 3 d starting 0.5 h after surgery. The infarct rate was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Motor and neurological function were evaluated using spinning rod experiment and Bederson score. Flow cytometry and ELISA were used to detect the whole blood platelet count and platelet factor 4 (PF4) level in serum. Bone marrow smears and immunohistochemistry were used to observe the proliferation, differentiation, apoptosis and phosphorylation levels of focal adhesion kinase (FAK) in bone marrow megakaryocytes. Immunofluorescence staining was used to detect platelet activation and FAK phosphorylation levels in brain tissue. A combination group of FAK agonist ZINC40099027 (ZIN) and medium-dose DGMI was set up to verify whether DGMI exerts a protective effect by inhibiting FAK activation. Results DGMI significantly reduced cerebral infarction and megakaryocyte generation in tMCAO mice. Among them, the inhibitory effect of medium-dose DGMI on differentiation and proliferation of bone marrow megakaryocytes in tMCAO mice, as well as the improvement of cerebral infarction area, motor coordination ability and neurological function score were the most significant (P < 0.05, 0.01, 0.001). In addition, DGMI could significantly reduce the number of platelets and PF4 level in serum of tMCAO mice (P < 0.05, 0.001), and inhibit the phosphorylation of FAK in bone marrow megakaryocytes and infiltrating platelets in brain (P < 0.01, 0.001). The FAK agonist ZIN significantly reversed the protective effects of DGMI on tMCAO mice (P < 0.05, 0.01, 0.001). Conclusion DGMI exerts neuroprotective effects by inhibiting the excessive generation of megakaryocytes during the acute phase of IS and the phosphorylation of FAK in infiltrating platelets in the brain, thereby suppressing platelet generation and activation.

Diterpene Ginkgolides Meglumine Injection  /  ischemic stroke  /  platelet  /  megakaryocyte  /  focal adhesion kinase