The Synergistic Effect of Ginkgo biloba Extract 50 and Aspirin Against Platelet Aggregation.

PURPOSE: We aimed to investigate potential synergistic antiplatelet effects of Ginkgo biloba extract (GBE50) in combination with aspirin using in vitro models. METHODS: Arachidonic acid (AA), platelet activating factor (PAF), adenosine 5'-diphosphate (ADP) and collagen were used as inducers. The antiplatelet effects of GBE50, aspirin and 1:1 combination of GBE50 and aspirin were detected by microplate method using rabbit platelets. Synergy finder 2.0 was used to analyze the synergistic antiplatelet effect. The compounds in GBE50 were identified by UPLC-Q/TOF-MS analysis and the candidate compounds were screened by TCMSP database. The targets of candidate compounds and aspirin were obtained in TCMSP, CCGs, Swiss target prediction database and drugbank. Targets involving platelet aggregation were obtained from GenCLiP database. Compound-target network was constructed and GO and KEGG enrichment analyses were performed to identify the critical biological processes and signaling pathways. The levels of thromboxane B2 (TXB2), cyclic adenosine monophosphate (cAMP) and PAF receptor (PAFR) were detected by ELISA to determine the effects of GBE50, aspirin and their combination on these pathways. RESULTS: GBE50 combined with aspirin inhibited platelet aggregation more effectively. The combination displayed synergistic antiplatelet effects in AA-induced platelet aggregation, and additive antiplatelet effects occurred in PAF, ADP and collagen induced platelet aggregation. Seven compounds were identified as candidate compounds in GBE50. Enrichment analyses revealed that GBE50 could interfere with platelet aggregation via cAMP pathway, AA metabolism and calcium signaling pathway, and aspirin could regulate platelet aggregation through AA metabolism and platelet activation. ELISA experiments showed that GBE50 combined with aspirin could increase cAMP levels in resting platelets, and decreased the levels of TXB2 and PAFR. CONCLUSION: Our study indicated that GBE50 combined with aspirin could enhance the antiplatelet effects. They exerted both synergistic and additive effects in restraining platelet aggregation. The study highlighted the potential application of GBE50 as a supplementary therapy to treat thrombosis-related diseases.

A combination of neostigmine and anisodamine protects against ischemic stroke by activating α7nAChR.

BACKGROUND: Increasing endogenous acetylcholine by neostigmine decreased the ischemic cerebral injury. The off-target action on muscarinic receptor produced a variety of adverse effects and limited the clinical application on stroke. AIM: We combined neostigmine with anisodamine and investigated the neuroprotection and mechanism. METHODS: Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion. Neuroprotective action of neostigmine in combination with anisodamine at varying ratios was examined to determine the optimal combination as well as ideal therapeutic window. Potential involvement of α7 nicotinic acetylcholine receptor was examined by measuring the infarct size, the expression of proinflammatory cytokines, and the biomarkers of apoptosis in α7 nicotinic acetylcholine receptor knockout mice. A set of in vitro experiments was conducted in RAW264.7 cells to probe into potential molecular mechanisms. RESULTS: The neostigmine/anisodamine combination conferred neuroprotection. The protection was most potent at a ratio of 1:500. At such a ratio, the combination increased the binding of acetylcholine to α7 nicotinic acetylcholine receptor and reduced proinflammatory cytokines. The neuroprotection was evident only in wild-type and not in α7 nicotinic acetylcholine receptor knockout mice. The combination significantly decreased the expression of Bad and Bax, and increased Bcl-2 and Bcl-xl in α7 nicotinic acetylcholine receptor wild-type mice but not in knockout mice. The combination did not affect caspase-8, cleaved caspase-8, or caspase-12. CONCLUSIONS: Current study identified the optimal combination of neostigmine and anisodamine against ischemic stroke, and indicated that the acetylcholine-α7 nicotinic acetylcholine receptor is involved in the protective effects.