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

Abstract

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. We combined neostigmine with anisodamine and investigated the neuroprotection and mechanism. 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. 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. 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.