The Combination Of Neostigmine And Anisodamine Protects Against Ischemic Stroke And Related Mechanism

OBJECTIVEStroke is the second most common cause of death and a major cause of chronic disability in the adults. Stroke has high morbidity and high mortality rate. Ischemic stroke occupies the most part of stroke, which is about 70%. In China, with the development of aging and lifestyle changes, stroke causes severe mental and economic burden to patients, families and society. Approved treatments for acute ischemic stroke include thrombolysis and anti-platelet therapy. Intervention focusing on neuroprotection is promising, but has achieved only limited success upon translation into clinical practice. Therefore, prevention and therapy of ischemic stroke is a very important subject for human, more safe and effective anti-stroke drugs are needed to be discovered and researched.In a recent study, we noticed increasing endogenous ACh can significant attenuates the ischemic cerebral injury by the anticholinesterase inhibitor neostigmine. Preliminary evidence in this study also indicated that the neuroprotective action of neostigmine is mediated by nicotinic ACh receptor (nAChR) but not muscarinic ACh receptor (mAChR). The off-target action of neostigmine on mAChR, however, could produce a variety of adverse effects, including bradycardia, abdominal pain, hydrostomia, and so on. These side effects severely limited the clinical application on stroke. Anisodamine is a belladonna alkaloid that antagonizes mAChR non-selectively. A previous study from our laboratory also revealed that anisodamine could decrease the levels of inflammatory cytokines and produce antishock effects. Since inflammation is intrinsically implicated in neuronal damage caused by stroke, we conducted a series of experiments to examine the potential benefits of combining neostigmine with anisodamine.METHODS:Experiment one:Male Sprague-Dawley (SD) rats were anesthetized with 2.0% isoflurane, rats were subjected to MCAO before the drugs injection by vein. The animals were randomly divided into 7 groups (n=10 in each group) and treated by the following drugs:10,20 and 40 μ/kg of neostigmine; 10,20 and 40 mg/kg of anisodamine. Twenty-four hours after MCAO, the neurological score was measured. Then the animals were sacrificed for various examinations. The coronal slices of brains were incubated in 1%TTC solution. Then the slices were photographed with a digital camera. The infarct size were traced and quantified with ImageJ software. We also designed 4 different doses of neostigmine (10,20,40and 80 μg/kg) combined with anisodamine (20 mg/kg) to investigate their optimal dose ratios.Experiment two:At a ratio of 1:500 (neostigmine 40 μg/kg+anisodamine 20 mg/kg), the combination conferred the most efficient protection against stroke. So all experiments described below used such a combination ratio (1:500). In the series of experiments,40 rats were divided into 4 groups and performed MCAO, included 3 doses of the neostigmine/anisodamine combination (10μg/kg/5 mg/kg,20μ/kg/10 mg/kg and 40 μg/kg/20 mg/kg,1:500). To determine the protective time window for the combination, the neostigmine/anisodamine combination (40 μg/kg/20 mg/kg, n=10 in each group) was injected by vein at 1,1.5 and 2 h after MCAO.Experiment three:RAW264.7 cells (1×105) were inoculated in 35-mm culture dish, the combination (1:500:neostigmine,0.2μg/ml+anisodamine 100μ/ml) or ACh (750 μM) for 20 minutes prior to staining with 1.5 μg/ml fluorescein isothiocyanate-labeleda-Bgt. After fixation, the cells were washed and examined under the fluorescent confocal microscope. The data were analyzed using ImageJ software.Experiment four:The a7nAChR KO mice (3 months old, n=7 in each group) were subjected to MCAO then administrated intravenously by the combination (40 μg/kg/20 mg/kg) or the vehicle (normal saline).Experiment five:The serum levels of TNF-α, IL-6 and IL-la were examined using an ELISA system. To further ensure the anti-apoptosis effect of the combination and clarify the apoptosis pathway, we measured the expression of caspase-8, cleaved caspase-8 (related to death receptors pathway), caspase-12 (endoplasmic reticulum-specific pathway) and some biomarkers (Bcl-2, Bax, Bcl-xl and Bad) involved in mitochondrial pathway.RESULTS:1. At a ratio of 1:500 (neostigmine 40 μg/kg+anisodamine 20 mg/kg), the combination conferred the most efficient protection against stroke. The combination reduced the infarct size within 2 h.2. The combination can bind to a7nAChR, it also increase the binding of ACh to αnAChR. The a7nAChR involved in the protective effect of the combination.3. The infarct size was significantly larger in a7nAChRKO mice than in the littermate WT control. The combination decreased the infarct size in WT mice but not in a7nAChRKO mice.4. The combination significantly decreased the serum levels of TNF-α, IL-1α and IL-6 in a7nAChR WT mice, but not in the KO mice.5. The combination treatment did not affect the expression of caspase-8, cleaved caspase-8 or caspase-12 in a7nAChR WT mice. The combination decreased the expression of Bad and Bax, and increased the expression of Bcl-2 and Bcl-x1, but not in the KO mice. In α7nAChR KO mice subjected to MCAO, Bad and Bax were significantly increased.CONCLUSION:Current study identified the optimal combination of neostigmine and anisodamine against ischemic stroke, and indicated that the acetylcholine-a7nAChR is involved in the protective effects.