The Anti-shock Effect Of Anisodamine Involves The Novel Ways For The Activating Of α7 Nicotinic Acetylcholine Receptor And The Increasing Of β-arrestins Expression

Backgrounds and Objectives:Anisodamine is a naturally occurring atropine derivative that has been isolated,synthesized and characterized by scientists in China in 1965.Like atropine and scopolamine,anisodamine is a non-specific cholinergic antagonist exhibiting the usual spectrum of pharmacological effects such as inhibition of salivation,gastrointestinal and sweat secretion,gastrointestinal motility,respiratory secretion and urinary bladder contraction.Other effects include mydriasis,cycloplegia, bronchodilation,neurological/cognitive impairment and alteration of cardiovascular function.Anisodamine significantly reduced the mortality rate for fulminant epidemic meningitis from 66.9%to 12.4%and for toxic bacillary dysentery from 20-30%to 0.5%. The putative and non-specific mechanisms proposed for the anti-shock effects of anisodamine include vasodilation of the microcirculation,anti-thrombotic and fibrinolytic effects,reversal of endotoxin-induced vascular leakage,stabilization of lysosomes and cathepsin inhibition,resulting to the ultimate improvement of blood flow in the microcirculation,but little molecular mechanism is available.It had been reported that acetylcholine inhibits the production of pro-inflammatory cytokines from endotoxin-stimulated macrophages through a mechanism dependent on theα7 nicotinic acetylcholine receptor subunit in 2000.Because acetylcholine is the principal vagus neurotransmitter,the central nervous system also regulates proinflammatory cytokine production through the efferent vagus nerve,termed the "cholinergic anti-inflammatory pathway".It had been reported that activation of this mechanism via vagus nerve stimulation can control the production of pro-inflammatory cytokines in experimental models of systemic inflammation,including lethal endotoxemia, hemorrhagic shock,and ischemia-reperfusion injury.Thus,the "cholinergic anti-inflammatory pathway" can directly modulate the systemic response to pathogenic invasion.Anisodamine is a non-specific cholinergic antagonist,so we supposed that whether anisodamine could indirectly activated the cholinergic anti-inflammatory pathway through its blockade of muscarinic receptor.Upon their discovery,β-arrestins were named for their capacity to sterically hinder the G protein coupling of agonist-activated seven-transmembrane receptors,ultimately resulting in receptor desensitization.Surprisingly,recent evidence shows thatβ-arrestins can also function to activate signaling cascades independently of G protein activation.By serving as multiprotein scaffolds,they direct the recruitment,activation,and scaffolding of cytoplasmic signaling complexes and thereby regulate aspects of cell motility,chemotaxis, apoptosis,and likely other cellular functions through a rapidly expanding list of signaling pathways.In 2006,Pei et al reported that association ofβ-arrestin and TRAF6 negatively regulated Toll-like receptor-interleukin 1 receptor signaling.Witherow et al reported thatβ-arrestin-1 inhibited NF-κB activity by means of its interaction with the NF-κB inhibitor IκBα.Gao et al had identified thatβ-arrestin-2 is a G protein coupled receptor stimulated regulator in NF-κB signal pathways and stimulation ofβ2-adrenergic receptor significantly enhancesβ-arrestin2-IκBαinteraction and greatly promotesβ-arrestin-2 stabilization of IκBα,indicating thatβ-arrestin-2 mediates a crosstalk betweenβ2-adrenergic receptor and NF-κB signal pathways and thus present a novel mechanism for regulation of the norepi-immune system by the sympathetic nervous system.Moreover,Luan et al identifiedβ-arrestin-2 as a phosphorylation-regulated suppressor of UV response andβ-arrestin-2 played a functional role in the response of epidermal cells to UV.Soβ-arrestins,which not only were important cross-talk point for several signal pathways,but also played an important role in the activation of NF-κB signal pathway.Ruan et al showed that in human umbilical vein endothelial cells,anisodamine counteracts endothelial cell activation induced by LPS through its inhibitory effect on the expression of PAI-1 and TF in these cells.Anisodamine also completely abolished LPS-induced NF-κB DNA binding activity in nuclear extracts from human umbilical vein endothelial cells.Our purpose is to identify whetherβ-arrestins was involved in the anti-shock effect of anisodamine. Methods:The conscious rats were given LPS to make a model ofendotoxemia.The results of receptor binding lebelled FITC-taggedα-bungarotoxin were ovserved under a confocal microscope.Quantitative PCR and Western Blotting were respectively used to measure the mRNA and protein expression of the objective protein.The genotypes ofα7 nicotinic acetylcholine receptor andβ-arrestin-2 knock-out mice were confirmed by PCR. Knock-down ofβ-arrestin-1 and muscurinic acetylcholine receptor 1 were attained by siRNA.Results:PartⅠ1.Anisodamine blunted both the acute drop in BP and the mortality in LPS-induced shock,resulting in significantly reduced TNFa and IL-1βexpression in response to LPS. Methyllycaconitine,a selective inhibitor of alpha7 nicotinic acetylcholine receptor subunit, antagonized the above effects of anisodamine.The anti-shock effects of anisodamine were markedly attenuated inα7nAchR knockout mice and by unilateral vagotomized mice. These results showed thatα7nAchR was involved in the anti-shock effect of anisodamine.2.In RAW264.7 macrophage cells,pretreated with anisodamine significantly augmented the acetylcholine-induced fluorescence density stained with FITC-taggedα-bungarotoxin compared with cells incubated with acetylcholine alone observed under the cofocal microscope.In the peritoneal macrophages from WT mice,anisodamine significantly augmented the inhibitory effect of acetylcholine on TNFαproduction induced by LPS.PartⅡ:1.It was showed that incubation with anisodamine dose- and time-dependently increased the mRNA and protein expression ofβ-arrestin-1 in RAW264.7 macrophage cells stimulated with LPS.This effect of anisodamine was significantly prevented by the inhibitor of protein synthesis cycloheximide and actinomycin D.Anisodamine also significantly increased the mRNA and protein expression ofβ-arrestin-1 in spleen in shocked-mice induced by LPS.Moreover anisodamine could significantly reverse the decrease of cAMP induced by LPS in RAW264.7 macrophage cells.H-89,a specific inhibitor of protein kinase A,could significantly prevent the effect of anisodamine on the expression ofβ-arrestin- 1 in RAW264.7 macrophage cells.2.Anisodamine could significantly inhibit the production of TNFαand IL-1βfrom RAW264.7 macrophage cells challenged by LPS.But inβ-arrestin-1 siRNA RAW264.7 maerophage cells,the effect of anisodamine were significantly attenuated compared with those WT controls.Conclusion:Our results provide evidences that the anti-shock effect of anisodamine is intimately linked to theα7nAchR-dependent pathway through blockade of muscarinic receptors and thus allowing more endogenous aeetylcholine binding to theα7nAchR. Moreover,anisodarnine increased the expression ofβ-arrestin-1 through the cAMP-PKA signal pathway in septic shock,and accordinglyβ-arrestin-1 plays an important role in the anti-shock effect of anisodamine.