【作者】 刘冲；

【导师】 苏定冯；

【作者基本信息】 第二军医大学 ， 药理学， 2008， 博士

【摘要】 目的:山莨菪碱是中国科学家1965年从茄科植物唐古特莨菪中提取出的生物碱,其人工合成品为654-2。山莨菪碱是托品酸和有机碱形成的酯类,结构与阿托品和东莨菪碱相类似,在托品基的6位炭原子上有不对称的羟基。山莨菪碱属于非选择性的毒蕈碱受体阻断剂,能够阻断M1—M5受体亚型。因此,山莨菪碱有一系列相似于阿托品的药理作用如抑制唾液分泌、抑制胃酸和汗液分泌,抑制胃肠蠕动及膀胱收缩、散瞳、扩张支气管。临床上已经用于由平滑肌痉挛、胃、十二指肠溃疡、胆道痉挛而引起的疼痛,类风湿性关节炎,胃溃疡,急性肾小球肾炎,偏头痛,过敏性紫癜,肺炎、有机磷酸酯类中毒的解救等多种疾病的治疗。自1965年以来,山莨菪碱被用于治疗循环性休克,特别是感染性休克的治疗取得了很好的治疗效果,如北京友谊医院儿科使用山莨菪碱治疗爆发型流行性脑脊髓膜炎,使死亡率从66.9%下降到12.4%,用山莨菪碱治疗中毒性细菌性痢疾,使死亡率从20-30%下降到0.5%。目前关于山莨菪碱抗休克的机制主要包括改善微循环;抑制血栓形成和纤维蛋白溶解作用;稳定溶酶体及抑制组织蛋白酶等,但是其相关的分子生物学机制很少报道。Wang等报道乙酰胆碱与巨嗜细胞上尼古丁受体结合能够抑制内毒素诱导的炎性因子TNFα,IL-1β,IL-6,IL-18等分泌。进一步研究发现乙酰胆碱的作用主要是通过与巨嗜细胞上的α7尼古丁受体结合,通过细胞内一系列的信号传递,最终抑制了Jak2-STAT3和NF-κB信号的激活,从而抑制了炎症因子的产生。在内毒素休克大鼠模型中,切断迷走神经能够明显加重内毒素诱导的休克反应,动物对内毒素的致死效应更加敏感。直接刺激迷走神经能够降低内毒素血症大鼠血浆和组织中TNFα和IL-6的含量,阻止血压的持续下降,减缓内毒素引起的低血压休克的发生时相。由于乙酰胆碱是迷走神经末梢分泌的主要神经递质,因此,他们把这条通路命名为胆碱能抗炎通路(cholinergic anti-inflammatory pathway)。胆碱能抗炎通路是通过神经来调节炎症反应程度的重要通路。山莨菪碱是非选择性的M受体阻断剂,因此,我们的第一个假设是:山莨菪碱通过阻断M受体,使迷走神经分泌的乙酰胆碱更多的与α7尼古丁受体结合激活了胆碱能抗炎通路,发挥了抗休克作用。β-arrestins是一组多功能的小分子蛋白质,它的经典作用是参与G蛋白偶联受体的脱敏。但是近年来的研究发现,β-arrestins作为脚手架蛋白,也参与了许多非G蛋白偶联受体激活的信号传导,如ERK,JNK,p38,Akt和PI3激酶信号通路等,因此β-arrestins也参与了趋化作用,凋亡作用及肿瘤转移等过程。2006年,Nature上报道了β-arrestins可以和TRAF6结合,负向调节内毒素休克时的信号传导通——TLR-IL-1R(toll-likereceptor-interleukin-1 receptor)信号通路,最终抑制转录因子NF-κB及AP-1的活化,从而抑制炎症因子的产生。此外,Witherow等报道了β-arrestins,特别是β-arrestin-1能够直接与NF-κB的抑制因子IKBα结合,使得IKBα-NF-κB复合体滞留在胞浆中,从而阻止NF-κB进入细胞核而抑制其活化。Gao等研究发现β-arrestin-2能够与IkBα结合,抑制IkBα的磷酸化降解,从而抑制NF-κB信号的激活及炎症因子的表达,并发现刺激β2肾上腺素能受体能够加强β-arrestin2-IkBα复合体的稳定,从而为交感系统的抗炎作用提供了理论依据。Luan等的研究发现β-arrestin-2能够与IkBα结合,抑制紫外线照射引发的NF-κB信号活化。阮等的研究发现在人脐静脉内皮细胞,山莨菪碱能够抑制内毒素诱导的内皮细胞活化,山莨菪碱也能够完全阻断内毒素诱导的NF-κB活化。因此,我们的第二个假设是:山莨菪碱增加了β-arrestins蛋白的表达,β-arrestins蛋白表达增加抑制了NF-κB信号的活化,参与了与山莨菪碱的抗休克作用。方法:本研究以内毒素诱导的休克为模型,研究了α7尼古丁受体阻断剂甲基牛扁碱对山莨菪碱抗休克作用的影响;在受体结合实验中,利用激光共聚焦观察了山莨菪碱对乙酰胆碱与α7尼古丁受体结合的影响;在内毒素诱导的RAW264.7巨嗜细胞,利用分子生物学技术研究了山莨菪碱对RAW264.7巨嗜细胞β-arrestin-1蛋白表达的影响及β-arrestin-1蛋白在山莨菪碱抗休克中的作用。结果:第一部分:山莨菪碱通过激活胆碱能抗炎通路发挥了抗休克作用1、α7尼古丁受体特异性阻断剂甲基牛扁碱显著地阻断了山莨菪碱对大鼠内毒素休克低血压的改善作用,同时也阻断了山莨菪碱降低内毒素休克大鼠血清和肝脏炎性细胞因子TSFα和IL-1β水平的作用;甲基牛扁碱也能显著地阻断山莨菪碱改善内毒素休克小鼠存活率的作用;在单侧迷走神经切断的小鼠模型上,山莨菪碱的抗休克作用下降;α7尼古丁受体基因敲除的小鼠与其野生对照小鼠相比,山莨菪碱降低内毒素休克小鼠血清中TNFα和IL-1β的作用显著下降。这些结果说明α7尼古丁受体参与了山莨菪碱的抗休克作用。2、受体结合实验结果说明,在RAW264.7巨嗜细胞,山莨菪碱能够通过阻断M受体,增加乙酰胆碱与α7尼古丁受体结合的数量;以α7尼古丁受体基因敲除小鼠和其野生对照小鼠的腹腔巨嗜细胞为模型,发现在野生小鼠的腹腔巨嗜细胞,山莨菪碱能够加强乙酰胆碱抑制内毒素诱导TNFα分泌的作用,但是在α7尼古丁受体基因敲除小鼠的腹腔巨嗜细胞,山莨菪碱则没有此作用。第二部分:山莨菪碱通过增加β-arrestin-1蛋白的表达发挥了抗休克作用1、在培养的RAW264.7巨嗜细胞,山莨菪碱能够剂量和时间依赖性地上调β-arrestin-1蛋白和mRNA表达;山莨菪碱的这个作用能够被蛋白质合成抑制剂放线菌酮和防线菌素D阻断;山莨菪碱也能够显著的逆转由内毒素引起的细胞内cAMP含量下降;用cAMP-PKA信号通路的特异性阻断剂H-89预处理细胞后,发现山莨菪碱上调β-arrestin-1的作用被阻断;以内毒素诱导的休克小鼠为模型,山莨菪碱能够显著增加脾脏β-arrestin-1的表达。2、在β-arrestin-1干扰的RAW264.7巨嗜细胞,山莨菪碱抑制内毒素诱导的TNFα和IL-1β分泌的作用显著下降。结论:山莨菪碱通过阻断M受体,使更多的迷走神经分泌的乙酰胆碱与巨嗜细胞上的α7尼古丁受体结合,从而激活了胆碱能抗炎通路,发挥了抗休克作用。此外,山莨菪碱能够通过cAMP-PKA信号通路增加β-arrestin-1蛋白的表达,β-arrestin-1蛋白在参与了山莨菪碱的抗休克作用。更多还原

【Abstract】 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.更多还原