Methyllycaconitine citrate   中文名称: 甲基牛扁亭柠檬酸盐

α7 neuronal nicotinic acetylcholine receptor/α7nAChR

和 10 μM 柠檬酸甲基乌头碱 (MLA) 的抑制可防止 Aβ25-35 引起的细胞活力降低。暴露于甲基乌头碱柠檬酸盐（2.5、5、10、20 μM）后，细胞活力没有变化。 Mmethylaconitine citrate 可抑制 aβ25-35 治疗引起的 LC3-II 水平升高。在 SH-SY5Y 细胞中，柠檬酸甲基乌头碱还可防止 Aβ 诱导的自噬体积累。流式细胞术证明，甲基乌头碱柠檬酸盐治疗导致 MDC 标记的空泡减少 [1]。

单独腹腔注射甲基乌头碱柠檬酸盐（MLA）（6 mg/kg）并不能刺激攀爬行为。使用柠檬酸甲基乌头碱可以大大减少甲基苯丙胺 (METH) 诱导的攀爬行为，大约可减少 50% 的转录位点。甲基乌头碱柠檬酸盐不会改变基础运动活性或冰毒引起的过度运动。在用甲基乌头碱柠檬酸盐（250±43 fmol/mg，n=7）复制的模型中，METH诱导的多巴胺神经元完全耗尽被完全阻止。由于柠檬酸甲基乌头碱对体温有直接影响，因此被省略。甲基乌头碱柠檬酸盐不会改变基础体温（37.0±0.5°C，n=5）或减轻 METH 引起的高热（38.2±0.4 °C，n=6，MLA+METH 组，ns 与 METH 组）[1] 。

细胞培养和药物治疗[1]
人神经母细胞瘤细胞系SH-SY5Y在37°C下在添加了10%FBS的RPMI-1640中培养。将60-70%融合的细胞用浓度为Aβ25-35、methyllycaconitine/甲基牛扁亭（MLA）、雷帕霉素或有或没有MLA的Aβ25-35处理。对照细胞在正常条件下培养。

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细胞活力测定[1]
将细胞铺在含有完整培养基的96孔板上并培养24小时。然后用指定浓度的化合物处理细胞指定时间。药物处理后，通过MTT法测定细胞存活率。简而言之，向每个孔中加入10µl MTT溶液（5mg/mL），并在37°C下孵育4小时。去除上清液后，向每个孔中加入100µL DMSO。用酶标仪在570nm处测量吸光度。所有实验重复3次。

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单丹磺酰尸胺染色（MDC）[1]
为了检测SH-SY5Y细胞中的自噬，将细胞铺在6孔板的盖玻片上。24小时后，用指定浓度的化合物处理细胞，在室温下用4%多聚甲醛固定15分钟，然后在37°C的黑暗中用MDC（1µg/mL的磷酸盐缓冲盐水[PBS]）染色，并立即用荧光显微镜观察。为了量化带有酸性囊泡的细胞数量，将细胞接种到6孔板中并培养过夜，然后在37°C下用1µg/mL MDC染色15分钟。孵育后，用PBS洗涤细胞，用胰蛋白酶-EDTA去除，重新悬浮，并通过流式细胞仪分析。

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Hoechst 33258染色检测细胞凋亡[1]
Hoechst 33258染色用于检测凋亡细胞核。将细胞镀在24孔板上。药物处理后，用10µg/mL Hoechst 33258对细胞染色15分钟。用PBS轻轻洗涤一次后，在荧光显微镜下观察细胞并拍照。

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流式细胞术检测细胞凋亡[1]
将细胞铺在六孔板中并孵育24小时，暴露于所需浓度的Aβ25-35中24小时，然后通过胰蛋白酶消化收获，并在PBS中洗涤两次。用AnnexinV/异硫氰酸荧光素（FITC）和碘化丙啶（PI）的组合染色后，立即通过流式细胞术分析细胞。

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免疫细胞化学[1]
如所述进行免疫细胞化学染色。简而言之，细胞在一夜之间被接种在盖玻片上。药物处理后，将细胞在4%多聚甲醛中固定30分钟。阻断后，将细胞与第一抗体（抗LC3）在4°C下孵育过夜。用PBS洗涤后，将细胞与PE标记的二抗（1∶500；Invitrogen）在室温下孵育1小时，然后用4-6-二脒基-2-苯基吲哚（DAPI）复染10分钟。通过激光扫描显微镜获得图像。

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蛋白质印迹分析[1]
处理后，收集细胞并用PBS轻轻洗涤两次，然后用蛋白质裂解缓冲液（25 mM Tris-HCl中的1%SDS，pH 7.5，4 mM EDTA，100 mM NaCl，1 mM PMSF，1%鸡尾酒蛋白酶抑制剂）裂解。样品在4°C下以12000 g离心15分钟，收集上清液。通过考马斯亮蓝蛋白测定法测定蛋白质的浓度。通过SDS-PAGE分离等量的蛋白质（50µg），并将其转移到硝化纤维膜上，在室温下用TBS中的5%脱脂奶粉封闭1小时，然后在4°C下与一抗（1∶1000）一起孵育过夜。洗涤膜，并在室温下用适当的二抗处理1小时。免疫复合物用增强化学发光试剂盒检测。

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电子显微镜（EM）[1]
用2%四氧化锇对细胞进行后固定，然后用乙醇和环氧丙烷进行递增梯度脱水步骤。然后将细胞包埋在LX-112培养基（Ladd）中，将切片切成超薄（90 nm），放置在未涂覆的铜网格上，用0.2%柠檬酸铅和1%醋酸铀酰染色。在80 kV的JEOL-1010电子显微镜（JEOL）下检查图像。

In a previous study, we demonstrated that in rat striatal synaptosomes, methamphetamine (METH)-induced reactive oxygen species (ROS) production was prevented by methyllycaconitine (MLA), a specific antagonist of alpha7 neuronal nicotinic acetylcholine receptors (alpha7 nAChR). The aim of this study was to test the influence of MLA on acute METH effects and neurotoxicity in mice, using both in vivo and in vitro models. MLA inhibited METH-induced climbing behavior by 50%. Acute effects after 30-min preincubation with 1 microM METH also included a decrease in striatal synaptosome dopamine (DA) uptake, which was prevented by MLA. METH-induced neurotoxicity was assessed in vivo in terms of loss of striatal dopaminergic terminals (73%) and of tyrosine hydroxylase levels (by 90%) at 72 h post-treatment, which was significantly attenuated by MLA. Microglial activation [measured as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide binding] was also present at 24 h post-treatment and was fully prevented by MLA, tending to confirm its neuroprotective activity. MLA had no effect on METH-induced hyperthermia. Additionally, flow cytometry assays showed that METH-induced ROS generation occurs inside synaptosomes from mouse striatum. This effect implied release of vesicular DA and was calcium-, neuronal nitric-oxide synthase-, and protein kinase C-dependent. MLA and alpha-bungarotoxin, but not dihydro-beta-erythroidine (an antagonist that blocks nAChR-containing beta2 subunits), fully prevented METH-induced ROS production without affecting vesicular DA uptake. The importance of this study lies not only in the neuroprotective effect elicited by the blockade of the alpha7 nicotinic receptors by MLA but also in that it proposes a new mechanism with which to study METH-induced acute and long-term effects. [2]

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Methyllycaconitine (MLA) is reported to be a selective antagonist for the nicotinic acetylcholine receptor alpha7 subtype and has been found in animal behavioral studies to reduce nicotine self-administration and attenuate nicotine withdrawal symptoms. While MLA crosses the blood-brain barrier (BBB), no studies have assessed brain uptake in animals subjected to chronic nicotine exposure. Given that chronic nicotine administration has been reported to alter BBB parameters that may affect the kinetic BBB passage of MLA, we evaluated MLA brain uptake in naive and S-(-)nicotine-exposed rats (4.5 mg/kg/day for 28 days; osmotic minipumps) using in situ rat brain perfusions. Our results demonstrate that in situ(3)H-MLA brain uptake rates in naive animals approximate to intravenous kinetic data (K(in), 3.24 +/- 0.71 x 10(-4) mL/s/g). However, 28-day nicotine exposure diminished (3)H-MLA brain uptake by approximately 60% (K(in), 1.29 +/- 0.4 x 10(-4) mL/s/g). This reduction was not related to nicotine-induced (3)H-MLA brain efflux or BBB transport alterations. Similar experiments also demonstrated that the passive permeation of (14)C-thiourea was diminished approximately 24% after chronic nicotine exposure. Therefore, it appears that chronic nicotine exposure diminishes the blood-brain passive diffusion of compounds with very low extraction rates (i.e. permeability-limited compounds). These findings imply that the pharmacokinetics of neuropharmaceutical agents that are permeability limited may need to be re-evaluated in individuals exposed to nicotine. [3]

[1]. Methyllycaconitine alleviates amyloid-β peptides-induced cytotoxicity in SH-SY5Y cells. PLoS One. 2014 Oct 31;9(10):e111536.

[2]. Methyllycaconitine prevents methamphetamine-induced effects in mouse striatum: involvement of alpha7 nicotinic receptors. J Pharmacol Exp Ther. 2005 Nov;315(2):658-67.

[3]. Chronic nicotine exposure alters blood-brain barrier permeability and diminishes brain uptake of methyllycaconitine. J Neurochem. 2005 Jul;94(1):37-44.

Alzheimer's disease (AD) is a chronic progressive neurodegenerative disorder. As the most common form of dementia, it affects more than 35 million people worldwide and is increasing. Excessive extracellular deposition of amyloid-β peptide (Aβ) is a pathologic feature of AD. Accumulating evidence indicates that macroautophagy is involved in the pathogenesis of AD, but its exact role is still unclear. Although major findings on the molecular mechanisms have been reported, there are still no effective treatments to prevent, halt, or reverse Alzheimer's disease. In this study, we investigated whether Aβ25-35 could trigger an autophagy process and inhibit the growth of SH-SY5Y cells. Furthermore, we examined the effect of methyllycaconitine (MLA) on the cytotoxity of Aβ25-35. MLA had a protective effect against cytotoxity of Aβ, which may be related to its inhibition of Aβ-induced autophagy and the involvement of the mammalian target of rapamycin pathway. Moreover, MLA had a good safety profile. MLA treatment may be a promising therapeutic tool for AD. [1]

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Several signaling pathways regulate the autophagy process with the mTOR pathway playing a key role. We paid attention to the downstream targets. 4E-binding protein 1 and p70S6K are directly phosphorylated by activated mTORC1 to negatively regulate autophagy. In our previous study, we showed that Aβ could induce autophagy in PC12 cells through an mTOR-dependent pathway. Here, we also found that Aβ induced autophagy in SH-SY5Y cells via mTOR signaling as evidenced by the downregulation of phosphorylated p70S6K levels. Moreover, Aβ-decreased p70S6K phosphorylation was attenuated by administration of MLA. The upregulation of mTOR signaling by MLA may inhibit Aβ-induced autophagy and contribute to its protective effect against Aβ-related cytotoxicity. In conclusion, we showed that Aβ25–35 inhibited SH-SY5Y cell growth and induced autophagy. Furthermore, MLA could provide neuroprotection against the cytotoxity of Aβ, which may be related to its inhibition of Aβ-induced autophagy via an mTOR pathway. MLA may be a safe and promising drug candidate for treatment of AD.[1]

C43H58N2O17

874.923834323883

874.373

351344-10-0

45073440

White to yellow solid powder

276

6

18

15

62

1610

14

CCN1C[C@@]2(CC[C@@H]([C@@]34[C@@H]2[C@@H]([C@@]([C@H]31)([C@]5(C[C@@H]([C@H]6C[C@@H]4[C@@H]5[C@H]6OC)OC)O)O)OC)OC)COC(=O)C7=CC=CC=C7N8C(=O)C[C@@H](C8=O)C.C(C(=O)O)C(CC(=O)O)(C(=O)O)O

INBLZNJHDLEWPS-OULUNZSJSA-N

InChI=1S/C37H50N2O10.C6H8O7/c1-7-38-17-34(18-49-32(42)20-10-8-9-11-23(20)39-26(40)14-19(2)31(39)41)13-12-25(46-4)36-22-15-21-24(45-3)16-35(43,27(22)28(21)47-5)37(44,33(36)38)30(48-6)29(34)36;7-3(8)1-6(13,5(11)12)2-4(9)10/h8-11,19,21-22,24-25,27-30,33,43-44H,7,12-18H2,1-6H3;13H,1-2H2,(H,7,8)(H,9,10)(H,11,12)/t19-,21+,22+,24-,25-,27+,28-,29+,30-,33-,34-,35+,36-,37+;/m0./s1

[(1S,2R,3R,4S,5R,6S,8R,9S,10S,13S,16S,17R,18S)-11-ethyl-8,9-dihydroxy-4,6,16,18-tetramethoxy-11-azahexacyclo[7.7.2.12,5.01,10.03,8.013,17]nonadecan-13-yl]methyl 2-[(3S)-3-methyl-2,5-dioxopyrrolidin-1-yl]benzoate;2-hydroxypropane-1,2,3-tricarboxylic acid

Methyllycaconitine (citrate); 351344-10-0; 20-ethyl-1alpha,6beta,14alpha,16beta-tetramethoxy-4-[[[2-[(3S)-3-methyl-2,5-dioxo-1-pyrrolidinyl]benzoyl]oxy]methyl]-aconitane-7,8-diol,2-hydroxy-1,2,3-propanetricarboxylate; [(1S,2R,3R,4S,5R,6S,8R,9S,10S,13S,16S,17R,18S)-11-ethyl-8,9-dihydroxy-4,6,16,18-tetramethoxy-11-azahexacyclo[7.7.2.12,5.01,10.03,8.013,17]nonadecan-13-yl]methyl 2-[(3S)-3-methyl-2,5-dioxopyrrolidin-1-yl]benzoate;2-hydroxypropane-1,2,3-tricarboxylic acid; MLA; G12422

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~142.87 mM)
H2O : ~2.18 mg/mL (~2.49 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (2.38 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (2.38 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (2.38 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。