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15 protocols using methyllycaconitine mla

1

Methyllycaconitine Modulates Electroacupuncture

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Methyllycaconitine (MLA; 5 mg/kg; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was diluted in 0.9% saline and administered intraperitoneally 30 min prior to each EA treatment (32 (link)). In the EA + NS group, 0.9% saline was injected, with a volume equivalent to that of the methyllycaconitine solution.
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2

Investigating Inflammatory Signaling Pathways

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LPS isolated from Escherichia coli(serotype 055:B5; Sigma-Aldrich, St. Louis, MO, USA) and methyllycaconitine (MLA; Sigma-Aldrich) were dissolved in physiological saline (0.9% sodium chloride). TQS (Tocris Bioscience, Ellisville, MO, USA) was reconstituted in physiological saline containing 1% dimethyl sulfoxide (DMSO) and 0.5% tween 80. All chemicals were prepared freshlyand administered intraperitoneally.
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3

Comparative Effects of Nicotinic Agents on Behavior

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(−)-Nicotine hydrogen tartrate [0.4 mg/kg, 5-min injection-to-placement interval (IPI)], varenicline dihydrochloride (0.1 and 1.0 mg/kg; 30-min IPI), dihydro-β-erythoidine (DHβE; 1.0 and 3.0 mg/kg; 45-min injection-to-placement interval; IPI), and methyllycaconitine (MLA; 3.0 and 10 mg/kg; 45-min IPI) were obtained from Sigma Aldrich (St. Louis, MO; nicotine, DHβE, and MLA) or NIDA (Bethesda, MD; varenicline) and dissolved in 0.9% saline. All injections were at 1 mL/kg. As per field standards, nicotine dose is reported as base form; all other drug doses are reported as salt form. The pH for nicotine was adjusted to 7.0±0.2 with a NaOH solution. All doses and IPIs were based on published research, including previous work from our laboratory (Levin et al., 2012 (link), Liu et al., 2010; Pittenger et al., 2017 (link); Wooters et al., 2009 (link)). Nicotine was injected s.c; all other drugs were injected i.p.
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4

Targeting α7nAchR Modulates CPB Outcomes

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Rats were randomly divided into four groups (n=24/group): i) The Sham group (S group), in which intubation and mechanical ventilation were performed in the right femoral artery only and the right internal jugular vein was catheterized without bypass; ii) the CPB surgery group (C group), which received the CPB surgery aforementioned; iii) the α7nAchR agonist group (P group), which received an i.p. injection of the α7nAchR agonist PHA568487 (0.8 mg/kg; Tocris Bioscience; Bio-Techne, Minneapolis, MN, USA) 30 min prior to CPB establishment; and iv) the PHA568487 + α7nAchR antagonist group (M group), which were also pretreated with PHA568487 (0.8 mg/kg) for 30 min, followed by i.p. injection of the α7nAchR antagonist methyllycaconitine (MLA; 6 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). CPB surgery was performed 60 min after MLA injection.
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5

Vagus Nerve Stimulation in Hemorrhagic Shock

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The right cervical vagus nerve was hooked with platinum electrodes, and a platinum electrode was attached to a neurostimulator. The stimulation parameters (pulse, width, frequency, and period) were set as follows: 1.0 mA, 1 Hz, 0.1 ms, and 15 min. Rats were divided into four groups (n = 6 each). Rats in the sham shock (SS) group received anesthesia and an identical procedure without hemorrhage shock, THS/R group received traumatic hemorrhage shock and fluid resuscitation; the VNS (THS/R + VNS) group received VNS before fluid resuscitation to detect the effects of VNS on THS/R rats, whereas the VNS-methyllycaconitine (THS/R + VSM) group received 10 mg/kg methyllycaconitine (MLA, Sigma–Aldrich, St. Louis, MO) (18 ) intraperitoneally before VNS to block α7nAchR, which mediates the anti-inflammatory effects of the efferent vagal CAP.
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6

Pharmacological Characterization of Nicotinic and Transient Receptor Potential Channels

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AITC, capsaicin, cytisine, HC-0300310031(HC-030031), nicotine, hexamethonium, mecamylamine, methyllycaconitine (MLA) were purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions of capsaicin (10 mM) and mecamylamine (10 mM) were made in 100% ethanol; stock solutions of AITC (100 mM), cytisine (100 mM) and HC-030031 (100 mM) were made in DMSO. A stock solution of MLA (1mM) was made in water. On the day of recording all stock solutions were diluted in bath solution so as to keep the final DMSO concentration less than 0.1%. nicotine was made on the experiment day with bath solution and used at concentrations between 1 μM and 1 mM. The α7 nAChR subunit selective antagonist MLA was used at a concentration of 20 nM (Genzen et al., 2001 (link)). The TRPA1 selective antagonist HC-030031 was used at a final concentration of 10μM based on results from previous studies (Sculptoreanu et al., 2010 (link)). The α3β4 nAChR subunit selective agonist cytisine was applied at a concentration of 100 μM (Genzen et al., 2001 (link)). The TRPA1 selective agonist AITC was applied at a final concentration of 100 μM based on results from previous studies (Zhang et al., 2014 (link)). The TRPV1 selective agonist capsaicin was used at a final concentration of 500 nM (Lu et al., 2006 (link)).
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7

Antioxidant Enzyme Assays for Compound APG

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The test compound APG, (10–30 mg/kg, i.p.), the reference drug ARP (1 mg/kg, i.p.) hydrochloride, and the CNS-penetrant methyllycaconitine (MLA, 1 µg/kg, i.p.) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The assay kits for superoxide dismutase (SOD) and catalase (CAT) were procured from Cayman Chemical (Ann Arbor, MI, USA).
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8

Selective α-7 nAchR Modulation

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PHA 568487 (PHA), a selective α-7 nAchR agonist, was purchased from Tocris Bioscience (Ellisville, MO), and methyllycaconitine (MLA), an α-7 nAchR antagonist, from Sigma (St Louis, MO). Both were diluted with 0.9% saline prior to the experiment, then injected intra-peritoneally as shown in Figure 1.
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9

Cigarette Smoke Extract Preparation

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Nicotine hydrogen tartrate (Sigma, St. Louis, MO) was dissolved in sterile saline and adjusted to pH 7.2–7.4. All nicotine doses were calculated as free base amounts. CSE was made daily by bubbling the smoke from eight unfiltered commercial cigarettes (Camel unfiltered, R.J. Reynolds Co.; 1.7 mg nicotine per cigarette; Ouyang et al., 2000 (link)) through 35 ml of sterile saline solution with a 50 ml glass syringe (35ml puffs over 2 s, repeated every 30 s) and the final solution was adjusted to pH 7.2–7.4, as described previously (Costello et al., 2014 (link); Gellner et al., 2016a (link)). The desired nicotine concentration of CSE is 150 μg/ml (Gellner et al., 2016a (link)). Methyllycaconitine (MLA) (Sigma, St. Louis, MO) and cytisine (Sigma, St. Louis, MO) were dissolved in sterile H2O.
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10

Alzheimer's Disease Biomarker Detection

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All mouse monoclonal (MMAb)/rabbit polyclonal (RPAb)/goat polyclonal (PGAb) antibodies used for immunostaining, ELISA and Western blot analyses were as follows: (A) PRAb against α7 nAChR (1:60 for immunostaining, 1:500 for Western blot, Genescript, Piscataway, NJ); (B) PRAb against S100B (1:60 for immunostaining, 1:500 for Western blot, LifeSpan BioScience); (C) Rhodamine green-labeled MMAb recognizing β-Amyloid(1–40), MMAb against β-Amyloid(1–42)(1:1000 for ELISA) and PRAb recognizing Tau (Pser199/202) (1: 1000 for ELISA, 1:500 for Western blot) from Ana Spec Inc (Fremont, CA); (D) MMAb against Aβ (17–24) (1:1000 for Western blot, Covance, Emeryville, CA); (E) PRAb against UCHL1(1: 1000 for ELISA, Protein Tech); and (F) PGAbs against RAGE (sc-8230)) (1:500 for Western blot) and PRAb against β-actin (sc-7210) (1:2000 for Western blot) from Santa Cruz Biotechnology (CA). Nicotine tartrate (NT), methamphetamine (METH) and methyllycaconitine (MLA) were purchased from Sigma-Aldrich (St. Louis, MO). RAGE inhibitor (FPS-ZM1) was obtained from Calbiochem and mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Vector (Buringame, CA). Gp120 was purchased from Immunodiagnostics (Bedford, MA).
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