Pyridostigmine bromide

Recombinant mouse TNF, RAGE-Fc chimera protein and enzyme-linked immunosorbent assay (ELISA) kits were obtained from R & D System Inc. (Minneapolis, MN). Triton X-114, Lipopolysaccharide (LPS, E. coli. 0111:B4), peptidoglycan, pyridostigmine bromide, human macrophage-colony stimulating factor (M-CSF), acetylcholine chloride, GTS-21, 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) substrate solution, lactate dyhydrogenase (LDH) cytotoxicity assay kit, non-immune rabbit IgG (Cat# I5006) and mouse IgG (Cat# I5381) were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure E. coli LPS (Cat # tlrl-pelps), poly I:C, and type B CpG oligonucleotide were obtained from InvivoGen (San Diego, CA). Thioglycollate medium was purchased from Becton Dickinson Co., (Sparks, MD). Fluorescent labeling kits were purchased from Molecular Probes (Eugene, OR). Alexa 568 labeled LPS was obtained from Invitrogen (Waltham, MA). Dynasore was purchased from Tocris Bioscience (Bristol, UK). Microscope cover glasses were obtained from Fisher Scientific (Cat# 12–545-82, Waltham, MA). Dako Fluorescence mounting medium was purchased from Agilent (Santa Clara, CA).

We purchased pyridostigmine bromide (PB), permethrin (Per), Neomycin trisulfate hydrate, Enrofloxacin, and Ribavirin from Sigma-Aldrich (St. Louis, MO, USA). Anti-claudin-2, anti-MyD88, anti-MCP-1, and anti-β-actin primary antibodies were purchased from Abcam (Cambridge, MA, USA). anti-β-tubulin, anti-TLR3, anti-TLR7, anti-IKKα, anti-p65, and anti-IL6 primary antibodies were purchased from Santacruz Biotechnology (Dallas, TX, USA) while anti-TRAF6 was purchased from Abclonal Technology (Woburn, MA, USA). Species-specific biotinylated conjugated secondary antibodies and Streptavidin-HRP (Vectastain Elite ABC kit) were purchased from Vector Laboratories (Burlingame, CA, USA). Fluorescence-conjugated (Alexa Fluor) secondary antibodies and ProLong Diamond antifade mounting media with DAPI were purchased from Thermofisher Scientific (Grand Island, NY, USA). All other chemicals used in this study were purchased from Sigma unless otherwise specified. Animal tissues were paraffin-embedded and sectioned into slides by AML laboratories (Baltimore, MD, USA).

All animal procedures were approved by the Committee on the Ethics of Animal Experimentation at the University of Nove de Julho (CEUA protocol No. 7612011118). Animal care was performed following the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. Adult male SHR (2–3 months old, 200–250 g) were housed in collective plastic cages (four animals per cage) with controlled temperature (23 °C), a 12:12-h light–dark cycle with rat chow provided ad libitum and unlimited access to water. Animals were randomly assigned to one of three groups, with 10–12 animals in each group: sham rats (Sham), untreated infarcted rats (AMI), and pyridostigmine-treated infarcted rats (AMI + PY). All animals were monitored for 7 days. The AMI + PY Group received pyridostigmine bromide (Sigma-Aldrich, St Louis, MO), as described previously (40 mg/kg once a day, by gavage) started one h after surgery and continued for seven days after this procedure^{8 (link), 63 (link)}. According to a prior study, the dose and period of pyridostigmine administration chose were appropriate to inhibit approximately 40% of plasma acetylcholinesterase activity^{29 (link)}. All methods were reported in accordance with ARRIVE guidelines.

AChE, acetylthiocholine iodide (ATCI), 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), reduced glutathione (GSH), pyridostigmine bromide, 1-chloro-2,4-dinitrobenzene (CDNB) and Folin’s reagent were procured from Sigma chemical Co. (St. Louis, MO, USA). Trichloroacetic acid (TCA), hydrogen peroxide, sodium di-hydrogen phosphate, sodium hydroxide and sodium carbonate were procured from Sisco Research Laboratory, Mumbai, India. 2,3-dimethylmaleic anhydride is isolated and characterised (purity, 96%) from root stock of Colocasia esculenta var esculenta (L.) Schott as reported earlier^{21 (link)}.

AChE, acetylthiocholine iodide, Coumaran, and Pyridostigmine bromide were procured from Sigma chemical Co. (St. Louis, MO, USA). Ammonium molybdate, ascorbic acid, trichloroacetic acid (TCA), hydrochloric acid (HCl) and other chemicals were procured from Sisco Research Laboratory, Mumbai, India.

Pyridostigmine bromide and IMP A were purchased from Sigma-Aldrich Chemie GmbH, Germany, their purities were investigated to be 99.98% and 99.90 for PR and IMP A, respectively, according to the reference method [1 ] for PR and the published HPLC method [19 ] for IMPA. Alkaline degradation of PR under specified condition was done resulting in IMP B [27 (link), 28 ] with purity of 99.80% according the published HPLC method [19 ].

1,3-bis[5(diethyl-o-nitrobenzylammonium)pentyl]-6-methyluracil dibromide (C547) was synthesized in the A. E. Arbuzov Institute of Organic and Physical Chemistry, Kazan, Russia.
Bambuterol, pyridostigmine bromide, tetra-isopropyl pyrophosphoramide, [1,5-bis (4-allyldimethylammo-niumphenyl) pentan-3-one dibromide], acetylthiocholine chloride, acetylcholine chloride, (5,5′-dithiobis-(2-nitrobenzoic acid) were purchased from Sigma-Aldrich.
Peptide DGDFAIVKFTKVLLDYTGHI was synthesis by “ATG Service Gene” company (St. Petersburg, Russia), quality of peptide synthesis was controlled by HPLC.