Nicotine

Based on previous studies [8 (link), 18 (link)], this study designed that nicotine (Sigma) was administrated to hPDLCs either at variable concentrations (10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷ mol/L) for 12 h, or a fixed concentration (10⁻⁵ mol/L) for a variable period of time (3, 6, 9, 12 and 24 h). In order to prove that nicotine-induced autophagy of hPDLCs was mediated by α7 nAChR, according to previous studies [6 (link), 8 (link)], we pretreated hPDLCs with α-BTX (10⁻⁸ mol/L, α7 nAChR specific receptor antagonist) (Tocris Bioscience), for 30 min before nicotine (10⁻⁵ mol/L, 12 h) stimulation. To further examine whether autophagy played a role in the secretion of inflammatory factors in hPDLCs, according to the literature report [19 (link), 20 (link)], we pretreated hPDLCs with 3-methyladenine (3-MA) (10⁻³ mol/L, a PI3K inhibitor that effectively blocks autophagy) (Sigma), for 30 min before nicotine (10⁻⁵ mol/L, 12 h) stimulation, the expression of autophagy and the secretion of IL-1β and IL-8 were detected.

The dFBr, CMPI, and nicotine were purchased from Tocris Bioscience (Minneapolis, MN, USA). Tween 80, propylene glycol, and normal saline (0.9% NaCl) were purchased from Acros Organic (part of Thermo Fisher Scientific, NJ), Amresco (Solon, OH.), and BDHR VWR analytical (Radnor, PA), respectively. For i.p. injection, CMPI and dFBr were dissolved in a vehicle mixture consisting of saline, Tween 80, and propylene glycol, at a ratio of 18:1:1, respectively [38 (link)]. For s.c. injection, nicotine was dissolved in physiological saline.

The cholinergic receptor ligands: Nicotine (0.05, 0.1 mg/kg) (Tocris, Minneapolis, MN, USA), a cholinergic nicotinic receptor agonist, Scopolamine (1 mg/kg) (Tocris, USA), a cholinergic muscarinic receptor antagonist,
The CB2-receptor ligands: JWH 133 (0.25, 0.5, 1 mg/kg) (Tocris, USA), a potent selective CB2-receptor agonist, AM 630 (0.25, 0.5, 1 mg/kg) (Tocris, USA), a competitive CB2-receptor antagonist.
Cholinergic receptor ligands were dissolved in a saline solution (0.9% NaCl). In turn, CB2-receptor ligands were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) in a saline solution. Nicotine was administered subcutaneously (s.c.), and Scopolamine and CB2-receptor ligands were administered intraperitoneally (i.p.) at a volume of 10 mL/kg. Fresh drug solutions were prepared on each day of experimentation. Control groups received injections of saline with Tween 80 at the same volume and by the same route of administration.
Experimental doses of cholinergic and CB2-receptor ligands used for behavioral experiments and procedures were chosen accordingly to those frequently used in literature and our previous experiences [6 (link),7 (link),12 (link),74 (link)].