Astressin

LPS obtained from Escherichia coli with the serotype 055:B5, human Ucn2 (Sigma-Aldrich, St. Louis, MO, USA), anakinra (Swedish Orphan Biovitrum, Stockholm, Sweden), IL-1β and IL-6 (Wako Pure Chemical Industries, Osaka, Japan) were dissolved in normal saline. Astressin, Astressin2-B (Sigma-Aldrich) and cortagine (PolyPeptide Laboratories, Torrance, CA, USA) were dissolved in double-distilled water. Goat anti-rat IL-6 neutralizing antibody and normal goat IgG (R&D Systems, Minneapolis, MN, USA) were dissolved in sterile phosphate buffered saline. The doses of the chemicals were determined according to the previous reports [13, [17] [18] [19] [20] [21] [22] . The volume of injection was 0.2 ml/rat.

Recombinant human IL-1 receptor antagonist, anakinra (Swedish Orphan Biovitrum, Stockholm, Sweden) and IL-1β (Wako Pure Chemical Industries, Osaka, Japan) were dissolved in normal saline. Astressin, CRF receptor antagonist (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in double-distilled water. All drugs were administered though intraperitoneal (ip) route. Girard et al. [16] reported that lipopolysaccharide (LPS)-induced cytokine expression in rat placenta was dose-dependently inhibited by ip anakinra at doses of 2-20 mg/kg. Moreover, we previously showed that ip anakinra (20 mg/kg) blocked LPS-induced suppressed gastric contractility in rats [17] . In addition, we demonstrated that Astressin (200 µg/kg, ip) successfully blocked CRDinduced visceral sensitization, and IL-1β (10 µg/kg, ip) are known to induce visceral allodynia in rats [15] . The doses of chemicals used in the present study were selected according to the above evidence.

A rat/human CRF (Peptide Institute Inc., Asagi, Japan), human urocortin 2, a selective CRF 2 agonist (Bachem AG, Bubendorf, Switzerland), LPS obtained from Escherichia coli with the serotype 055:B5 (Sigma-Aldrich) and anakinra, an IL-1 receptor antagonist (Swedish Orphan Biovitrum, Stockholm, Sweden) were dissolved in normal saline. Astressin, a non-selective CRF receptor antagonist, Astressin 2 -B, a selective CRF 2 antagonist (Sigma-Aldrich) and cortagine, a selective CRF 1 agonist (PolyPeptide Laboratories, Torrance, CA, USA) were dissolved in double-distilled water. The doses of the chemicals were determined according to the previous reports (Santos et al. 1999 (link), Nozu et al. 2017b,c) . The volume of injection was 0.2 mL/rat. Additionally, eritoran tetrasodium, a TLR4 antagonist (a kind gift from Eisai Inc., Andover, MA, USA) was dissolved in PBS with the concentration of 3.5 mg/mL. LPS was subcutaneously injected. Other drugs were administered via intraperitoneal route.