Sivelestat

Deionized water Milli-Q (conductivity < 0.1 μS cm⁻¹) (Millipore system, Molsheim, France) and analytical grade chemicals were used without further purification unless otherwise stated. PSU in pellets with average molecular weight (MW) 35,000 Da (CAS Number 25135-51-7); 1-methyl-2-pyrrolidone (NMP) (CAS Number 872-50-4); Polyvinylpyrrolidone (PVP) K30 (CAS Number 9003-39-8); HEPES sodium salt (CAS Number 75277-39-3); phorbol-12-myristate-13-acetate (PMA) (CAS Number 16561-29-8); acetonitrile (CAS Number 75-05-8); Elastase, Human Neutrophil (CAS 9004-06-2); Elastase Substrate V, Fluorogenic (CAS 72252-90-5); and single-side polished N-type silicon wafer (CAS Number 7440-21-3) were all purchased from Sigma-Aldrich Co., Saint Louis, MO, USA. Sivelestat, neutrophil elastase inhibitor (CAS Number 127373-66-4), and Human PMN Elastase ELISA Kit were acquired from Abcam, Cambridge, UK. Dimethyl sulfoxide (DMSO) (CAS Number 127373-66-4) was purchased from VWR Chemicals, Radnor, PA, USA. Monoclonal antibodies CD42a (GRP-P), PE, and CD62P (P-Selectin), and APC, used for the determination of platelet activation, were purchased from Invitrogen, ThermoFisher Scientific, Waltham, MA, USA.

A total of 180 male Swiss mice (Janvier Labs, France, 25–37 g, 5–7 weeks old) were used in this study. A single intraperitoneal (i.p.) injection of LPS (22 mg/kg, from Escherichia coli O55:B5, L2880, Merck, Darmstadt, Germany) was used to create our preclinical endotoxemic shock model. Mice were divided into different groups: the LPS group (n = 49); the LPS + atenolol group: a selective β1-adrenergic blocker (atenolol, 0.1 mg/kg i.p, AstraZeneca, Courbevoie, France) was injected 6 hours post LPS injection (n = 33); and the LPS + sivelestat group: a selective ET inhibitor (sivelestat, ab14618, Abcam, Cambridge, UK) was injected 4 hours (10 mg/kg, i.p.), 8 hours (20 mg/kg, i.p.), and 12 hours (20 mg/kg, i.p.) post LPS injection (n = 16). Control groups were also used for each condition: the saline group (NaCl 0.9%, i.p.; n = 56), the atenolol group (atenolol, 0,1 mg/kg i.p., 6 hours post saline injection; n = 14), and the sivelestat group (NaCl 0.9%, i.p. + 3 sivelestat injections at 4, 8, and 12 hours post saline injection; n = 12) (Supplemental Figure 1).
Twenty-two hours post LPS injection, the mice were used for gastrocnemius MEP recordings or harvested for tissue analysis. This delay is sufficient to induce a severe inflammatory response but does not cause the death of the animals which occurs from the twenty-fourth hour.