Animal model of endotoxemia and sepsis

Endotoxemia was induced in Balb/C mice (male and female, 7–8 weeks old, 20–25 g) by intraperitoneal (i.p.) injection of bacterial endotoxin (LPS, 5 mg kg⁻¹)^1,65. Sepsis was induced in Balb/C mice (male and female, 7–8 weeks old, 20–25 g) by caecal ligation and puncture^1,65. Mice were randomly allocated into treatment groups and no blinding was done. Shikonin and C16 were dissolved in vehicle (10% DMSO, 20% cremophor:ethanol [3:1] and 70% phosphate-buffered saline) and administered i.p. to mice at the indicated time points. Blood was collected at indicated time points, allowed to clot for 2 h at room temperature, and then centrifuged for 15 min at 1,500g. Serum samples were stored at −20 °C before analysis. Mortality was recorded for up to 2–3 weeks after the onset of lethal endotoxemia or sepsis to ensure that no additional late deaths occurred.

Myeloid cell-specific PKM2-knockout mice were bred by crossing PKM2^flox/flox (strain name: B6;129S-Pkm^tm1.1Mgvh/J; #024048; The Jackson Laboratory) and LysM-Cre (strain name: B6.129P2-Lyz2^{tm1(cre)Ifo/J}; #004781; The Jackson Laboratory) transgenic mice.

We conducted all animal care and experimentation in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines ( http://www.aaalac.org/) and with approval from the Institutional Animal Care and Use Committees from the Xiangya Hospital, University of Pittsburgh, or Third Military Medical University.