Lps l2630

The LPS modeling was performed according to the method reported previously [19 (link)]. Briefly, Mice were anesthetized with pentobarbital sodium (80 mg/kg, i.p.) and restrained onto a stereotaxic apparatus. A skin incision was made to expose the skull, and a small burr hole was drilled perpendicularly to the skull. Using an infusion pump, 1 μl saline or 1 μl of 10 mg/ml LPS (L2630; serotype O111:B4; Sigma) in saline was ICV administrated (flow rate, 0.3 μl/min) to mice stereotactically via the coordinates: − 2.5 mm dorsal/ventral, − 1.0 mm lateral, and − 0.5 mm anterior/posterior from bregma. The needle remained in place for 5 min for proper dispersal of the drug.

Animals were singly housed from the time of weaning (Calcr^LepRbKO and Calcr^Sim1KO) or beginning 7 days after surgery (Calcr^NTSKO). Food intake and body weight were monitored weekly. Calcr^NTSTetTox mice and their controls were monitored from the time of surgery, and were also studied for meal patterns in metabolic chambers (TSE Systems Inc) during the final week of chow feeding.
For stimulation studies, KO mice, DREADD-expressing mice and their controls that were either at least two-months old or two months post-surgery were treated with saline or drugs (CNO, 4936, Tocris; sCT, 4033011, Bachem; Davalintide and Leptin, AstraZenica/MedImmune from original lots produced at Amylin Pharmaceuticals; PYY3-36, 4018889, Bachem; LPS, L2630, Sigma; Exendin 4, 6355, Tocris; Cholecystokinin Octapeptide (sulfated) ammonium, H-2080, Bachem) at the onset of dark cycle, food intake was monitored over four h. For chronic food intake and body weight changes, mice were given saline for two to three days prior to injecting saline or drugs twice per day (approximately 5:30 PM and 8:00 AM) for 3 or 4 days, followed by saline injections for another two or three days to assess recovery from the treatment.

Scorpion defensin BmKDfsin3, His-BmKDfsin3 and ST2345 peptide were synthesized by GL Biochem (Shanghai, China). TNF-α (96-300-01A-10) and IRAK-1/4 inhibitor I (HY-13329) were purchased from the PeproTech (Rocky Hill, CT, USA) and MedChemExpress (Monmouth Junction, NJ, USA), respectively. IL-1β (SRP6169) and LPS (L2630) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against P-p38 MAPK (4511), and p38 MAPK (9212) were from the Cell Signaling Technology (Beverly, MA, USA). Anti-HepC cAg (C7-50) (sc-57800) antibody was purchased from Santa Cruz Biotechnology (Delaware Ave Santa Cruz, CA, USA). Antibodies against GAPDH (60004-1-lg), Tubulin (11224-1-AP), and Flag (20543-1-AP) were from ProteinTech Group (Wuhan, China). Alexa Fluor 488 Affinipure donkey anti-mouse IgG (H+L) (34106ES60) was purchased from Yeasen (Shanghai, China).