Drug Administration and Animal Experimental Procedures

The experimental design and protocol used for the animal experiment in this study are illustrated in Figure 1A . The mice were divided into three groups randomly; (1) the control vehicle (normal saline, NP) administered group; (2) the LPS + NP treated group; (3) the LPS + rhFGF21 treated group. Mice were pretreated intraperitoneally (i.p.) with vehicle or rhFGF21 (0.75, 1.5, and 3 mg/kg) twice daily for three consecutive days. These doses were chosen because it has previously been shown that rhFGF21 with the dose of 1.5 mg/kg dramatically attenuated locomotor function deficits in mice (Jiang et al., 2018). One hour after the last rhFGF21 administration on day 3, the mice were treated i.p. with LPS (0.83 mg/kg) dissolved in sterile saline. The concentration of LPS was based on the results of previous studies (Tomaz et al., 2014; Taniguti et al., 2019; Wang et al., 2019a; Wang et al., 2019b). The animals were subjected to following behavioral tests 24 h after LPS. Then, the animals were deeply anesthetized with isoflurane and euthanized by decapitation. The hippocampal tissue was rapidly removed and stored at -80°C until assays. NF-κB, iNOS, and brain-derived neurotrophic factor (BDNF) protein levels were assessed by western blot analysis (n = 5). Levels of the proinflammation factors IL1-β, TNF-α, and IL-6 were determined by RT-PCR (n = 5). To detect Iba1, NF-κB, and FGFR1 by immunofluorescence localization (n = 4), mice were deeply anesthetized and subjected to cardiac perfusion with saline followed by perfusion with 4% paraformaldehyde (PFA). Their brains were rapidly removed and post-fixed in 4% PFA overnight for further immunofluorescence analysis ( Figure 1A ).

Endogenous FGF21 expression level in the hippocampus of LPS-induced mice was reduced. (A) Schematic illustration of animal experimental procedures showing the duration of the lipopolysaccharide (LPS) and/or exogenous rhFGF21 administration in adult mice and analysis. (B) Representative bands of endogenous FGF21 expression detected by western blot. (C) Densitometric analysis for the protein expression of FGF21. (D) Endogenous FGF21 mRNA expression level in the hippocampus of LPS-induced mice. Data are means ± SEM (n = 5). ^###p < 0.001 compared to control group.