Fig. 5

Requirement of high-dose spermidine for pain relief in ApoE4-TR mice due to limited spermidine retention. (A) Schematic of experimental design and spermidine treatment (200 µl 44.7 mM or 22.35 mM). (B, C) Mechanical thresholds of ApoE3-TR and ApoE4-TR mice before and after SNI, with or without spermidine gavage treatment at high dose (B) or low dose (C). n = 10 mice per group. Cohen’s d for ApoE3-TR vs. ApoE4-TR post-SNI following 22.35 mM SPD gavage: 2.29 (D7), 3.58 (D14), and 3.09 (D28). (D, E) ELISA measurement of pro-inflammatory cytokine levels in the spinal dorsal horn of ApoE3-TR and ApoE4-TR mice 14 days after 44.7 mM spermidine (D) or 22.35 mM spermidine (E) treatment. n = 3 biologically independent samples. (F) Experimental scheme of CYM and spermidine treatment. (G) ELISA detection of pro-inflammatory cytokine levels in the supernatant of ApoE3 and ApoE4 astrocytes with or without 10 mM spermidine treatment. n = 3 biologically independent samples. (H) ELISA detection of pro-inflammatory cytokine levels in the supernatant of ApoE3 and ApoE4 astrocytes with or without 5 mM spermidine treatment. n = 3 biologically independent samples. (I) An experimental scheme for the detection of intracellular spermidine content. (J) ELISA analysis of the spermidine levels in the ApoE3 and ApoE4 astrocytes after treatment with 5 mM (right) or 10 mM (left) spermidine. n = 3 independent biological samples. (K) Experimental scheme for the determination of spermidine retention capacity in astrocytes. (L) ELISA analysis of intracellular spermidine levels in ApoE3 and ApoE4 astrocytes after 10 mM spermidine treatment and subsequent medium replacement. n = 3 independent biological samples. All data are expressed as mean ± SEM. Statistical comparisons were conducted with two-way ANOVA followed by Bonferroni’s post hoc test (B, C); one-way ANOVA followed by Tukey’s post hoc test (D, E, G, H); and unpaired Student’s t-test (J, L). *P < 0.05, and **P < 0.01