Fig. 6.

TPPU attenuates mechanical allodynia, ER stress, and neuroinflammation by EET signaling in CPSP rats. a The time schedule of the present experiment. After 7-day acclimation, rats were subjected to thalamic hemorrhage and were then treated with the vehicle (0.1% DMSO) or TPPU (1 mM) alone, or in combination with exogenous 14,15-EET (0.1 μg) or 14,15-EEZE (3.2 ng) once daily within the first 5 days after stroke. This was followed by a 6-day treatment-free interval and repeat doses at days 12, 13, and 14. Rats in each treatment group were analyzed by von Frey tests for PWMT at 1 day before (baseline, BL) and at 4, 7, 10, and 14 days post stroke. Besides, Western blot (WB), immunofluorescent staining (IF), and electron microscopic (EM) detection were performed on day 14 after lesion to measure the effects of TPPU/EETs on ER stress and neuroinflammation. b Both TPPU and TPPU+14,15-EET-treated CPSP rats exhibited increased PWMT in both hind paws on day 4 during the first drug delivery phase, with this analgesic activity still effective on day 7 during the treatment-free interval until day 10, the effect of which completely disappeared given that the PWMT was longer different from the vehicle-treated group. However, the repeat doses on days 12, 13, and 14 regained their efficacy on allodynia on day 14. These ameliorations were completely blocked by 14,15-EEZE application. Data are expressed as mean ± SD, n = 10 per group, two-way ANOVA, followed by Bonferroni tests, **P < 0.01, ***P < 0.001, ****P < 0.0001. c EM observation of the subcellular morphological change of the neurons around the lesion site on day 14 after CPSP. The red rows of the left panel indicate the ribosomes associated with highly dilated ER membranes in the vehicle-treated CPSP rats, whereas this dilation was alleviated by TPPU administration (as shown by the red arrows of the right panel). Scale bars = 1 μm. d, e Representative Western blot bands and quantification of ER stress markers and JNK/p38 in the perilesion site of vehicle, TPPU, TPPU + EET, and TPPU + EEZE group were presented. Except for ATF6, the expression of ER stress markers and the phosphorylation of JNK and p38 around the lesion site were significantly decreased in both TPPU and TPPU + 14,15-EET-treated CPSP rats on day 14 after lesion compared with the vehicle-treated rats, shown by Western blot. This effect was completely eliminated by co-application with 14,15-EEZE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the vehicle-treated group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with the TPPU+EEZE group, n = 5 per group, one-way ANOVA with Bonferroni’s post hoc test. f, g Immunostaining and quantification of GFAP⁺ and IBA1⁺ cells around the injured thalamic-VPL region on day 14 after lesion. n = 4 rats per group, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the vehicle group, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 compared with the TPPU + EEZE group. Scale bar = 500 μm