Fig 2: Involvement of JNK and p38 mitogen-activated protein kinase pathways in the anti-apoptotic effects of MFA. (A) The expression levels of JNK, p-JNK, p38 and p-p38 were analyzed by western blot analysis, using GAPDH as a reference. (B) The ratio of pJNK/JNK and p-p38/p38MAPK. **P<0.01 compared with control group; ##P<0.01 compared with CCl4-treated model group. MFA, methyl ferulic acid; DDB, dimethyl diphenyl bicarboxylate; p-JNK, phosphorylated c-Jun N-terminal kinase.

Fig 3: 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

Fig 4: Effect of LPS on the P38 MAPK pathway in INS-1 cells. The effects of LPS on the P38 MAPK signaling pathway in INS-1 cell was assessed by reverse transcription-quantitative polymerase chain reaction and western blotting. (A) LPS had little influence on the mRNA expression of P38 (Mapk14). (B) The cells were stimulated with LPS (5 µg/ml) for 24 h. The protein expression levels of HMGB1, P38 and p-P38 were analyzed by western blotting. HMGB1, high mobility group box 1; LPS, lipopolysaccharide; p-, phosphorylated.

Fig 5: The MAPK/ERK signaling pathway is involved in the regulation of MMP-1 expression. (A) Serum-deprived SiHa cells were incubated for 1 h in the presence or absence of MßCD and cholesterol, and were then incubated with 10 ng/ml EGF for 2 h. The cell lysates were analyzed by western blotting with anti-AKT, anti-phospho-AKT, anti-ERK1/2, anti-phospho-ERK1/2, anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK and anti-ß-actin antibodies. The results shown are from representative experiments performed in triplicate. (B) Serum-deprived SiHa cells were incubated with 10 ng/ml EGF in the presence of the corresponding pharmacological inhibitors for 1 h before exposure to EGF. Total RNA was extracted 2 h later and then analyzed by reverse transcription-quantitative polymerase chain reaction, in which the MMP-1 or GAPDH mRNA levels were measured. Cell lysates were analyzed 24 h later by western blotting, in which anti-MMP-1 or anti-ß-actin antibodies were used as probes. The laser densitometry results (the data are the mean of three independent experiments ±SD) are shown in the bar graph and are expressed as a ratio. #P<0.05 compared with EGF-stimulated cells with MßCD treatment. *P<0.05 compared with EGF-stimulated cells with MßCD treatment. (C) Cells were infected with recombinant adenoviruses for expression of CA-MEK and DN-MEK for 48 h, and then treated with EGF (10 ng/ml) for 24 h in the absence of MßCD treatment for 1 h before exposure to EGF. Cell lysates were analyzed by western blotting, in which anti-ERK1/2, anti-phospho-ERK1/2, anti-MMP-1 or anti-ß-actin antibodies were used as probes. The laser densitometry results (the data are the mean of three independent experiments ± SD) are shown in the bar graphs and are expressed as a ratio. P<0.05 compared with each other. Phospho, phosphorylated; MMP, matrix metalloproteinase; EGF, epidermal growth factor; MßCD, methyl-ß-cyclodextrin; mRNA, messenger RNA; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; CA, constitutively active; DN, dominant negative; SD, standard deviation.