Fig 2: Reduced M1- and M2-type macrophages with rapamycin. (A) Representative Western blot showing iNOS, TNF-a, IL-10 and CD206 protein expression levels in AVF treated with rapamycin or control (day 3 and 7). (B) Graphs showing densitometry measurements of iNOS, TNF-a, IL-10 and CD206 expression in the AVF after control or rapamycin treatment, normalized to GAPDH. iNOS: p = 0.0011 (ANOVA). *p = 0.0241, day 3; *p = 0.0054, day 7 (post hoc); n = 2–3. TNF-a: *p = 0.0020 (ANOVA). *p = 0.0223, day 3; *p = 0.0250, day 7 (post hoc); n = 2–3. IL-10: *p < 0.0001 (ANOVA). *p = 0.0011, day 3; *p = 0.0006, day 7 (post hoc); n = 2–3. CD206: p = 0.0013 (ANOVA). *p = 0.0126, day 3; *p = 0.0200, day 7 (post hoc); n = 2–3. (C) Photomicrographs showing representative dual IF for CD68 (red) and iNOS (green, top row) or CD68 (red) and TNF-a (green, bottom row) in AVF after control or rapamycin treatment (day 7). Scale bar, 25 µm. L, lumen. (D) Bar graphs showing quantification of dual IF after control or rapamycin treatment. Left graph, iNOS-CD68: p < 0.0001 (ANOVA). *p = 0.0006, day 3; *p = 0.0004, day 7; *p = 0.0073, day 21 (post hoc); n = 5. Right graph, TNF-a-CD68: p < 0.0001 (ANOVA). *p < 0.0001, day 3; *p < 0.0001, day 7 (post hoc); n = 5. (E) Photomicrographs showing representative dual IF for CD68 (red) and IL-10 (green, top row) and CD68 (red) and CD206 (green, bottom row) in control or rapamycin treated AVF (day 7). (F) Bar graphs showing quantification of dual IF after control or rapamycin treatment (day 7). Left graph, IL-10-CD68: p < 0.0001 (ANOVA). *p < 0.0001, day 3; *p < 0.0001, day 7 (post hoc); n = 5. CD206-CD68: p < 0.0001 (ANOVA). *p < 0.0001, day 3; *p < 0.0001, day 7 (post hoc); n = 5. (G) Photomicrograph of representative of CD45+ cells in control or rapamycin treated mice AVF (day 7). (H) Bar graph showing number of CD45 immunoreactive cells in AVF after control vs rapamycin treatment; p < 0.0001 (ANOVA); *p < 0.0001, day 3; *p = 0.0020, day 7; *p = 0.2110, day 21 (post hoc); n = 5. (I) Representative photomicrographs showing VCAM-1 (top row) and ICAM-1 (bottom row) IF in AVF after control or rapamycin treatment (day 7). (J) Bar graphs showing relative quantification of VCAM-1 and ICAM-1 intensity in AVF, normalized to sham vessels. VCAM-1: p = 0.3162 (ANOVA); n = 6. ICAM-1: p = 0.9280 (ANOVA); n = 4–6. Data represent mean ± SEM.

Fig 3: Effects of ethanol extracts of C. rutidosperma and E. thymifolia on the mRNA expression of iNOS and COX-2 in LPS-treated BV2 cells. BV2 cells were cultured with indicated reagent in six-well plates for 4 h followed by RNA extraction and RT-Q-PCR for measuring mRNA levels of iNOS (a) and COX-2 (b), as described in Materials and Methods. Data are represented as the mean ± SD (n = 3). Statistical differences are presented ** p < 0.01 compared with the vehicle control (without LPS) and # p < 0.05; ## p < 0.01 compared with the LPS-treated vehicle.

Fig 4: Extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and nitric oxide synthase 2 (NOS2) expression induced by interferon-? (IFN-?) and/or interleukin-17 (IL-17) in RAW 264.7 cells. (A-a) Representative western blot analysis of p-ERK1/2 in RAW 264.7 cells treated with indicated cytokines for 30 min. (B-a, C-a and D-a) Representative western blot analysis of NOS2, I?Ba degradation and p-p38 in RAW 264.7 cells pretreated with p-ERK1/2 inhibitor PD98059 for 30 min prior to cytokine exposure for 24 h, 4 h or 5 min. ß-actin was used as a loading control. (A-b, B-b, C-b and D-b) Average quantification obtained by densitometric analysis for western blot analysis. Data are expressed as the density ratio of target protein to its non-treated level in arbitrary units. Data are presented as the means ± SD from three independent experiments. **p<0.01.

Fig 5: Effects of ethanol extracts of C. rutidosperma and E. thymifolia on iNOS and COX-2 expression in LPS-induced BV2 cells. (a) BV2 cells were cultured with indicated reagent in 6-well plates for 16 h and Western blotting of iNOS, COX-2 and a-tubulin were performed, as described in Materials and Methods. Representative blots from three independent experiments are shown; (b,c) Band intensities were quantified by ImageJ software and indicated as relative folds of iNOS/a-tubulin and COX-2/a-tubulin. Data are represented as the mean ± SD (n = 3). Statistical differences are presented ** p < 0.01 compared with the vehicle control (without LPS) and ## p < 0.01 compared with the LPS-treated vehicle.