Figure 5.
17R-RvD1 prevents H/R-induced activation of Nfr2 system and modulates miRNA related to proinflammatory and profibrotic pathways in the hearts of sickle cell mice. (A) Immunoblot analysis using specific antibodies against p-Nrf2 and Nrf2 in the hearts of AA and SS mice under normoxia and treated with vehicle or 17R-RvD1 (100 ng) and exposed to H/R: hypoxia (8% oxygen; 10 hours), followed by reoxygenation (21% oxygen; 3 hours); 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. One representative gel from 4 gels with similar results is shown. Densitometric analysis of immunoblots is shown on the right. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); §P < .05 (compared with AA normoxia); °P < .05 (compared with vehicle-treated mice). (B) Immunoblot analysis using specific antibodies against HO-1 and Nqo1 in the hearts of AA and SS mice treated, similar to panel A; 75 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (C) Immunoblot analysis, using specific antibodies against Prx2 and Prx3, in the hearts of AA and SS mice treated similar to panel A; 30 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); §P < .05 (compared with AA normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (D) miRNAs regulated by 17R-RvD1 in the hearts of SS mice undergoing H/R. MicroRNA expression was determined using RNU5G, RNU1A1, and SNORD61 as housekeeping small noncoding RNAs. ∗P < .05; ∗∗P < .01 (1-way ANOVA). Wb, Western blot.

17R-RvD1 prevents H/R-induced activation of Nfr2 system and modulates miRNA related to proinflammatory and profibrotic pathways in the hearts of sickle cell mice. (A) Immunoblot analysis using specific antibodies against p-Nrf2 and Nrf2 in the hearts of AA and SS mice under normoxia and treated with vehicle or 17R-RvD1 (100 ng) and exposed to H/R: hypoxia (8% oxygen; 10 hours), followed by reoxygenation (21% oxygen; 3 hours); 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. One representative gel from 4 gels with similar results is shown. Densitometric analysis of immunoblots is shown on the right. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); §P < .05 (compared with AA normoxia); °P < .05 (compared with vehicle-treated mice). (B) Immunoblot analysis using specific antibodies against HO-1 and Nqo1 in the hearts of AA and SS mice treated, similar to panel A; 75 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (C) Immunoblot analysis, using specific antibodies against Prx2 and Prx3, in the hearts of AA and SS mice treated similar to panel A; 30 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); §P < .05 (compared with AA normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (D) miRNAs regulated by 17R-RvD1 in the hearts of SS mice undergoing H/R. MicroRNA expression was determined using RNU5G, RNU1A1, and SNORD61 as housekeeping small noncoding RNAs. ∗P < .05; ∗∗P < .01 (1-way ANOVA). Wb, Western blot.

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