Fig. 6.

MC-ELNs reduce DOX-induced cardiomyocytes apoptosis via regulating p62/Keap1/Nrf2 pathway. (A) H9c2 cells or NRVM were pre-treated with 10 µg/mL MC-ELNs for 6 h, and then cultured with or without 1 µM doxorubicin (DOX) for another 24 h. Western blot analysis of Keap1 levels. (B) Immunoprecipitation assay (IP) and western blot analysis of cell lysate derived from PBS- or DOX-treated H9c2 cells in the absence or presence of 10 µg/mL of MC-ELNs (DOX, DOX + MC-ELNs). IP was performed with an antibody against p62. The blot was probed with antibodies against p62 and Keap1. β-actin was employed as a loading control. (C) Confocal microscopy analysis of the co-localization of Keap1 and p62. Scale bar: 10 μm. (D) Representative fluorescence images of Nrf2 expressions in H9c2 cells with indicated treatment. Scale bar: 20 μm. (E, F) Western blot analysis of Nrf2 and HO-1 in H9c2 cells treated with 1 µM DOX in the absence or presence of 10 µg/mL MC-ELNs. (G) After treatment and 2-day incubation with Nrf2 siRNA, H9c2 cells were treated with 1 µM DOX in the absence or presence of 10 µg/mL MC-ELNs. Western blot analysis of Nrf2, HO-1, Cleaved capsase3, and Cleaved PARP. (H) H9c2 cells were treated with 1 µM DOX in the absence or presence of 10 µg/mL MC-ELNs or K67 (100 µM, Keap1-p62 interaction inhibitor) as indicated. Western blot analysis of Nrf2, Keap1, Cleaved capsase3, and Cleaved PARP. All data are presented as mean ± SD (n = 3 experiments per group for A, B, E-H; n = 1 experiments per group for C, D). Comparisons among more than two groups were performed by ordinary one-way analysis of variance (ANOVA) followed by the Tukey’s multiple comparisons test