Astragalus polyphenols attenuates doxorubicin-induced cardiotoxicity by activating the PI3K/AKT/NRF2 pathway

Fig 1. ASP alleviates DOX-induced cardiotoxicity and oxidative stress in AC16 and H9C2 cells. (A) The CCK-8 assay was utilized to assess cell viability in DOX-treated AC16 cells (n = 4); CCK-8 assay for detecting the protective effect of ASP on DOX-injured AC16 (B) and H9C2 (C) cells (n = 4); Flow cytometry was used to detected DCFH-DA/FITC along with statistical analysis in AC16 (D&F) and H9C2 (E&G) cells (n = 3); (H&I) The effects of ASP on the reduction of MMP potential induced by DOX in AC16 cells were measured (red/green ratio) (Scale bar = 100 μm, n = 150); (J) Quantitative q-PCR analysis of ANP and BNP mRNA expression in AC16 and H9C2 cells was conducted (n = 3); For the data presented above, one-way ANOVA (Tukey post-test), means ± SD. P > 0.05 indicates nonsignificant (ns), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 2. Effects of ASP on PI3K/AKT/Nrf2 pathway in AC16 cells. (A) Representative protein levels of p-PI3K, p-AKT, AKT, Nrf2, and ferroptosis-related signaling pathways in different concentrations of ASP-treated AC16 cells; (B-H) Statistical analysis of relative protein expression shown in Fig 2A (n = 3); One-way ANOVA (Tukey post-test), means ± SD. P > 0.05, nonsignificant (ns), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 3. Combined treatment of ASP rescues DOX-induced suppression of the PI3K/AKT/Nrf2 pathway in AC16 cells. (A) The protein levels of p-PI3K, p-AKT, AKT, Nrf2, and the ferroptosis-related signaling pathway in AC16 cells; (B-F) WB analysis demonstrates that ASP restores the expression of p-PI3K, p-AKT, AKT, Nrf2 and the downstream pathway in a dose-dependent manner in DOX-treated cells (n = 3); One-way ANOVA (Tukey post-test), means ± SD. P > 0.05, nonsignificant (ns), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 4. PI3K inhibition abolishes the protective effect of ASP against DOX-induced cardiac injury and mitochondrial damage. (A) Western blots of p-AKT, AKT, Nrf2, and downstream signaling pathways in AC16 cells; (B-F) The statistical results of relative protein expression (n = 3); (G, H) Lipid ROS tested by flow cytometry using DCFH-DA staining (n = 3); (I, J) Quantitative q-PCR analysis of cardiac injury markers, specifically ANP and BNP mRNA expression (n = 3); (K, L) Representative images of JC-1 aggregates (red), JC-1 monomers (green) and merged images of both (merge) (Scale bar = 100 μm), andstatistical analysis of red/green ratio (n = 150); One-way ANOVA (Tukey post-test), means ± SD. P > 0.05, nonsignificant (ns), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 5. ASP protects the AC16 cells from DOX-induced cardiotoxicity via Nrf2 activation. (A) Representative images of protein expression detected by Western blot of p-PI3K,p-AKT, AKT, and the Nrf2 downstream signaling pathways in AC16 cells; (B, C) ML385 does not affect PI3K/AKT (n = 3); (D-F) The statistical results show that ML385 significantly represses NRF2 activation, leading to a decrease in its downstream gene expression (n = 3); (G, H) Lipid ROS levels (n = 3); (I, J) Quantitative q-PCR analysis of relative ANP and BNP mRNA expression (n = 3); (K, L) Representative images (Scale bar = 100 μm) and statistical analysis of JC-1 (n = 150); One-way ANOVA (Tukey post-test), means ± SD. P > 0.05, nonsignificant (ns), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.