Hesperetin alleviates doxorubicin-induced cardiotoxicity by regulating the AMPK/NLRP3 pathway

doi:10.12122/j.issn.1673-4254.2025.09.05

Abstract

Abstract:

Objective To verify whether hesperetin (Hes) alleviates doxorubicin (DOX)-induced cardiotoxicity by reducing inflammation via regulating the AMPK/NLRP3 pathway. Methods C57/bl6 mice and H9c2 cells treated with DOX to mimic cardiotoxicity were randomly divided into Sham (or control) group, DOX group, DOX+Hes group, DOX+Hes+compound C (CC, an AMPK inhibitor) group. Cardiac function and myocardial pathologies of the mice were evaluated, and the changes in H9c2 cell morphology and viability were assessed. Lactate dehydrogenase (LDH) activity in mouse myocardial tissues and H9c2 cells was measured using ELISA, and H9c2 cell apoptosis was detected with TUNEL staining. In both H9c2 cells and the myocardial tissues of the mice, cellular expression levels of TNF-α, IL-6 and IL-1β mRNAs and cleaved caspase-3, Bcl2, Bax, IL-1β, IL-18, p-AMPK, AMPK, p-mTOR, mTOR, NLRP3, ASC and caspase-1 proteins were detected using RT-PCR and Western blotting. Results DOX treatment caused cell swelling, decreased cell viability and increased LDH activity in H9c2 cells, resulting also in significantly increased cell apoptosis and cleaved caspase-3 expression and decreased Bcl2/Bax ratio. The DOX-treated mice showed obvious myocardial fiber swelling and inflammatory infiltration, decreased cardiac function and significantly increased myocardial LDH activity. In H9c2 cells, DOX treatment significantly increased the mRNA expressions of TNF-α, IL-6 and IL-1β and protein expressions of IL-1β and IL-18, lowered the expressions of p-AMPK and p-mTOR, and increased the expressions of NLRP3, ASC and caspase-1. Hes treatment obviously reduced these toxic effects of DOX in H9c2 cells, but its protective effects were blocked by application of compound C. Conclusion Hes reduces DOX-induced cardiotoxicity by inhibiting inflammation via regulating the AMPK/NLRP3 pathway.

Key words: hesperetin, doxorubicin, AMPK/NLRP3, inflammatory response, H9c2 cells

Aili YAN, Mengyao LUO, Jinrui CHANG, Xinhua LI, Juanxia ZHU. Hesperetin alleviates doxorubicin-induced cardiotoxicity by regulating the AMPK/NLRP3 pathway[J]. Journal of Southern Medical University, 2025, 45(9): 1850-1858.

Figures/Tables 7

Fig.1 Compound C blocks the protective effects of Hes against DOX-induced cell injury in H9c2 cells. A: Cell morphology in each group (Original magnification: ×100). B: Comparison of cell viability among the 4 groups. C: LDH activity in the cells in the 4 groups. **P<0.01 vs Control group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

Fig.2 Compound C reduces the protective effect of Hes against DOX-induced cell apoptosis.A: Representative Western blots of Bcl2, Bax, cleaved caspase-3 and GAPDH in each group. B: Bcl2/Bax ratios in each group. C: Cleaved caspase-3 expression levels in each group. D: TUNEL staining of the cells in each group (Scale bar=100 μm). **P<0.01 vs Control group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

Fig.3 Compound C attenuates the protective effect of Hes against DOX-induced inflammation. A-C: Relative mRNA expressions of TNF‑α, IL-6 and IL-1β in each group. D: Representative Western blots of TNF‑α, IL-6, IL-1β and GAPDH in each group. E, F: Relative expression levels of IL-1β and IL-18 in the cells in each group. **P<0.01 vs Control group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

Fig.4 Compound C blocks the effect of Hes against DOX-induced changes in protein expressions of AMPK/NLRP3 signaling. A: Representative Western blots of p-AMPK, AMPK, p-mTOR, mTOR, NLRP3, ASC, caspase-1 and GAPDH in each group. B-F: Relative expression levels of p-AMPK, p-mTOR, NLRP3, ASC, and caspase-1 in each group. **P<0.01 vs Control group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

Fig.5 Compound C blocks the protective effect of Hes against DOX-induced myocardial injury in mice. A: HE staining of the myocardial tissues in each group (×200). B: Myocardial LDH activity in each group. C: LVEF in each group. D: LVFS in each group. **P<0.01 vs Sham group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

Fig.6 Compound C attenuates the protective effect of Hes against DOX-induced inflammation in the myocardial tissues of the mice. A-C: Relative mRNA expressions of TNF-α, IL-6 and IL-1β in each group. D: Representative Western blots of IL-1β, IL-18 and GAPDH. E, F: Relative expression levels of IL-1β and IL-18 in each group. **P<0.01 vs Sham group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

Fig.7 Compound C blocks the effect of Hes against DOX-induced changes in expression of AMPK/NLRP3 signaling in the mice. A: Representative Western blots of p-AMPK, AMPK, p-mTOR, mTOR, NLRP3, ASC, caspase-1 and GAPDH. B-F: Relative expression levels of p-AMPK, p-mTOR, NLRP3, ASC, and caspase-1 in the myocardial tissues of the mice in each group. **P<0.01 vs Sham group; ##P<0.01 vs DOX group; &&P<0.01 vs DOX+Hes group (n=6).

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