Protective effect of limonin against doxorubicin-induced cardiotoxicity via activating nuclear factor - like 2 and Sirtuin 2 signaling pathways

Abstract

The anti-tumor and anti-inflammatory effects of limonin have been established, here, we aim to explore whether limonin can induce protective effects against doxorubicin (DOX)-mediated cardiotoxicity which limits its clinical application. We found that limonin attenuated DOX-mediated cytoxicology of myocardial cell line H9C2 by measuring cell viability and reactive oxygen species (ROS) level. Additionally, limonin ameliorates DOX-induced cardiac injury in rat by examining the activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) concentration, and histopathological changes. Mechanistically, it was shown that limonin partially abrogated the inhibition of Nuclear factor - like 2 and Sirtuin 2 signaling induced by DOX. Furthermore, limonin-mediated protective effects on DOX-mediated cytoxicology of H9C2 were rescued by a Sirt2-specific inhibitor or siRNA against Sirt2. Thus, this work reveals that limonin can suppress DOX-mediated cardiotoxicity by activating Nrf2 and Sirt2 signaling.

Keywords: Limonin; Nrf2; Sirt2; cardiotoxicity; doxorubicin.

Figure 1.

Limonin improves cell viability of H9C2 cells suppressed by DOX treatment. (a) Cell viability of H9C2 cells was evaluated after being treated with different concentrations of DOX. (b) Cell viability of H9C2 cells was determined after being treated with different concentrations of limonin or limonin plus DOX. (c) LDH activity was detected in H9C2 cells with DOX treatment as well as limonin or not. (d) CK level was measured in H9C2 cells with DOX treatment as well as limonin or not. *p < 0 05, **p < 0 01 vs control

Figure 2.

Limonin attenuates DOX-mediated apoptosis on H9C2 cells. (a and b) TUNEL-positive cells were examined in H9C2 cells with or without DOX treatment as well as limonin or not (a), and quantified (b). (c and d) The protein expression of apoptotic executors (Cleaved caspase 3, Bax, Bcl-2) was detected in H9C2 cells (c), and quantified (d) as depicted in (A). (e) ROS level was determined in H9C2 cells as depicted in (A). **p < 0 01 vs control

Figure 3.

Limonin ameliorates DOX-induced cardiac injury in rat. (a) CAT activity was detected in heart tissues from rat treated differently as indicated. (b) SOD activity was examined in heart tissues from rat treated differently as indicated. (c) ROS level was determined in heart tissues from rat treated differently as indicated. (d) MDA activity was evaluated in heart tissues from rat treated differently as indicated. (e) GSH-Px activity was measured in heart tissues from rat treated differently as indicated. **p < 0 01 vs control. (f) Myocardial cell injury was detected heart tissues from rat treated differently as indicated. Control group: showing branched striated muscle fibers with central vesicular nuclei and light eosinophilic cytoplasm. Some blood capillaries present in between fibers. DOX group: showing area of degenerated wavy muscle fibers with absent striations (black arrows). Some fibers appear with pyknotic nuclei (red arrows), others with absent nuclei (*). Notice marked congested blood capillaries with inflammatory cells infiltration (&) and hemosiderin pigment (#) between muscle fibers. Limonin group: showing branched striated muscle fibers with central vesicular nuclei and light eosinophilic cytoplasm. Some blood capillaries present in between fibers. DOX + limonin (5 mg/kg): showing some areas of degenerated muscle fibers with absent striations (black arrows), while others are showing preserved muscle fiber with central oval nuclei (red arrows). Notice marked scattered dilated congested capillaries (c) with cloudy fatty degeneration and intermuscular edema (#). DOX + limonin (10 mg/kg) group: showing areas of preserved muscle fiber striations with central oval nuclei (red arrows). Notice some dilated congested capillaries (c) with minimal areas of edema between fibers. (HE× 200, scale bar = 20 µm)

Figure 4.

Limonin attenuates the Nrf2 and Sirt2 signaling inactivated by DOX. (a) The protein expression of Nrf2 and Sirt2, and their downstream effectors was examined in H9C2 cells with or without DOX treatment plus limonin or not. DOX: 1 μM, Limonin: 5 μM. (b) The protein expression of Nrf2 and Sirt2, and their downstream effectors was examined in heart tissues from rat treated differently as indicated. DOX: 10 mg/kg, Limonin: 10 mg/kg

Figure 5.

Limonin ameliorates DOX-induced cardiotoxicity dependent on Sirt2. (a) LDH activity was detected in H9C2 cells treated differently as indicated. (b) CK level was examined in H9C2 cells treated differently as indicated. (c) Sirt2 activity was measured in H9C2 cells with or without AGK2 treatment. (d) Sirt2 mRNA level was detected in H9C2 cells transfected with or without Sirt2 siRNA. (e and f) TUNEL-positive cells were determined in H9C2 cells treated differently as indicated (e) and quantified (f). (g) The protein expression of apoptotic executors was evaluated in H9C2 cells treated differently as indicated. (h) ROS level was measured in H9C2 cells treated differently as indicated. *p < 0 05, **p < 0 01 vs control. DOX: 1 μM, Limonin: 5 μM, AGK2: 1 μM, si-Sirt2: 50 nM