Protective Effects of Oroxylin A against Doxorubicin-Induced Cardiotoxicity via the Activation of Sirt1 in Mice

Abstract

Doxorubicin- (DOX-) related cardiac injury impairs the life quality of patients with cancer. This largely limited the clinical use of DOX. It is of great significance to find a novel strategy to reduce DOX-related cardiac injury. Oroxylin A (OA) has been identified to exert beneficial effects against inflammatory diseases and cancers. Here, we investigated whether OA could attenuate DOX-induced acute cardiotoxicity in mice. A single dose of DOX was used to induce acute cardiac injury in mice. To explore the protective effects, OA was administered to mice for ten days beginning from five days before DOX injection. The data in our study indicated that OA inhibited DOX-induced heart weight loss, reduction in cardiac function, and the elevation in myocardial injury markers. DOX injection resulted in increased oxidative damage, inflammation accumulation, and myocardial apoptosis in vivo and in vitro, and these pathological alterations were alleviated by treatment of OA. OA activated the sirtuin 1 (Sirt1) signaling pathway via the cAMP/protein kinase A, and its protective effects were blocked by Sirt1 deficiency. OA treatment did not affect the tumor-killing action of DOX in tumor-bearing mice. In conclusion, OA protected against DOX-related acute cardiac injury via the regulation of Sirt1.

Figure 1

OA treatment attenuated cardiac dysfunction in DOX-treated mice. (a) Bodyweight in the indicated group (n = 10). (b) The ratio of heart weight to tibial length (n = 10). (c, d) Serum and cardiac CK-MB (n = 6). (e, f) Serum and cardiac LDH (n = 6). (g) Heart rate in the indicated groups (n = 6). (h) EF in the indicated groups (n = 6). (i, j) ±dP/dt in DOX-treated mice (n = 6). (k, l) LVSP and LVEDP in DOX-treated mice (n = 6). Differences between multiple groups were determined by one-way ANOVA followed by Tukey's test. ^∗P < 0.05 vs. control group, ^#P < 0.05 vs. DOX group.

Figure 2

OA treatment improved contractile function in cardiomyocytes. (a) Resting cell length (n = 6). (b) Peak shortening (n = 6). (c, d) ±dL/dt (n = 6). Differences between multiple groups were determined by one-way ANOVA followed by Tukey's test. ∗P < 0.05 vs. control group, ^#P < 0.05 vs. DOX group.

Figure 3

OA treatment prevented oxidative damage induced by DOX in cardiac tissue. (a, b) Myocardial 4-HNE and nitrotyrosine (n = 6). (c) GSH levels (n = 6). (c, d) Gpx and SOD activity (n = 6). (f–i) The mRNA levels of gp91phox, Nox4, p47phox, and p67phox (n = 6). (j) The protein expression of Nrf2 and downstream targets (n = 6). Differences between multiple groups were determined by one-way ANOVA followed by Tukey's test. ^∗P < 0.05 vs. control group, ^#P < 0.05 vs. DOX group.

Figure 4

OA treatment attenuated inflammatory response and myocardial apoptosis in DOX-treated hearts. (a–d) Relative mRNA levels of inflammatory factors (n = 6). (e, f) The mRNA levels of MMP-2 and MMP-9 (n = 6). (g) The protein expression of p-IκBα and nuclear p65 (n = 6). (h) The protein expression of Bcl-2 (n = 6). (i) TUNEL staining and apoptotic rate (n = 6). (j, k) Caspase3/7 and PARP activities (n = 6). Differences between multiple groups were determined by one-way ANOVA followed by Tukey's test. ^∗P < 0.05 vs. control group, ^#P < 0.05 vs. DOX group.

Figure 5

OA treatment exerted protection via Sirt1. (a, b) The Sirt1 protein expression and activity in vivo (n = 6). (c, d) The Sirt1 protein expression and activity in vitro (n = 6). (e, f) The production of ROS and superoxide (n = 5). (g) Nrf2 DNA binding activity (n = 5). (h, i) The mRNA levels of HO-1 and NQO1 (n = 6). (j) NF-κB binding activity (n = 5). (k) The mRNA level of TNF-α (n = 5). (l) Cell viability (n = 5). Differences between multiple groups were determined by one-way ANOVA followed by Tukey's test. For (a–i), ^∗P < 0.05 vs. matched control; for (j, k), ^∗P < 0.05 vs. control group, ^#P < 0.05 vs. DOX group, ^†P < 0.05 vs. DOX+OA group.

Figure 6

Sirt1 depletion abolished the protection provided by OA. (a, b) The cardiac CK-MB and LDH in DOX-treated mice (n = 6). (c) EF in the indicated groups (n = 10). (d, e) Myocardial 4-HNE and nitrotyrosine (n = 6). (f) The protein expression of nuclear Nrf2 and NF-κB (n = 6). (g, h) Caspase3/7 and PARP activities (n = 6). Differences between multiple groups were determined by one-way ANOVA followed by Tukey's test. ^∗P < 0.05 vs. matched control.

Figure 7

OA treatment activated Sirt1 via cAMP/PKA signaling axis. (a, b) Relative NAD+ levels (n = 6). (c, d) Relative cAMP levels (n = 6). (e) Relative PKA activity in cells (n = 6). (f) Relative PKA activity in the hearts (n = 6). (g) Relative Sirt1 activity in cells (n = 6). (h) Cell viability of cells (n = 6). Differences between multiple groups were determined by one-way ANOVA followed by Tukey's test. For (a–e), ^∗P < 0.05 vs. control/PBS group, ^#P < 0.05 vs. DOX group. For others, ^∗P < 0.05 versus the matched group.

Figure 8

OA treatment did not increase tumor growth. (a) Schedule of the experiment. (b, c) Tumor size and tumor tissue mass (n = 6). (d–f) DOX concentrations in vivo in tissue (n = 6). Differences between multiple groups were determined by one-way ANOVA followed by Tukey's test. ^∗P < 0.05 vs. matched control.