Protection against Doxorubicin-Induced Cytotoxicity by Geniposide Involves AMPKα Signaling Pathway

Figure 1. GE improved cardiac function and alleviated cardiac injury in mice with DOX treatment. (a) Body weight of four groups (n = 10). (b) Statistical results of the heart weight (HW)/tibia length (TL) (n = 10). (c) Heart rate of mice in four groups. (d) Maximum pressure. (e) Ejection fraction (EF) of mice (n = 10). (f, g) Hemodynamic analysis of mice with or without GE treatment (n = 10). (h) The mRNA expressions of hypertrophic markers (n = 10). (i) Plasma cardiac troponin-I (cTnI) levels. (j, k) Levels of creatine kinase (CK) and lactate dehydrogenase (LDH). Values represent the mean ± SD. ∗P < 0.05 versus saline, #P < 0.05 versus DOX.

Figure 2. GE protected against DOX-induced oxidative damage in vivo. (a) The mRNA expressions of several antioxidant enzymes (n = 10). (b) Activity of superoxide dismutase (SOD) (n = 10). (c) Levels of glutathione (GSH) (n = 10). (d) Activity of glutathione peroxidase (Gpx) (n = 10). (e–g) Levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE)-protein adducts, and 3-nitrotyrosine (3-NT) (n = 10). (h) Western blot and quantitative results in the indicated groups (n = 10). Values represent the mean ± SD. ∗P < 0.05 versus saline, #P < 0.05 versus DOX.

Figure 3. GE attenuated DOX-induced cell death in vivo. (a) The mRNA expressions of apoptosis markers (n = 10). (b, c) Western blot and quantitative results (n = 10). (d) Activity of caspase 3 of mice in four groups (n = 10). (e) Representative images of TUNEL and the quantitative results (n = 10). Arrows indicate TUNEL-positive cells. Values represent the mean ± SD. ∗P < 0.05 versus saline, #P < 0.05 versus DOX.

Figure 4. GE inhibited DOX-induced toxicity in vitro. (a) Cell viability after the treatment of DOX and GE with three different doses and durations (n = 3). (b) Levels of LDH in H9C2 cells (n = 3). (c) ROS level detected by DCF-DA in the indicated group (n = 3). (d) Activity of SOD in four groups (n = 3). (e) Levels of MDA (n = 3). (f) The activity of caspase 3 after GE (100 μmol/l) for 12 h (n = 3). Values represent the mean ± SD. ∗P < 0.05 versus PBS, #P < 0.05 versus DOX.

Figure 5. GE activated AMPKα to prevent DOX-induced toxicity in vitro. (a, b) Western blot and quantitative results in vivo (n = 3) and in vitro (n = 3). (c) Representative images of TUNEL and the quantitative results (n = 3). Arrows indicate TUNEL-positive cells. (d) Cell viability after GE (100 μmol/l) for 12 h (n = 3). (e) LDH levels of four groups (n = 3). (f) ROS level detected by DCF-DA in the indicated group (n = 3). (g) Levels of MDA (n = 3). (h) Western blot and quantitative results of Nrf2 and GAPDH in H9C2 cells (n = 6). Values represent the mean ± SD. In (a), ∗P < 0.05 versus saline, #P < 0.05 versus DOX; in (b), ∗P < 0.05 versus PBS. In others, ∗P < 0.05 versus DOX.

Figure 6. GE lost protection in mice with AMPKα deficiency. (a) The protein levels of t-AMPKα2 of C57 and AMPKα2 knockout mice (n = 10). (b) EF of mice (n = 10). (c, d) The levels of cTnI and 3-NT (n = 10) after the treatment of DOX and GE (50 mg/kg). (e) Activity of caspase 3 in the indicated group (n = 10). (f) The protein levels of caspase 3 and quantitative results (n = 10). Values represent the mean ± SD. ∗P < 0.05 versus DOX.