Protective Effects of Dioscin Against Doxorubicin-Induced Hepatotoxicity Via Regulation of Sirt1/FOXO1/NF-κb Signal

Figures

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  Figure 1. Dioscin inhibits Dox-induced AML-12 cell damage and liver injury in mice. (A) Chemical structure of dioscin. (B) Dox-induced nephrotoxicity on AML-12 cell. (C) Cytotoxicity of dioscin on AML-12 cells and the effects of dioscin on cell viability induced by Dox. (D) Effects of dioscin (50, 100, and 200 ng/ml) for 24-h pretreatment on the cellular morphology and structure of AML-12 cells by bright image (200× magnification) investigation. (E) Effects of dioscin on AST and ALT levels in mice. (F) H&E staining (200× original magnification) of the liver tissue in mice. All data are expressed as the mean ± SD (n = 5 for in vitro test and n = 8 for in vivo test). *p < 0.05, **p < 0.01 compared with the model group. ## p < 0.01 compared with the control group. ALT, alanine transaminase; AML-12, alpha mouse liver 12; AST, aspartate transaminase; Dox, doxorubicin; H&E, hematoxylin and eosin.

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  Figure 2. Dioscin attenuates oxidative damage in AML-12 cells and mice. (A) Effects of dioscin on intracellular ROS level in AML-12 cells treated with Dox. (B) Effects of dioscin on MDA, SOD, GSH, and GSH-Px levels in mice. Data are presented as the mean ± SD (n = 3 for in vitro test and n = 8 for in vivo test). *p < 0.05 and **p < 0.01 compared with the Dox group. # p < 0.05, ## p < 0.01 compared with the control group. AML-12, alpha mouse liver 12; Dox, doxorubicin; GSH, glutathione; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; ROS, reactive oxygen species; SOD, superoxide dismutase.

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  Figure 3. Effects of dioscin on the apoptosis of AML-12 cells and mice using TUNEL assay. (A) Dioscin attenuates Dox-induced apoptosis in AML-12 cells. (B) Dioscin inhibits Dox-induced liver cell apoptosis in mice. Data are presented as the mean ± SD (n = 5 for in vitro test and n = 8 for in vivo test). *p < 0.05 and **p < 0.01 compared with the model group. ## p < 0.01 compared with the control group. AML-12, alpha mouse liver 12; Dox, doxorubicin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

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  Figure 4. Sirt1 levels are activated in AML-12 cells and mice by dioscin. (A – D) Effects of dioscin on the protein levels of Sirt1 in AML-12 cells and mice. (E – F) Effects of dioscin on the expression levels of Sirt1 based on immunofluorescence staining in AML-12 cells and mice (200× magnification). Data are presented as the mean ± SD (n = 3 for in vitro test and n = 8 for in vivo test). *p < 0.05 and **p < 0.01 compared with the model group. # p < 0.05, ## p < 0.01 compared with the control group. AML-12, alpha mouse liver 12; Sirt1, silent information regulator 1.

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  Figure 5. Dioscin suppresses the levels of protein in oxidative stress caused by Dox. (A) Effects of dioscin on the protein levels of FOXO1, Keap1, Nrf2, and HO-1 in AML-12 cells. (B) Effects of dioscin on the protein levels of FOXO1, Keap1, Nrf2, and HO-1 in mice. Data are presented as the mean ± SD (n = 3 for in vitro test and n = 8 for in vivo test). *p < 0.05 and **p < 0.01 compared with the model group. # p < 0.05, ## p < 0.01 compared with the control group. AML-12, alpha mouse liver 12; Dox, doxorubicin; FOXO1, forkhead box protein O1; HO-1, heme oxygenase-1; Keap1, kelch-like ECH-associated protein-1; Nrf2, NF-E2-related factor 2.

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  Figure 6. Dioscin ameliorates inflammation in vitro and in vivo. (A – D) Effects of dioscin on the expression levels of NF-κB in AML-12 and mice. (E – F) Effects of dioscin on the mRNA levels of IL-1β, IL-6, and TNF-α in AML-12 and mice. Data are presented as the mean ± SD (n = 3 for in vitro test and n = 8 for in vivo test). *p < 0.05 and **p < 0.01 compared with the model group. # p < 0.05, ## p < 0.01 compared with the control group. AML-12, alpha mouse liver 12; IL-1β, interleukin 1β; IL-6, interleukin 6; NF-κB, nuclear factor κB; TNF-α, tumor necrosis factor alpha.

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  Figure 7. Dioscin regulates P53-mediated apoptosis pathway in vivo and in vitro. (A – B) Effects of dioscin on the expression levels of BAX and BCL-2 in AML-12 cells. (C – D) Effects of dioscin on the expression levels of P53, BAX, BCL-2 in mice. All data are presented as the mean ± SD (n = 3 for in vitro test and n = 8 for in vivo test). *p < 0.05 and **p < 0.01 compared with the model group. # p < 0.05, ## p < 0.01 compared with the control group. AML-12, alpha mouse liver 12; BAX, BCL-2-associated X; P53, tumor suppressor P53.

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  Figure 8. Sirt1 siRNA reverses the effects of dioscin on Dox-induced cell damage. (A) Effects of dioscin on Sirt1 expression, ROS level, and cell apoptosis with or without transfection of Sirt1 siRNA in vitro. (B) Effects of dioscin on the expression levels of Sirt1, FOXO1, Keap1, Nrf2, HO-1, NF-κB, BAX, and BCL-2 after transfection with Sirt1 in AML-12 cells. Data are presented as the mean ± SD (n = 5 for in vitro test and n = 8 for in vivo test). # p < 0.05 and ## p < 0.01 versus the Dox group; *p < 0.05 and **p < 0.01 versus the Dox group transfected with Sirt1 siRNA. AML-12, alpha mouse liver 12; BAX, BCL-2-associated X; Dox, doxorubicin; FOXO1, forkhead box protein O1; HO-1, heme oxygenase-1; Keap1, kelch-like ECH-associated protein-1; NF-κB, nuclear factor κB; Nrf2, NF-E2-related factor 2; ROS, reactive oxygen species; Sirt1, silent information regulator 1.

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  Figure 9. Diagram of the possible mechanism in which dioscin inhibits Dox-induced hepatotoxicity. Dox inhibits the expression of Sirt1, which could trigger cell oxidative stress, inflammation, and apoptosis via Sirt1/FOXO1/NF-κB signal pathway to induce hepatotoxicity. However, dioscin promotes the expression of Sirt1 and inhibits the oxidative stress, inflammation, and apoptosis to rescue Dox-induced hepatotoxicity through the Sirt1/FOXO1/NF-κB signal. Blue color indicates the function of Dox, and red color indicates the function and effect of dioscin. Dox, doxorubicin; FOXO1, forkhead box protein O1; NF-κB, nuclear factor κB; Sirt1, silent information regulator 1.