Dioscin Protects against Cisplatin-Induced Acute Kidney Injury by Reducing Ferroptosis and Apoptosis through Activating Nrf2/HO-1 Signaling

Abstract

Acute kidney injury (AKI) is a clinical syndrome with high morbidity and mortality worldwide, and there is currently no effective means to prevent it. Dioscin is naturally present in the dioscoreaceae plants and has antioxidant and anti-inflammatory effects. Here, we found that dioscin is protective against cisplatin-induced AKI. Pathological and ultrastructural observations revealed that dioscin reduced renal tissue lesions and mitochondrial damage. Furthermore, dioscin markedly suppressed reactive oxygen species and malondialdehyde levels in the kidneys of AKI rats and increased the contents of glutathione and catalase. In addition, dioscin dramatically reduced the number of apoptotic cells and the expression of pro-apoptotic proteins in rat kidneys and human renal tubular epithelial cells (HK2). Conversely, the protein levels of anti-ferroptosis including GPX4 and FSP1 in vivo and in vitro were significantly enhanced after dioscin treatment. Mechanistically, dioscin promotes the entry of Nrf2 into the nucleus and regulates the expression of downstream HO-1 to exert renal protection. However, the nephroprotective effect of dioscin was weakened after inhibiting Nrf2 in vitro and in vivo. In conclusion, dioscin exerts a reno-protective effect by decreasing renal oxidative injury, apoptosis and ferroptosis through the Nrf2/HO-1 signaling pathway, providing a new insight into AKI prevention.

Keywords: Nrf2/HO-1 signaling; acute kidney injury; apoptosis; dioscin; ferroptosis; oxidative stress.

Figure 1

Dioscin relieves cisplatin-induced AKI. (A) Schematic representation of animal study design in the present study. SPSS, stroke-physiological saline solution. (B) Schematic diagram of cell experiment design. (C) Transmission electron microscope (scale bar = 1 μm) and H&E staining (scale bar = 20 μm) of rat kidney tissue (n = 5). Yellow pentagram, normal mitochondria; red pentagram, damaged mitochondria; red arrow, abnormal nucleus; yellow arrow, inflammatory cell infiltrated; blue arrow, renal tubular epithelial cell degeneration; black arrow, renal tubular epithelial cell desquamation. (D) Histological damage score. (E,F) Bun and SCr levels in rat serum (n = 6). (G) HK2 cell activity. C, control group; Dio, dioscin group; CP, cisplatin group; Dio + CP, dioscin + cisplatin group. Results are presented as Mean ± SD. Statistical significance was obtained by one-way ANOVA. ** p < 0.01 compared with C group. ^# p < 0.05, ^## p < 0.01 compared with CP group.

Figure 2

Dioscin ameliorates oxidative damage in rat kidneys and HK2 cells. (A,B) Representative images (scale bar = 50 μm) and quantification of ROS (red) in rat kidney tissue (n = 5). (C,D) Levels of MDA and GSH in rat kidney tissue (n = 6). (E,F) Representative images (scale bar = 20 μm) and quantification of CAT immunohistochemistry in rat kidney (n = 5). (G) Representative images (scale bar = 100 μm) and quantification of ROS (green) in HK2 cells (n = 5). C, control group; Dio, dioscin group; CP, cisplatin group; Dio + CP, dioscin + cisplatin group. Results are presented as Mean ± SD. Statistical significance was obtained by one-way ANOVA. ** p < 0.01 compared with C group. ^## p < 0.01 compared with CP group.

Figure 3

Dioscin reduces cisplatin-induced apoptosis in rat kidney and HK2 cells. (A,B) Representative images and quantification of TUNEL staining (scale bar = 20 μm) in rat kidney (n = 5). (C–E) The protein expression of CytC and Caspase3 in rat kidney (n = 3). (F,G) Representative images and quantification of AO/EB staining (scale bar = 100 μm) in HK2 cells (n = 5). (H–J) The protein expression of CytC and Caspase3 in HK2 cells (n = 3). C, control group; Dio, dioscin group; CP, cisplatin group; Dio + CP, dioscin + cisplatin group. Results are presented as Mean ± SD. Statistical significance was obtained by one-way ANOVA. * p < 0.05, ** p <0.01 compared with C group. ^## p < 0.01 compared with CP group.

Figure 4

Dioscin improves cisplatin-induced ferroptosis in rat kidney and HK2 cells. (A–C) Representative images and quantification of GPX4 immunofluorescence (scale bar = 50 μm) and FSP1 immunohistochemistry (scale bar = 50 μm) in rat kidneys (n = 5). (D,E) The protein expressions of GPX4 and FSP1 in rat kidneys (n = 3). (F) Representative images and quantification of ferro orange staining (scale bar = 50 μm) in HK2 cells (n = 5). (G,H) The protein levels of GPX4 and FSP1 in HK2 cells (n = 3). C, control group; Dio, dioscin group; CP, cisplatin group; Dio + CP, dioscin + cisplatin group. Results are presented as Mean ± SD. Statistical significance was obtained by one-way ANOVA. * p < 0.05, ** p < 0.01 compared with C group. ^## p < 0.01 compared with CP group.

Figure 5

Dioscin upregulates Nrf2/HO-1 signaling in cisplatin-treated rat kidney and HK2 cells. (A–C) Representative images and quantification of Nrf2 and HO-1 immunofluorescence (scale bar = 20 μm) in rat kidneys (n = 5). (D–F) Representative images and quantification of Nrf2 and HO-1 immunofluorescence (scale bar = 15 μm) in HK2 cells (n = 5). C, control group; Dio, dioscin group; CP, cisplatin group; Dio + CP, dioscin + cisplatin group. Results are presented as Mean ± SD. Statistical significance was obtained by one-way ANOVA. ** p < 0.01 compared with C group. ^# p < 0.05, ^## p < 0.01 compared with CP group.

Figure 6

The protective effect of dioscin on rat kidneys is attenuated after Nrf2 inhibition. (A) Representative images of transmission electron microscope (scale bar = 1 μm), H&E staining (scale bar = 20 μm), and ROS staining (scale bar = 50 μm) in rat kidneys (n = 5). (B) Quantification of ROS staining in rat kidneys. (C,D) Representative images and quantification of TUNEL staining (scale bar = 20 μm) in rat kidneys (n = 5). (E–G) The protein levels of CytC and Caspase3 in rat kidneys (n = 3). (H–J) The protein levels of GPX4 and FSP1 in rat kidneys (n = 3). (K–M) Representative images and quantification of GPX4 immunofluorescence (scale bar = 50 μm) and FSP1 immunohistochemistry (scale bar = 50 μm) in rat kidneys (n = 5). (N–P) Representative images and quantification of Nrf2 and HO-1 immunofluorescence (scale bar = 20 μm) in rat kidneys (n = 5). C, control group; Dio, dioscin group; CP, cisplatin group; Dio + CP, dioscin + cisplatin group. ** p < 0.01 represents extremely significant difference. Results are presented as Mean ± SD. Statistical significance was obtained via an unpaired Student’s t-test.

Figure 7

The protective effect of dioscin on HK2 cells is attenuated after Nrf2 inhibition. (A–D) Representative images and quantification of AO/EB staining (scale bar = 100 μm), ROS staining (scale bar = 100 μm) and ferro orange staining (scale bar = 50 μm) in HK2 cells (n = 5). (E–G) The protein expressions of CytC and Caspase3 in HK2 cells (n = 3). (H–J) The protein expressions of GPX4 and FSP1 in HK2 cells (n = 3). (K–M) Representative images and quantification of Nrf2 and HO-1 immunofluorescence (scale bar = 15 μm) in HK2 cells (n = 5). C, control group; Dio, dioscin group; CP, cisplatin group; Dio + CP, dioscin + cisplatin group. ** p < 0.01 represents extremely significant difference. Results are presented as Mean ± SD. Statistical significance was obtained by an unpaired Student’s t-test.

Figure 8

The schematic diagram of regulatory of Dioscin in the Nrf2/HO-1signaling to improve cisplatin-induced kidney injury. Cisplatin increases the accumulation of ROS and MDA and the levels of CytC and Caspase3 as well as reduces levels of GPX4 and FSP1 in kidneys, resulting in oxidative stress, apoptosis and ferroptosis in renal tissue. Dioscin exerts a protective effect on the kidney by upregulating Nrf2/HO-1 signaling.