Protective effects of dioscin against cisplatin-induced nephrotoxicity via the microRNA-34a/sirtuin 1 signalling pathway

Abstract

Background and purpose: Dioscin exhibits a range of pharmacological actions but little is known of its effects on cisplatin (CDDP)-induced nephrotoxicity. Here, we have assessed the effects and the possible mechanisms of dioscin against CDDP-induced nephrotoxicity.

Experimental approach: We used an in vivo model of CDDP-induced nephrotoxicity in rats and mice and, in vitro, cultures of NRK-52E and HK-2 cells. The dual luciferase reporter assay was used to demonstrate modulation, by dioscin, of the targeting of sirtuin 1 (Sirt1) by microRNA (miR)-34a. Molecular docking assays were used to analyse the effects of dioscin with Sirt1, Keap1 and NF-κB.

Key results: Dioscin attenuated cell damage in vitro and decreased renal injury in rats and mice, treated with CDDP. In terms of mechanisms, dioscin reversed CDDP-induced up-regulation of miR-34a and thus up-regulated Sirt1 levels. In addition, dioscin altered levels of haem oxygenase 1, glutathione-cysteine ligase subunits (GCLC, GCLM) and Keap1, along with increased nuclear translocation of Nrf2, thus decreasing oxidative stress. Also, dioscin affected levels of AP-1, COX-2, HMGB1, IκB-α, IL-1β, IL-6 and TNF-α and decreased the ratio of acetylated NF-κB and normal NF-κB, to suppress inflammation. From molecular docking assays, dioscin directly bound to Sirt1, Keap1 and NF-κBp65 by hydrogen bonding and/or hydrophobic interactions.

Conclusions and implications: Our results have linked CDDP-induced nephrotoxicity and the miR-34a/Sirt1 signalling pathway, which was modulated by dioscin. This natural product could be developed as a new candidate to alleviate CDDP-induced renal injury.

Figure 1

Effects of dioscin on protecting against CDDP‐induced cytotoxicity in NRK‐52E and HK‐2 cells. (A) Cytotoxicity of dioscin on NRK‐52E cells. (B) Cytotoxicity of dioscin on HK‐2 cells. (C) CDDP‐induced toxicity in NRK‐52E and HK‐2 cells. (D) Effects of dioscin on loss of cell viability in NRK‐52E cells, induced by CDDP. (E) Effects of dioscin on loss of cell viability in HK‐2 cells, induced by CDDP. (F) Effects of incubation with dioscin (50, 100 and 200 ng∙mL⁻¹) for 12 h, on the cellular morphology and structure of NRK‐52E and HK‐2 cells by bright image (100× magnification) investigation. (G) Effects of dioscin on the levels of ROS in NRK‐52E cells after CDDP exposure. (H) Effects of dioscin on the levels of ROS in HK‐2 cells after CDDP exposure. Data are presented as the mean ± SD (n = 5). ^# P < 0.05, significantly different from control group; *P < 0.05, significantly different from CDDP groups.

Figure 2

Effects of dioscin on CDDP‐induced nephrotoxicity in rats and mice. (A) Effects of dioscin on RI and serum Cr and BUN levels in rats. (B) Effects of dioscin on RI and serum Cr and BUN levels in mice. (C) H&E staining (200× original magnification) of the kidney tissue in rats and mice. (D) Effects of dioscin on MDA, SOD, GSH and GSH‐Px levels in renal tissues from rats. (E) Effects of dioscin on MDA, SOD, GSH and GSH‐Px levels in renal tissues from mice. Data are presented as the mean ± SD (n = 8). ^# P < 0.05, significantly different from control group; *P < 0.05, significantly different from CDDP groups.

Figure 3

Dioscin regulates the Sirt1 signalling pathway in a miR‐34a‐dependent manner. (A) Effects of dioscin on miR‐34a levels in vitro and in vivo. Data are presented as the mean ± SD (n = 5). ^# P < 0.05, significantly different from control group; *P < 0.05, significantly different from CDDP groups. (B) Dioscin modulated Sirt1 via miR‐34a, based on dual luciferase reporter assay in NRK‐52E and HK‐2 cells. Data are presented as the mean ± SD (n = 5). ^# P < 0.05, significantly different from the negative control group; *P < 0.05, significantly different from the group transfected with miR‐34a mimic.

Figure 4

Dioscin activates Sirt1 levels in vitro and in vivo. (A) Effects of dioscin on Sirt1 levels based on immunofluorescence staining in NRK‐52E and HK‐2 cells (200× magnification). (B) Effects of dioscin on Sirt1 levels based on immunofluorescence staining in renal tissues from rats and mice (200× magnification). Data are presented as the mean ± SD (n = 5). ^# P < 0.05, significantly different from control group; *P < 0.05, significantly different from CDDP groups.

Figure 5

Dioscin regulates the Sirt1/Nrf2‐mediated pathway in vivo and in vitro. (A) Effects of dioscin on the protein levels of Keap1, total Nrf2, nuclear Nrf2, cytoplasmic Nrf2, HO‐1, GCLC, GCLM in NRK‐52E and HK‐2 cells (n = 5). (B) Effects of dioscin on the protein levels of Keap1, total Nrf2, nuclear Nrf2, cytoplasmic Nrf2, HO‐1, GCLC and GCLM in renal tissues from rats and mice (n = 5).

Figure 6

Dioscin suppresses inflammation caused by CDDP in vitro and in vivo. (A) Effects of dioscin on the protein levels of AP‐1, COX‐2, IκB‐α degradation, HMGB1 and acetylated NF‐κB in vitro and in vivo. (B) Effects of dioscin on the mRNA levels of IL‐1β, IL‐6 and TNF‐α in vitro and in vivo. Data are presented as the mean ± SD (n = 5). ^# P < 0.05, significantly different from control group; *P < 0.05, significantly different from CDDP groups.

Figure 7

Dioscin reverses the effects of miR‐34a mimics. (A) Effects of miR‐34a on the protein levels of Sirt1 in CDDP‐treated NRK‐52E and HK‐2 cells. (B) Effects of dioscin on the viability of NRK‐52E and HK‐2 cells with or without transfection of miR‐34a mimic in vitro. (C) Effects of dioscin on the protein levels of Sirt1, Keap1, Nrf2 and acetylated NF‐κB after the transfection of miR‐34a mimic in NRK‐52E and HK‐2 cells. (D) Effects of dioscin on the mRNA levels of IL‐1β, IL‐6 and TNF‐α after transfection of miR‐34a mimic in NRK‐52E and HK‐2 cells. (E) Effects of dioscin on the expression levels of Sirt1 in NRK‐52E and HK‐2 cells with or without transfection of miR‐34a mimic, based on immunofluorescence staining in vitro (200× magnification). Data are presented as the mean ± SD (n = 5). ^# P < 0.05, significantly different from CDDP groups; *P < 0.05, significantly different from CDDP group transfected with miR‐34a mimic.

Figure 8

Dioscin reverses the effects of Sirt1 siRNA. (A) Effects of dioscin on the viability of NRK‐52E and HK‐2 cells with or without transfection of Sirt1 siRNA in vitro. (B) Effects of dioscin on the protein levels of Sirt1, Keap1, Nrf2 and acetylated NF‐κB after the transfection of Sirt1 siRNA in NRK‐52E and HK‐2 cells. (C) Effects of dioscin on the mRNA levels of IL‐1β, IL‐6 and TNF‐α after transfection of Sirt1 siRNA in NRK‐52E and HK‐2 cells. (D) Effects of dioscin on the expression of Sirt1 in NRK‐52E and HK‐2 cells with or without transfection of Sirt1 siRNA based on immunofluorescence staining in vitro (200× magnification). Data are presented as the mean ± SD (n = 5). ^# P < 0.05, significantly different from CDDP groups; *P < 0.05, significantly different from CDDP group transfected with Sirt1 siRNA.

Figure 9

Targets of dioscin against CDDP‐induced nephrotoxicity. (A) The schematic and the three‐dimensional diagram of the hydrogen bond interaction between dioscin and Sirt1. (B) Hydrophobic effect between dioscin and Sirt1. The strong hydrophobic regions are orange, and the strong hydrophilic regions are blue. The stick model of dioscin is coloured in grey. (C) The schematic diagram and the three‐dimensional diagram of the hydrogen bond interaction between dioscin and Keap1. The three‐dimensional diagram of the hydrogen bond interaction between dioscin and Keap1. (D) Hydrophobic interactions between dioscin and Keap1. The strong hydrophobic regions are orange, and the strong hydrophilic regions are blue. The stick model of dioscin is coloured in light yellow. (E, F) The schematic diagram and the three‐dimensional diagram of the hydrogen bond interaction between dioscin and NF‐κBp65.