Sulforaphane Protect Against Cadmium-Induced Oxidative Damage in mouse Leydigs Cells by Activating Nrf2/ARE Signaling Pathway

Int J Mol Sci. 2019 Feb 1;20(3):630. doi: 10.3390/ijms20030630.

Abstract

Cadmium (Cd) is harmful for humans and animals, especially for the reproductive system. However, the mechanism of its toxicity has not been elucidated, and how to alleviate its toxicity is very important. This study aimed to explore the role and mechanism of action of sulforaphane (SFN) in protecting mouse Leydigs (TM3) cells from cadmium (Cd)-induced damage. The half-maximal inhibitory concentration (IC50) of Cd and the safe doses of SFN were determined using a methyl thiazolyl tetrazolium (MTT) assay. The testosterone secretion from TM3 cells was measured using the enzyme-linked immunosorbent assay. The intracellular oxidative stress was evaluated using corresponding kits. The cell apoptosis was detected using flow cytometry. The mRNA expression of genes associated with NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling was detected using reverse transcription⁻polymerase chain reaction, including Nrf2, heme oxygenase I (HO-1), glutathione peroxidase (GSH-Px), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS). The protein expression of Nrf2, GSH-Px, HO-1, γ-GCS, and NQO1 was detected using Western blot analysis. The results showed that the IC50 of Cd to TM3 cells was 51.4 µmol/L. SFN reduced the release of lactate dehydrogenase from Cd-exposed cells. Cd + SFN 2.5 treatment significantly elevated testosterone concentration compared with the Cd group (p < 0.05). SFN significantly increased total superoxide dismutase (T-SOD) and GSH-Px activity and GSH content in Cd-treated cells (p < 0.05; p < 0.01), inhibited the production of malondialdehyde or reactive oxygen species caused by Cd (p < 0.05; p < 0.01), and reduced the apoptotic rate of Cd-induced TM3 cells (p < 0.01). SFN upregulated the mRNA expression of Nrf2, GSH-Px, HO-1, NQO1, and γ-GCS in Cd-treated cells, indicating the protective effect of SFN against Cd-induced oxidative stress or cell apoptosis by activating the Nrf2/ARE signaling pathway.

Keywords: Nrf2/ARE signaling pathway; SFN; TM3 cell; cadmium; oxidative damage.

MeSH terms

  • Animals
  • Antioxidant Response Elements / drug effects
  • Antioxidants / pharmacology*
  • Apoptosis / drug effects
  • Cadmium Chloride / antagonists & inhibitors*
  • Cadmium Chloride / toxicity
  • Cell Line
  • Cell Survival / drug effects
  • Dose-Response Relationship, Drug
  • Gene Expression Regulation
  • Glutamate-Cysteine Ligase / genetics
  • Glutamate-Cysteine Ligase / metabolism
  • Glutathione Peroxidase / genetics
  • Glutathione Peroxidase / metabolism
  • Heme Oxygenase-1 / genetics
  • Heme Oxygenase-1 / metabolism
  • Isothiocyanates / pharmacology*
  • Leydig Cells / cytology
  • Leydig Cells / drug effects*
  • Leydig Cells / metabolism
  • Male
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mice
  • NAD(P)H Dehydrogenase (Quinone) / genetics
  • NAD(P)H Dehydrogenase (Quinone) / metabolism
  • NF-E2-Related Factor 2 / agonists
  • NF-E2-Related Factor 2 / genetics*
  • NF-E2-Related Factor 2 / metabolism
  • Oxidative Stress / drug effects
  • Reactive Oxygen Species / antagonists & inhibitors
  • Reactive Oxygen Species / metabolism
  • Signal Transduction / drug effects*
  • Signal Transduction / genetics
  • Sulfoxides
  • Superoxide Dismutase / genetics
  • Superoxide Dismutase / metabolism
  • Testosterone / biosynthesis

Substances

  • Antioxidants
  • Isothiocyanates
  • Membrane Proteins
  • NF-E2-Related Factor 2
  • Nfe2l2 protein, mouse
  • Reactive Oxygen Species
  • Sulfoxides
  • Testosterone
  • Glutathione Peroxidase
  • Heme Oxygenase-1
  • Hmox1 protein, mouse
  • Superoxide Dismutase
  • NAD(P)H Dehydrogenase (Quinone)
  • Nqo1 protein, mouse
  • Glutamate-Cysteine Ligase
  • sulforaphane
  • Cadmium Chloride