Simvastatin-Mediated Nrf2 Activation Induces Fetal Hemoglobin and Antioxidant Enzyme Expression to Ameliorate the Phenotype of Sickle Cell Disease

Abstract

Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. Here, we report that the FDA-approved oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient primary erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammatory stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of the antioxidant reduced glutathione, and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of the critical histone methyltransferase enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant gene regulatory regions in an antioxidant response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression, modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD, and represents a therapeutic strategy for further development.

Keywords: Nrf2; enhancer of zeste homolog 2; fetal hemoglobin; histone methylation; oxidative stress; sickle cell disease; simvastatin.

Figure 1

Simvastatin activates γ-globin expression to suppress sickling of SCD erythroblasts: (A) Two-phase culture of SCD patients’ peripheral blood CD34⁺ stem cells. (B) SCD erythroblasts at day 10 of culture were treated with 0.1% DMSO, 50 µM hydroxyurea (HU), 100 µM dimethyl fumarate (DMF), 2.5–20 µM simvastatin (SIM), or untreated control (UT) for 48 h and protein expression of fetal hemoglobin (HbF) and adult sickle hemoglobin (HbS) was quantified. (C,D) The percentage of HbF-positive cells (F-cells %) in SCD erythroblasts from the same culture was determined by flow cytometry (C) and quantified (D). (E) Cell morphology was analyzed to measure the number of sickled erythroblasts under hypoxia for the different treatment conditions and the percentage of sickled cells quantified (F). Data represent the mean ± SD of three independent biological replicates. *, p < 0.05.

Figure 2

Simvastatin increases the expression of NRF2 and antioxidant proteins. Day 12 SCD erythroblasts after 48 h treatment with 0.1% DMSO, 50 µM HU, 100 µM DMF, 2.5–20 µM simvastatin (SIM), or untreated control (UT) were detected for the expression of antioxidant factors at the protein (A) and mRNA levels (B). Data represent the mean ± SD of three biological replicates. *, p < 0.05.

Figure 3

Simvastatin increased the antioxidant capacity of SCD erythroblasts. Incubation with H₂O₂ (40 µM) for 8 h was completed with day 12 SCD erythroblasts after 48 h treatment of 2.5–20 µM simvastatin (SIM), then cellular viability (A) and the cellular content of reduced (GSH), oxidative (GSSG) glutathione, and their ratio (B–D) was determined. The cellular NQO1 activity (E) and cystine contents (F) in the same cells cultured as described in panel A were measured with assay kits. (G) The cystine uptake efficacy of SCD erythroblasts was determined by a BioTracker Cystine-FITC Live Cell Dye followed by flow cytometry analysis. Data represent the mean ± SD of three biological replicates. *, p < 0.05.

Figure 4

Simvastatin attenuates EZH2 expression and histone H3K27Me3 levels to modify chromatin structure and gene expression: (A) Shown are the effects of SIM on histone H3 methylation (H3K27Me3), acetylation (AcH3), and EZH2 protein expression. Total histone H3 and β-ACTIN were loading controls. (B) Quantitative RT-PCR analysis of the relative mRNA levels of H3K27Me3 modifiers EZH1/2 and KDM6a/b in SCD erythroblasts treated with 2.5–20 µM simvastatin (SIM) or untreated control (UT). Data represent mean ± SD of three biological replicates. *, p < 0.05.

Figure 5

EZH2 regulates NRF2 expression to alter ARE motif chromatin structure in target genes: (A) The effects of shEZH2-mediated gene silencing (two different constructs) on protein levels of NRF2, HbF, antioxidant factors, and H3K27Me3 were investigated. Histone H3 and β-ACTIN were loading controls. (B–E) ChIP assay determined the association of Nrf2, H3K27Me3, TATA box-binding protein (TBP), and RNA polymerase II (Pol II) to the HBG1 (B), HMOX1 (C), NQO1 (D), and SLC7A11 (E) gene loci for SCD erythroblasts treated or untreated (UT) with simvastatin (SIM, 5 µM). Primers spanning the ARE motifs of each gene were used. Data represent the mean ± SD of three biological replicates. *, p < 0.05.

Figure 6

Simvastatin suppresses H3K27Me3 modifications to activate fetal γ-globin expression in preclinical SCD mice: (A) Peripheral blood samples of SCD mice after 4 weeks of simvastatin treatment were determined by the HbF-expressing cells (F-cells) by flow cytometry. The F-cell % and mean fluorescence intensity (MFI) of HbF-positive cells were quantified. (B,C) The spleen CD71⁺ cells from SIM-treated SCD mice were used to determine the expression of H3K27Me3, HbF, and HbS at the protein (B) and mRNA levels (C). (D) ChIP assay determined the association of H3K27Me3, TBP, and Pol II to the HBG1 and HBB gene loci in spleen CD71⁺ cells from SIM-treated SCD mice. One-way ANOVA with Bonferroni’s multiple comparison tests was used for statistical analysis (n = 5). *, p < 0.05.

Figure 7

Simvastatin reduces organ damage and iron content in preclinical SCD mice: (A,B) Representative images of the spleen (A) and liver (B) of SCD mice treated with or without simvastatin (SIM, 7.5 mg/kg daily) for 4 weeks. Quantification of spleen and liver weights are shown in the graph. (C) Representative H&E and Prussian blue staining of SIM-treated SCD mouse spleens. (D) The iron content in spleen tissue was quantified. (E) Representative H&E and Prussian blue staining of SIM-treated SCD mouse livers. (F) The iron content in liver tissues was quantified. Scale bar, 50 μm. One-way ANOVA and Bonferroni’s multiple comparison test were used for statistical analysis (n = 5). *, p < 0.05.

Figure 8

Simvastatin mitigates reactive oxygen species (ROS) and inflammatory stress in preclinical SCD mice. Cellular NADPH and NADP+ levels and their ratio (A), ROS levels by 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining (B), and relative expression of antioxidant genes (C) were determined in the spleen CD71⁺ cells of SCD mice. (D) ChIP assay determined the association of H3K27Me3, TBP, and Pol II to the antioxidant gene loci (Nqo1, Cat, Hmox1, Gclc, and Slc7a11) in spleen CD71⁺ cells of SIM-treated SCD mice. (E) Heatmap representation of pro-inflammatory factor gene transcripts in the peripheral blood of SCD mice between SIM and vehicle treatment control. One-way ANOVA with Bonferroni’s multiple comparison tests was used for statistical analysis (n = 5 mice). *, p < 0.05; ***, p < 0.001.