Withaferin A inhibits ferroptosis and protects against intracerebral hemorrhage

Abstract

Recent studies have indicated that suppressing oxidative stress and ferroptosis can considerably improve the prognosis of intracerebral hemorrhage (ICH). Withaferin A (WFA), a natural compound, exhibits a positive effect on a number of neurological diseases. However, the effects of WFA on oxidative stress and ferroptosis-mediated signaling pathways to ICH remain unknown. In this study, we investigated the neuroprotective effects and underlying mechanism for WFA in the regulation of ICH-induced oxidative stress and ferroptosis. We established a mouse model of ICH by injection of autologous tail artery blood into the caudate nucleus and an in vitro cell model of hemin-induced ICH. WFA was injected intracerebroventricularly at 0.1, 1 or 5 µg/kg once daily for 7 days, starting immediately after ICH operation. WFA markedly reduced brain tissue injury and iron deposition and improved neurological function in a dose-dependent manner 7 days after cerebral hemorrhage. Through in vitro experiments, cell viability test showed that WFA protected SH-SY5Y neuronal cells against hemin-induced cell injury. Enzyme-linked immunosorbent assays in vitro and in vivo showed that WFA markedly decreased the level of malondialdehyde, an oxidative stress marker, and increased the activities of anti-oxidative stress markers superoxide dismutase and glutathione peroxidase after ICH. Western blot assay, quantitative polymerase chain reaction and immunofluorescence results demonstrated that WFA activated the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling axis, promoted translocation of Nrf2 from the cytoplasm to nucleus, and increased HO-1 expression. Silencing Nrf2 with siRNA completely reversed HO-1 expression, oxidative stress and protective effects of WFA. Furthermore, WFA reduced hemin-induced ferroptosis. However, after treatment with an HO-1 inhibitor, the neuroprotective effects of WFA against hemin-induced ferroptosis were weakened. MTT test results showed that WFA combined with ferrostatin-1 reduced hemin-induced SH-SY5Y neuronal cell injury. Our findings reveal that WFA treatment alleviated ICH injury-induced ferroptosis and oxidative stress through activating the Nrf2/HO-1 pathway, which may highlight a potential role of WFA for the treatment of ICH.

Keywords: behavior; brain injuries; ferroptosis; heme oxygenase-1; hemorrhagic stroke; neuroprotection; nuclear factor E2-related factor 2; nuclear translocator; oxidative stress; stroke.

Figure 1

Timeline of experimental treatments and model design. GSH-Px: Glutathione peroxidase; HO-1: heme oxygenase-1; ICH: intracerebral hemorrhage; IF: immunofluorescence; LDH: lactate dehydrogenase; MDA: malondialdehyde; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nrf2: nuclear factor E2-related factor 2; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; ROS: reactive oxygen species; SOD: superoxide dismutase; WB: western blot; WFA: withaferin A.

Figure 2

The flow chart summarizing experimental setup and the number of mice analyzed at different stages of the study. GSH-Px: Glutathione peroxidase; HO-1: heme oxygenase-1; ICH: intracerebral hemorrhage; IF: immunofluorescence; LDH: lactate dehydrogenase; MDA: malondialdehyde; Nrf2: nuclear factor E2-related factor 2; qPCR: quantitative polymerase chain reaction; SOD: superoxide dismutase; WFA: withaferin A.

Figure 3

Withaferin A (WFA) induces heme oxygenase-1 (HO-1) expression and attenuates oxidative damage in the late stage of intracerebral hemorrhage (ICH) in vivo. (A) Quantitative analysis of relative gene level of HO-1 in the sham, ICH, and WFA-treated mouse groups on days 1, 3 and 7 after ICH, **P < 0.01 (two-way analysis of variance followed by Bonferroni correction). Representative western blot images (B) and quantitative analysis of relative protein levels of HO-1 (C) in the sham, ICH, and ICH + WFA (0.1, 1, and 5 μg/kg) groups on day 7 after ICH. (D) Malondialdehyde (MDA) content in the indicated groups. (E) Glutathione peroxidase (GSH-Px) bioactivity in the indicated groups. (F) Superoxide dismutase (SOD) level in the indicated groups. For B–F, *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Bonferroni correction). Data are expressed as the mean ± SEM (n = 4). ICH: Intracerebral hemorrhage; WFA: withaferin A.

Figure 4

WFA improves the pathological changes and decreases the neurobehavioral deficits in the late stage of ICH in vivo. Pathological changes in mouse brain from the indicated groups were evaluated by Nissl staining (A) and Perl’s staining (B) on day 7 after ICH. The blue particles represent the degree of iron deposition and black arrows indicate typical iron deposition. (C) Grid walking test results on days 1 and 7 after ICH. (D) Rotarod test results on days 1 and 7 after ICH. (E) Typical paw prints heat map in each group on day 7 after ICH. Statistical analysis of the percentage of correct steps (F), paw area (G), and speed (H) in each group on days 1 and 7 after ICH. n = 3 mice per group in (A) and (B); n = 10 mice per group in (C–H). *P < 0.05, **P < 0.01 (two-way analysis of variance followed by Bonferroni correction). Data are expressed as the mean ± SEM. ICH: Intracerebral hemorrhage; WFA: withaferin A.

Figure 5

WFA induces the expression of HO-1 and prevents oxidative damage in SH-SY5Y neuroblasts in vitro. Representative western blot images (A) and densitometric analysis (B) of HO-1 expression in SH-SY5Y neuroblasts treated as indicated. Cells were treated with 100 μM hemin alone or co-treated with WFA at various concentrations (1, 10 and 100 nM) for 24 hours. (C) MTT assay was used to analyze cell survival rate in cells treated as indicated. (D) MDA content in the indicated cell groups. (E) GSH-Px bioactivity in the indicated cell groups. (F) SOD level in the indicated cell groups. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Bonferroni correction). “ns” stands for non-significant. Data are expressed as the mean ± SEM (n = 6). GSH-Px: Glutathione peroxidase; HO-1: heme oxygenase-1; MDA: malondialdehyde; SOD: superoxide dismutase; WFA: withaferin A.

Figure 6

WFA upregulates nuclear factor E2-related factor 2 (Nrf2) expression and promotes nuclear translocation of Nrf2 after ICH in vivo and in vitro. (A) Venn analysis of overlapping genes that interact with both WFA and HO-1 in the CTD database and String database. (B) Representative western blot images and (C) quantitative analysis of relative protein level of cytoplasmic Nrf2 and nuclear Nrf2 in the sham, ICH, and WFA (0.1, 1, and 5 μg/kg)-treated groups on day 7 after ICH. (D) Quantification of relative nuclear and cytoplasmic Nrf2 protein levels in the sham, ICH, and WFA (0.1/1/5 μg/kg)-treated groups on day 7 after ICH. (E) Representative western blot images and (F and G) quantification of relative protein expression of cytoplasmic Nrf2, nuclear Nrf2, and ration of nuclear/cytoplasmic Nrf2 in the hemin-induced ICH model with or without WFA treatment. (H) Nrf2 localization was detected by immunofluorescence with an anti-Nrf2 antibody (green fluorescence). Nuclei were stained with DAPI (blue fluorescence). (I) Allocation of green and blue signals in the dual-channel overlay images was analyzed with the ImageJ “Co-localization Finder” plugin, and co-localization of the green and blue signals is shown in white spots. The x-axes indicate the intensities of the green signals from the green channel (Nrf2) and the y-axes from the blue channel (DAPI). For every scatterplot, the intensities are given as the pixel grey values ranging from 0 to 255. Co-localization clusters the pixels from two channels along the diagonal. Pearson’s correlation coefficient (Rr) reflects the intensity distribution relationship between the two channels. Manders overlap coefficient (MOC) reflects the true degree of co-localization of the two channels. The maximal theoretical value for the Rr and MOC is 1.0. Quantification analysis of Nrf2 nuclear location with parameter. (J) Pearson’s correlation coefficient (Rr) and (K) MOC. n = 6 mice per group in B–D; n = 3 per group in E–K. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Bonferroni correction). Data are presented as the mean ± SEM. HO-1: Heme oxygenase-1; ICH: intracerebral hemorrhage; Nrf2: nuclear factor erythroid-related factor 2; WFA: withaferin A.

Figure 7

Silencing Nrf2 depresses HO-1 expression and reverses WFA protection of hemin-induced SH-SY5Y neuroblast oxidative injury in vitro. (A) Representative western blot images and (B) quantitative analyses of Nrf2 at 24 hours after siRNA treatment in the ICH injury model induced by hemin with or without WFA exposure. (C) Quantification of relative gene expression of Nrf2 HO-1 after siRNA treatment in the ICH injury model induced by hemin with or without WFA exposure. (D) Representative western blot images and (E) quantitative analyses of HO-1 at 24 hours after siRNA treatment in ICH injury model induced by hemin with or without WFA exposure. (F) Cell survival was analyzed after siRNA treatment in the ICH injury model induced by hemin with or without WFA exposure. (G) MDA content in the indicated groups. (H) GSH-Px activity in the indicated groups. (I) SOD level in the indicated groups. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Bonferroni correction). Data are expressed as the mean ± SEM (n = 6). GSH-Px: Glutathione peroxidase; HO-1: heme oxygenase-1; MDA: malondialdehyde; Nrf2: nuclear factor erythroid-related factor 2; SOD: superoxide dismutase; WFA: withaferin A.

Figure 8

WFA induces HO-1 expression, resulting in ameliorated ferroptosis in hemin-induced SH-SY5Y neuroblast injury in vitro. (A) Representative western blot bands and (B) relative density of HO-1 in different groups. (C) Cell viability detection in different groups. (D) Typical BODIPY 581/591 C11 fluorescence images and (E) fluorescence density of BODIPY 581/591 C11 in different groups. Green indicates lipid ROS and blue indicates nuclei. (F) Representative western blot bands and (G–I) quantification of the relative density of 4-HNE, TF and FTH1 band densities in different groups. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Bonferroni correction). Data are expressed as the mean ± SEM (n = 3). 4-HNE: 4-Hydroxynonenal; FTH1: ferritin heavy chain 1; HO-1: heme oxygenase-1; TF: transferrin; WFA: withaferin A; ZnPP: zinc protoporphyrin, a specific HO-1 inhibitor.

Figure 9

WFA combined with ferrostatin-1 influences cell damage in response to hemin-induced cell injury in vitro. Quantification analysis of cell viability with MTT assay (A) and LDH release (B). (C) Quantification of BODIPY 581/591 C11 fluorescence density. (D) Representative images in the indicated groups. Green indicates lipid ROS and blue indicates nuclei. (E) Representative western blot bands and (F) quantification of the relative density of 4-HNE in the indicated groups. (G) MDA level in the indicated groups. (H) GSH-Px activity in the indicated groups. *P < 0.05, **P < 0.01, between the indicated groups (one-way analysis of variance followed by Bonferroni correction). Data are presented as the mean ± SEM (n = 3). 4-HNE: 4-Hydroxynonenal; Fer-1: ferrostatin-1; GSH-Px: glutathione peroxidase; LDH: lactic dehydrogenase; MDA: malondialdehyde; WFA: withaferin A.