TRIOL attenuates intracerebral hemorrhage injury by bidirectionally modulating microglia- and neuron-mediated hematoma clearance

Abstract

Intracerebral hemorrhage (ICH) represents the most severe subtype of stroke, and the lack of effective clinical pharmacotherapies poses a substantial threat to human health. Hematoma plays a crucial role in determining the prognosis of ICH patients by causing primary mechanical extrusion, followed by secondary brain injuries, such as cerebral edema, iron-mediated oxidative stress, and inflammation resulting from its degradation products. 5α-androst-3β,5α,6β-triol (TRIOL) is a neuroprotective steroid currently undergoing phase II clinical trial for acute ischemic stroke with anti-oxidative and anti-inflammatory properties. However, whether TRIOL can protect brain against ICH injury remains unclear. In this study, we found that TRIOL significantly improved neurological function while reducing hematoma volume, cerebral edema, and tissue damage after ICH. Moreover, TRIOL enhanced microglial hematoma clearance through promoting CD36-mediated erythrophagocytosis and CD163-associated hemoglobin scavenging, while simultaneously reducing the release of microglial inflammatory factors and activating the antioxidative transcription factor Nrf2. Additionally, TRIOL inhibited neuron mediated hematoma absorption by suppressing heme oxygenase 2 (HO-2) and protected neurons against ICH-induced damage in vitro and in vivo. TRIOL also mitigated neuronal iron-dependent oxidative damage by increasing ferritin levels but decreasing divalent metal transporter 1 (DMT1) expression. Overall, these findings highlight the promising potential of TRIOL as a drug candidate for treating ICH.

Keywords: Hematoma clearance; Heme oxygenase 1; Heme oxygenase 2; Intracerebral hemorrhage; Nrf2; TRIOL.

Graphical abstract

Fig. 1

TRIOL ameliorates ICH injuries caused by autologous blood injection. (A–B) The Clark score (A) and the beam-walking (B) was used to evaluate neurological function of mice at 24 hours after autologous blood injection. (C) Quantification of hemorrhagic volume of consecutive brain sections. (D) Changes of mice body weight. Sham group n = 7, ICH + HP-β-CD group n = 6, ICH + TRIOL group n = 5, TRIOL treatment (i.v.) at the dosage of 60 mg/kg. Statistics were performed with one-way ANOVA followed by Tukeys post-test. (Means ± S.D.). (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 2

TRIOL ameliorates ICH injuries and improves recovery of neurological function caused by collagenase injection. (A) The flow chart of experimental design. (B) Analysis of the effect of TRIOL on neurological function of ICH mice by the Clark scale and (C) motor ability by the rope grip test. (Sham, n = 12; ICH + HP-β-CD, n = 15; ICH + TRIOL, n = 13). (D–G) Representative consecutive SWI (D) or T2 MRIs (F) on day 1 after ICH. The SWI (E) or T2 (G) lesion volume was calculated. (n = 3). (H–I) HE staining showed the injury area of brain 3 days after ICH. (n = 4). (J) Mice body weight changes after ICH. (Sham, n = 12; ICH + HP-β-CD, n = 15; ICH + TRIOL, n = 13) Statistics were performed with (B–C, E-I) one-way ANOVA and (J) two-way ANOVA. Data are shown as means ± S.D. (B–C, E-I) and means ± S.E.M. in (J). (n.s. = no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 3

TRIOL significantly reduces hematoma volume, Hb content and prevents the BBB disruption in mice brain after ICH caused by collagenase injection. (A–D) Hematoma volume analysis on day 1 (A–B) or 7 (C–D) after ICH surgery. (day 1: each group n = 4, day 7: each group n = 6). (E–G) Hb content after 1 day of ICH by ELISA (E) or 3 days after ICH using immunoblotting (F–G). (H) Evans blue dye extravasation assessed BBB integrity 1 day following ICH. (n = 6) (I) Analysis of the effect of TRIOL on brain water content in mice 3 days after ICH. (Sham, n = 6; ICH + HP-β-CD, n = 8; ICH + TRIOL, n = 7). Statistics were performed with one-way ANOVA followed by Tukey's post-test. (Means ± S.D.). (n.s. = no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 4

TRIOL promotes the erythrophagocytosis ability of microglia. (A–B) TRIOL increased the protein contents of CD36 by immunoblotting. (C) Representative images showed the expression of CD36 and the engulfed RBCs on BV2 cells by immunofluorescence. (D) Quantification analysis of the mean fluorescence intensity (MFI) of CD36 in (C). (E) Erythrophagocytosis index showed the percentage number of RBC-BV2 cells. (F) Representative images showed TRIOL promoted BV2 erythrophagocytosis by flow cytometry. (G) Quantification analysis of CD11b⁺ and FITC⁺ BV2 cells. BV2 cultures were pretreated with 10, 20 and 40 μM TRIOL for 1 h and then were incubated for 12 h with CFSE-stained RBCs. (H) Immunofluorescence analysis of the effect of TRIOL on microglial CD36 in the perihematomal area of brain after 3 days of ICH induced by collagenase injection. (n = 4, above scale bar = 20 μm, below scale bar = 50μm). (I) The intensity of the red fluorescent signal of CD36 co-localized with the green IBA-1 fluorescent signal from (H) was counted using Image pro plus. Statistics were performed with one-way ANOVA followed by Tukey's post-test. (Means ± S.D.). (n.s. = no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 5

TRIOL selectively upregulates microglial CD163/HO-1 (A–D) Immunoblotting analyzed the expression level of primary microglia CD163, CD91 and HO-1 at 24 h after treatment with the different dose of TRIOL. (E–F) Immunofluorescence analysis of the engulfed Hb on BV2 cells treatment with TRIOL. (G–H) Immunoblotting analyzed the protein content of BV2 cells ferritin at 24 h after treatment with the different dose of TRIOL. (I–K) Immunoblotting detected the effect of TRIOL on CD163 and CD91 content after 3 days of ICH induced by collagenase injection. n = 3, respectively. (L–O) Immunofluorescence analysis of the effect of TRIOL on the expression of CD163 in microglia (L–M) and neurons (N–O) at 3 days post-ICH. (n = 3, above scale bar = 10 μm, below scale bar = 50 μm). The intensity of the red CD163 fluorescent signal co-localized with the green NeuN or IBA-1 fluorescent signal was counted using Image pro plus. Statistics were performed with one-way ANOVA followed by Tukey's post-test. (Means ± S.D.). (n.s. = no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 6

TRIOL inhibited microglial activation and neuroinflammation. (A) LDH release assay showed the effect of TRIOL on BV2 cells injured by Hb. (B) Representative phase-contrast morphological images of TRIOL on BV2 cells induced by Hb. After the pre-incubation of 40 μM TRIOL for 1 h, the cells were subjected to 10 μM Hb for 24 h (Scale bar = 25 μm). (C) Number of activated microglia were counted. (D–F) The mRNA expression of inflammatory cytokines IL-1β, TNF-α, and IL-6 was detected using qPT-PCR. (G) The concentrations of IL-1β, TNF-α, and IL-6 in hemorrhagic hemisphere at 3 day following ICH caused by collagenase injection were analyzed by ELISA. Statistics were performed with one-way ANOVA followed by Tukey's post-test. (Means ± S.D.). (n.s. = no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 7

TRIOL selectively inhibits the increased HO-2 on neurons after ICH. Immunoblotting analyzed the expression level of neuronal HO-1 and HO-2 at 24 h after treatment with (A–C) the different dose of TRIOL, and at different time points (6,12,24h) after treatment with hemin (D–F). (G–H) Immunofluorescence analysis of HO-2 in neuron under hemin stimulation. (Scale bar = 10 μm). Immunoblotting analysis showed HO-1 and HO-2 protein changes in the brain tissue on day 1 (I–J) or 3 (K–L) after ICH induced by collagenase injection. (M − N) Immunofluorescence analysis of neuronal HO-2 in cortex on 3 days after ICH. (O–P) Immunohistochemical analysis displayed the protein level of HO-2 in cortex and perihematoma on 3 days after ICH. (Above scale bar = 10 μm, below scale bar = 50 μm) Statistics were performed with one-way ANOVA followed by Tukey's post-test. (means ± S.D.). (n.s. = no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 8

TRIOL rescues neuronal iron accumulation and reduces MDA level. (A–B) Iron probe detection of Fe²⁺ in (A) primary cortical neurons pre-incubated of 40 μM TRIOL for 1 h and induced by 10 μM hemin for 24 h, and (B) hemorrhagic hemisphere brain tissue after 3 days of ICH caused by collagenase injection. (C) Perl's staining showed iron ions deposition in the perihematomal brain tissue after 3 days of ICH caused by collagenase injection. (Scale bar = 15 μm) (D) Statistical diagram of Perl's stain. Immunoblotting analysis Iron-related proteins ferritin, DMT1 and TfR in (E–H) the primary cortical neurons stimulated by hemin for 24 h and (I–L) the hemorrhagic brain of mice 3 days after ICH caused by collagenase injection. Colorimetric detection of MDA-thiobarbituric acid adduct in (M) the hemin induced neurons and (N) the hemorrhagic brain 3 days after ICH caused by collagenase injection. Statistics were performed with one-way ANOVA followed by Tukey's post-test. (means ± S.D.). (n.s. = no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 9

TRIOL alleviated neuronal injuries after ICH. (A) LDH release assay shows the effect of TRIOL on neurons injured by hemin. (B–C) Representative PI staining images of TRIOL on neurons injured by hemin. After the pre-incubation of 40 μM TRIOL for 1 h the cells were subjected to 10 μM hemin for 24 h (Scale bar = 25 μm). (D–E) Representative images showed Nissl-stained brain section of ICH mice caused by collagenase injection. Red arrows denoted damaged neurons. (n = 4, scale bar = 20 μm) (F–G) Representative image of FJC staining. Partial denatured neurons marked by red arrows. (n = 3, scale bar = 100 μm) Statistics were performed with one-way ANOVA followed by Tukey's post-test. (Means ± S.D.). (n.s. = no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).