Oxidized low-density lipoprotein receptor 1: a novel potential therapeutic target for intracerebral hemorrhage

Figures

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  Figure 1. Silencing OLR1 attenuates functional impairment, hematoma expansion, brain water content, and neuron loss in rat brain tissues 24 hours after ICH. After injection with OLR1 siRNA or scramble siRNA for 24 hours, rats were infused with collagenase type VII to induce ICH. (A, B) The mRNA (A) and protein (B) levels of OLR1 by quantitative reverse transcription-polymerase and western blot analysis. (C) mNSS score. A higher score was accompanied with severe deficit. (D) Corner turn score. (E) Brain water content. (F) Representative images of brain sections. Depletion of OLR1 decreased the increased hematoma area in serial brain sections of rats after ICH. (G) Fluro-Jade B-stained cells. The downregulation of OLR1 reverted the ICH-induced neuron loss of brain tissues. White arrows indicate neuron loss. Scale bar: 50 μm. Data in A, B and E are shown as mean ± SD (n = 6) and were analyzed by one-way analysis of variance following by Bonferroni’s post hoc test. Data in C and D are shown as median, interquartile range, maximum and minimum and were analyzed by Kruskal-Wallis test followed by Dunn’s post hoc test. The experiments were repeated 12 times in C and D, and six times in other…

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  Figure 2. Silencing of OLR1 alleviates the inflammation of rat brain tissues 24 hours after ICH. After injection with OLR1 siRNA or scramble siRNA for 24 hours, rats were infused with collagenase type VII to induce ICH. (A–C) Concentrations of IL-1β, IL-6 and TNF-α in the supernatant of rat brain tissues as detected by enzyme-linked immunosorbent assay. (D) Representative immunofluorescence staining to identify MPO-positive cells. The loss of OLR1 reversed the elevated MPO in brain tissues induced by ICH. MPO-positive cells (white arrows) were labeled red with Cy3. Nuclei were labeled blue by DAPI. Scale bar: 50 μm (top three rows), 10 μm (bottom row). Data are shown as mean ± SD and were analyzed by one-way analysis of variance following by Bonferroni’s post hoc test. The experiments were repeated six times. DAPI: 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; ICH: intracerebral hemorrhage; IL: interleukin; MPO: myeloperoxidase; OLR1: oxidized low-density lipoprotein receptor 1; siRNA: small interfering RNA; TNF-α: tumor necrosis factor-α.

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  Figure 3. Silencing of OLR1 mitigates oxidative stress in rat brain tissues after ICH. After injection with OLR1 siRNA or scramble siRNA for 24 hours, rats were infused with collagenase type VII to induce ICH. (A) Representative staining with dihydroethidium (a fluorescent ROS indicator; indicated by white arrows). The loss of OLR1 rescued the increased ROS levels in brain tissues induced by ICH. Scale bar: 50 μm. (B) SOD activity. (C–E) Contents of GSH, MDA, and 4-HNE. Data are shown as mean ± SD (n = 6) and were analyzed by one-way analysis of variance followed by Bonferroni’s post hoc test. The experiments were repeated six times. GSH: Glutathione; ICH: intracerebral hemorrhage; MDA: malondialdehyde; OLR1: oxidized low-density lipoprotein receptor 1; siRNA: small interfering RNA; ROS: reactive oxygen species; SOD: superoxide dismutase; 4-HNE: 4-hydroxynonenal.

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  Figure 4. Silencing of OLR1 suppresses ferroptosis of neurons in rat brain tissues 24 hours after ICH. After injection with OLR1 siRNA or scramble siRNA for 24 hours, rats were infused with collagenase type VII to induce ICH. (A) Protein expression of GPX4, FTH1, and COX-2. Protein expression was normalized by levels in the sham group. (B) Representative immunofluorescence staining with NeuN and GPX4. Knockdown of OLR1 reversed the ICH-induced decrease of GPX4 levels in NeuN-positive cells. NeuN-positive cells were labeled by FITC (green), and GPX-4-positive cells were labeled by Cy3 (red). White arrows indicate the NeuN-positive cells with GPX4 expression. Scale bar: 50 μm (upper three rows), 10 μm (lower row). Data are shown as mean ± SD (n= 6) and are analyzed by one-way analysis of variance followed by Bonferroni’s post hoc test. COX-2: Cyclooxygenase-2; FITC: fluorescein Isothiocyanate; FTH1: ferritin heavy polypeptide 1; GPX4: glutathione peroxidase 4; ICH: intracerebral hemorrhage; OLR1: oxidized low-density lipoprotein receptor 1; siRNA: small interfering RNA.

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  Figure 5. Silencing of OLR1 represses the p38 signaling pathway 24 hours after ICH. The protein levels of p-p38 and p38 were evaluated in brain tissues of rats injected with OLR1 siRNA or scramble siRNA, followed by infusion with collagenase type VII to induce ICH. Data are shown as mean ± SD (n = 6) and are analyzed by one-way analysis of variance following by Bonferroni’s post hoc test. The experiments were repeated six times. ICH: Intracerebral hemorrhage; OLR1: oxidized low-density lipoprotein receptor 1; siRNA: small interfering RNA.