Albumin Reduces Oxidative Stress and Neuronal Apoptosis via the ERK/Nrf2/HO-1 Pathway after Intracerebral Hemorrhage in Rats

Abstract

Background: Albumin has been regarded as a potent antioxidant with free radical scavenging activities. Oxidative stress and neuronal apoptosis are responsible for its highly damaging effects on brain injury after intracerebral hemorrhage (ICH). Here, the present study investigated the neuroprotective effect of albumin against early brain injury after ICH and the potential underlying mechanisms.

Methods: Adult male Sprague-Dawley rats were subjected to intrastriatal injection of autologous blood to induce ICH. Human serum albumin was given by intravenous injection 1 h after ICH. U0126, an inhibitor of extracellular signal-regulated kinase (ERK1/2), and ML385, an inhibitor of nuclear factor-E2-related factor 2 (Nrf2), were intraperitoneally administered 1 h before ICH induction. Short- and long-term neurobehavioral tests, western blotting, immunofluorescence staining, oxidative stress evaluations, and apoptosis measurements were performed.

Results: Endogenous expression of albumin (peaked at 5 days) and heme oxygenase 1 (HO-1, peaked at 24 h) was increased after ICH compared with the sham group. Albumin and HO-1 were colocalized with neurons. Compared with vehicle, albumin treatment significantly improved short- and long-term neurobehavioral deficits and reduced oxidative stress and neuronal death at 72 h after ICH. Moreover, albumin treatment significantly promoted the phosphorylation of ERK1/2; increased the expression of Nrf2, HO-1, and Bcl-2; and downregulated the expression of Romo1 and Bax. U0126 and ML385 abolished the treatment effects of albumin on behavior and protein levels after ICH.

Conclusions: Albumin attenuated oxidative stress-related neuronal death may in part via the ERK/Nrf2/HO-1 signaling pathway after ICH in rats. Our study suggests that albumin may be a novel therapeutic method to ameliorate brain injury after ICH.

Figure 1

Experimental design and animal groups. ICH: intracerebral hemorrhage; WB: western blot; IF: immunofluorescence; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; FJC: fluoro-Jade C staining; 8-OHdG: 8-hydroxy-2-deoxyguanosine; MDA: malondialdehyde; SOD: superoxide dismutase; DMSO: dimethyl sulfoxide; i.v.: intravenous injection; i.p.: intraperitoneal injection. ^∗Extra 2 rats were used for IF (albumin and HO-1 with neurons) at 72 h after ICH. ^∗∗Assumed the optimal dose group.

Figure 2

Time course of albumin and HO-1 expression as well as cellular localization of neurons after ICH. (a) Representative WB bands and (b, c) quantitative analysis of albumin and HO-1 expression after ICH; ^∗p < 0.05 vs. sham, mean ± SD, one-way ANOVA, Tukey′s test, n = 6/group. (d) Brain sample with schematic illustration showing one area in the perihematomal region and the small white square within the coronal section of the brain indicates the location of where the immunofluorescence staining images were taken. (e, f) Representative microphotographs of coimmunofluorescence staining of albumin (green) with neurons (NeuN, red), as well as HO-1 (green) with neurons (NeuN, red) in the ipsilateral perihematomal region at 72 h after ICH (n = 2/group), nuclei were stained with DAPI (blue), scale bar = 50 μm, HO-1: heme oxygenase 1; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 3

Effects of albumin on short-term neurobehavioral outcome and neuronal damage at 72 h after ICH. (a) Albumin improved short-term neurological function (modified Garcia test, left forelimb placement test, and corner turn test) at 24 h and 72 h after ICH (n = 6/group). Short-term neurological results showed that albumin treatment at the medium-dosage of 1.25 g/kg improved short-term neurological function significantly compared to the vehicle and low and high dosage groups. Therefore, we further chose the middle dosage for further study. (b) Representative micrographs of FJC-positive cells within the ipsilateral perihematomal region at 72 h after ICH. (c) Representative micrographs of TUNEL-positive neurons within the ipsilateral perihematomal region at 72 h after ICH. (d) Brain sample with schematic illustration showing one areas in the perihematomal region and the small white square in the coronal section of the brain indicates the area used for counting FJC and TUNEL-positive neurons. (e, f) Quantitative analyses of TUNEL and FJC-positive cells in the perihematomal area at 72 h after ICH (n = 4/group); scale bar = 50 μm, nuclei were stained with DAPI (blue). ^∗p < 0.05 vs. sham, ^@p < 0.05 vs. ICH+vehicle, mean ± SD, one-way ANOVA, Tukey's test, TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; FJC: Fluoro-Jade C staining.

Figure 4

Effects of albumin on oxidative stress levels at 72 h after ICH. (a) Representative micrographs of 8-OHdG (green) immunofluorescence staining in the ipsilateral perihematomal region at 72 h after ICH. Nuclei were stained with DAPI (blue). (b) Brain sample with schematic illustration showing one areas in the perihematomal region and the small white square in the coronal section of the brain indicates the area used for counting 8-OHdG-positive cells. (c) Quantitative analysis of 8-OHdG (green) immunofluorescence staining in the ipsilateral perihematomal region at 72 h after ICH (n = 4/group). (d) Superoxide dismutase (SOD) and malondialdehyde (MDA) levels (n = 6/group). ^∗p < 0.05 vs. sham, ^@p < 0.05 vs. ICH+vehicle, mean ± SD, one-way ANOVA, Tukey′s test. 8-OHdG: 8-hydroxy-2-deoxyguanosine; SOD: superoxide dismutase; MDA: malondialdehyde.

Figure 5

Albumin (1.25 g/kg, i.v.) treatment improved long-term motor and memory function after ICH. (a–d) Water maze tests performed from days 22-27 after ICH, (e) foot fault test, and (f) rotarod test performed at days 7, 14, and 21 after ICH. ^∗p < 0.05 vs. sham, #p < 0.05 vs. ICH+vehicle, mean ± SD, two-way repeated measures ANOVA (a, b), one-way ANOVA for (d–f), Tukey′s test, n = 8/group.

Figure 6

Blockade of ERK and Nrf2 reversed the effects of albumin on short-term neurobehavioral outcome and oxidative stress-induced neuronal death at 72 h after ICH. (a) Modified Garcia test, left forelimb placement test, and corner turn test. (b) Representative western blotting images. (c–h) Quantitative analyses of p-ERK/ERK, Nrf2, HO-1, Romo1, Bax, and Bcl-2 expression. ^∗p < 0.05 vs. sham, ^@p < 0.05 vs. ICH+vehicle, ^&p < 0.05 vs. ICH+albumin, ^#p < 0.05 vs. ICH+albumin+DMSO; mean ± SD, one-way ANOVA, Tukey's test, n = 6/group. p-ERK1/2: phosphorylated extracellular regulated protein kinases 1/2; ERK1/2: extracellular regulated protein kinases1/2; Nrf2: nuclear factor-E2-related factor 2; HO-1: heme oxygenase 1; Romo1: reactive oxygen species modulator 1; Bax: B-cell lymphoma-2 associated X protein; Bcl-2: B-cell lymphoma-extra large 2.