Aucubin alleviates oxidative stress and inflammation via Nrf2-mediated signaling activity in experimental traumatic brain injury

Abstract

Background: Aucubin (Au), an iridoid glycoside from natural plants, has antioxidative and anti-inflammatory bioactivities; however, its effects on a traumatic brain injury (TBI) model remain unknown. We explored the potential role of Au in an H₂O₂-induced oxidant damage in primary cortical neurons and weight-drop induced-TBI in a mouse model.

Methods: In vitro experiments, the various concentrations of Au (50 μg/ml, 100 μg/ml, or 200 μg/ml) were added in culture medium at 0 h and 6 h after neurons stimulated by H₂O₂ (100 μM). After exposed for 12 h, neurons were collected for western blot (WB), immunofluorescence, and M29,79-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. In vivo experiments, Au (20 mg/kg or 40 mg/kg) was administrated intraperitoneally at 30 min, 12 h, 24 h, and 48 h after modeling. Brain water content, neurological deficits, and cognitive functions were measured at specific time, respectively. Cortical tissue around focal trauma was collected for WB, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, Nissl staining, quantitative real time polymerase chain reaction (q-PCR), immunofluorescence/immunohistochemistry, and enzyme linked immunosorbent assay (ELISA) at 72 h after TBI. RNA interference experiments were performed to determine the effects of nuclear factor erythroid-2 related factor 2 (Nrf2) on TBI mice with Au (40 mg/kg) treatment. Mice were intracerebroventricularly administrated with lentivirus at 72 h before TBI establishment. The cortex was obtained at 72 h after TBI and used for WB and q-PCR.

Results: Au enhanced the translocation of Nrf2 into the nucleus, activated antioxidant enzymes, suppressed excessive generation of reactive oxygen species (ROS), and reduced cell apoptosis both in vitro and vivo experiments. In the mice model of TBI, Au markedly attenuated brain edema, histological damages, and improved neurological and cognitive deficits. Au significantly suppressed high mobility group box 1 (HMGB1)-mediated aseptic inflammation. Nrf2 knockdown in TBI mice blunted the antioxidant and anti-inflammatory neuroprotective effects of the Au.

Conclusions: Taken together, our data suggest that Au provides a neuroprotective effect in TBI mice model by inhibiting oxidative stress and inflammatory responses; the mechanisms involve triggering Nrf2-induced antioxidant system.

Keywords: Aubucin; Inflammation; Nuclear factor erythroid-2 related factor 2 (Nrf2); Oxidative stress; Traumatic brain injury (TBI).

Fig. 1

Effects of Au on the primary neurons exposed to 100 μM H₂O₂. a, b Representative western blot bands (a) and quantification of relative protein expression (b) for nuclear Nrf2, NQO-1, HO-1, Bcl2, Bax, and CC3. c, d Representative micrographs (c) and quantification (d) of DCFH-DA staining of Control group, H₂O₂ group, and H₂O₂ + Au (200 μg/ml) group in primary neurons. e Representative image of immunofluorescence staining of Control group, H₂O₂ group, and H₂O₂ + Au (200 μg/ml) group. Bars represent the mean ± SD. ^#P < 0.05 versus Control group; *P < 0.05 versus H₂O₂ group. Scale bars = 50 μm (n = 3 for each group)

Fig. 2

Au treatment reduced brain edema and improved neurologic functions. a, b Effects of TBI and Au on water content (a) and neurologic deficits (b). c, d Representative western blot bands (c) and quantification of relative protein expression (d) for MMP-9. e Effects of Au on the latency to fall in the rotarod test were analyzed using two-way ANOVA. The Bonferroni post hoc test was used to compare differences among several groups at 14 days post-TBI. f Typical swimming path of mice in all groups during the training days (upper) and the probe trial period (lower) of the MWM test. i Effects of Au on the latency to find platform in the MWM test. The two-way ANOVA was performed, and the Bonferroni post hoc test was utilized to compare differences among several groups on day 28. j, k Au did not affect the average swimming speed during the training days (j) or during the probe trial period (k). l Time spent in the correct quadrant during the probe trail. m Number of crossings over the platform position during the probe trail. Bars represent the mean ± SD. *P < 0.05 versus indicated groups

Fig. 3

Au decreased the neural apoptosis and neuronal loss caused by TBI. a, b Representative WB bands (a) and quantification of relative protein expression (b) for Bcl2, Bax, and CC3. c–f Representative photomicrographs and quantification of TUNEL staining (c, e Scale bars = 20 μm) and Nissl staining (d, f Scale bars = 50 μm) in cortex of the traumatized side. The red arrow indicated damaged neurons. g Diagram of mouse brain section showing the location of lesion cavity (red area) and photograph region (red squares). Bars represent the mean ± SD. *P < 0.05 versus indicated groups (n = 6 for each group)

Fig. 4

Au attenuates oxidative stress in TBI mice. a–d The ELISA analysis of MDA (a), SOD (b), GSH (c), and GSH-Px (d) in serum (upper) and brain tissue (lower) among four groups. e–g WB bands (e) and quantification of relative protein expression (f, g) for GPx1 and SOD1. i, j Representative immunohistochemical images and quantitative analysis of oxidative stress markers 8-OHdG. Bars represent the mean ± SD. *P < 0.05 versus indicated groups. Scale bars = 50 μm (n = 6 for each group)

Fig. 5

Effects of Au on protein level of Nrf2 in the perilesional cortex after TBI. a–d Representative WB bands (a) and quantification analysis of nuclear Nrf2 (b), cytoplasmic Nrf2 (c), and total Nrf2 (d). e Typical double immunofluorescence images of NeuN and Nrf2 (scale bars = 50 μm). Bars represent the mean ± SD. *P < 0.05 versus indicated groups (n = 6 for each group)

Fig. 6

Au upregulated the expression of HO-1 and NQO-1. a, b The mRNA levels of HO-1 (a) and NQO-1 (b) in cerebral cortex of the injured side. c, d WB bands (top) and quantitative analysis (bottom) of HO-1 (c) and NQO1 (d). e Representative IHC images of HO-1 (upper) and NQO-1 (lower). Bars represent the mean ± SD. *P < 0.05 versus indicated groups. Scale bars = 50 μm

Fig. 7

Effects of Au on the number of Iba-1+ cells in the perilesional cortex after TBI. a, b Representative photomicrographs (a) and quantitative analysis (b) of Iba-1-positive cells in the perilesional cortex. c, d Representative western blot bands (b) and quantification analysis of Iba-1 (c). Bars represent the mean ± SD. *P < 0.05 versus indicated groups. Scale bars = 50 μm (n = 6 for each group)

Fig. 8

Au reduced HMGB1-meditated inflammation after TBI. a–c Representative western blot bands (a) and quantification analysis of HMGB1, TLR4, MyD88 (b), nuclear NF-κB p65, and total NF-κB p65 (c). d, e Representative western blot bands (d) and quantification analyses (e) of iNOS, COX2, and IL-1β. Bars represent the mean ± SD. *P < 0.05 versus indicated groups

Fig. 9

Effects of Nrf2 shRNA delivery and Nrf2 shRNA with Au co-administration in TBI mice. a, b WB bands (a) and quantification (b) of Nrf2 protein in Sham, TBI, TBI + NC, and TBI + LV groups. c The mRNA expression levels of Nrf2 in all groups. d–i Representative western blot bands (d, g) and quantification analysis of nuclear Nrf2, HO-1, Bcl2, Bax, CC3 (e, f), HMGB1, TLR4, MyD88, nuclear NF-κB p65, and COX2 (h, i). Bars represent the mean ± SD. *P < 0.05 versus indicated groups; ns, not significant (n = 6, each group)