Docosahexaenoic Acid Alleviates Oxidative Stress-Based Apoptosis Via Improving Mitochondrial Dynamics in Early Brain Injury After Subarachnoid Hemorrhage

Abstract

Mitochondrial dysfunction is considered a crucial therapeutic target for early brain injury following subarachnoid hemorrhage (SAH). Emerging evidence indicates that docosahexaenoic acid (DHA), an essential omega-3 fatty acid, protects mitochondria in various chronic diseases. This study aimed to investigate the neuroprotective effects of DHA on mitochondrial dynamic dysfunction after EBI using in vivo and in vitro approaches. For in vivo experiments, the rat endovascular perforation SAH model was performed, whereby DHA was administered intravenously 1 h after induction of SAH. Primary cultured neurons treated with oxyhemoglobin (OxyHb) for 24 h were used to mimic SAH in vitro. Our results demonstrated that DHA improved neurological deficits and reduced brain edema in rats with SAH, and attenuated OxyHb-induced neuronal death in primary cultured cells. DHA reduced the amount of reactive oxygen species-positive cells and improved cell viability when compared to the SAH + vehicle group in vitro. DHA attenuated malondialdehyde levels and superoxide dismutase stress, increased Bcl2 and Bcl-xl, and decreased Bax and cleaved caspase-3 in vivo. Additionally, DHA ameliorated mitochondrial dysfunction, upregulated the mitochondrial fusion-related protein Optic Atrophy 1, and downregulated the mitochondrial fission-related protein Dynamin-Related-Protein 1 (Drp1) and Serine 616 phosphorylated Drp1 after SAH both in vitro and in vivo. Taken together, our current study demonstrates that DHA might prevent oxidative stress-based apoptosis after SAH. The characterization of the underlying molecular mechanisms may further improve mitochondrial dynamics-related signaling pathways.

Keywords: Apoptosis; Docosahexaenoic acid; Early brain injury; Mitochondrial dynamics; Mitochondrial dysfunction; Oxidative stress; Subarachnoid hemorrhage.

Fig. 1

DHA reduced brain damage by improving neurological deficits and reducing brain edema. a Schematic representation of Sham and SAH groups. b SAH grades and c mortality rates of all groups. Intravenous administration of DHA (100 μg/kg, 300 μg/kg) significantly improved d modified Garcia score, e beam balance score and f brain edema of the left hemisphere at 24 h after SAH. n = 6 for each group. Bars represent the mean ± SD. ^##p < 0.01, ^#p < 0.05 versus Sham group; **p < 0.01, *p < 0.05 versus SAH + vehicle group. Vehicle, 1% dimethylsulfoxide and sterile saline

Fig. 2

DHA attenuated SAH-induced cell death both in vitro and in vivo. a Quantitative analysis of cell viability among all cell experimental groups. DHA treatment significantly decreased OxyHb-induced cell death at concentrations of 100 μM, 300 μM, 500 μM, and 700 μM. b DHA administration markedly reduced the number of TUNEL-positive neurons at 24 h after SAH. n = 3 for each group. Scale bars = 50 µm. c Representative bands and d quantitative analysis showed the expression of Bcl-2, Bcl-xl, Bax, and cleaved caspase-3 in each group. DHA treatment significantly attenuated mitochondria-related apoptosis 24-h post-SAH. Bars represent the mean ± SD. ^#p < 0.05 versus Control or Sham group; **p < 0.01, *p < 0.05 versus OxyHb or SAH + vehicle group. Vehicle, 1% dimethylsulfoxide and sterile saline

Fig. 3

DHA reduced SAH-induced oxidative stress both in vitro and in vivo. a Representative immunofluorescence images to detect ROS reduction in OxyHb + DHA group compared with the OxyHb group in vitro. Scale bars = 50 µm. b Representative crest and c quantitative analysis of ROS release from each cell. DHA treatment markedly reduced the generation of cell ROS. H₂0₂ was added to one group for Rosup standard. d The level of MDA and e the activity of SOD in each group showed the antioxidant effect of DHA in vivo. n = 6 for each group. Bars represent the mean ± SD. ^##p < 0.01 versus Control or Sham group; **p < 0.01, *p < 0.05 versus OxyHb or SAH + vehicle group. Vehicle, 1% dimethylsulfoxide and sterile saline

Fig. 4

DHA improved mitochondrial morphology after SAH both in vitro and in vivo. a The representative images showed that OxyHb induced extensive mitochondrial fragmentation, which could be reversed by DHA treatment. b Quantification of mitochondrial length, mitochondrial circularity, and mitochondrial area in each in vitro group. n = 3 for each group. Scale bars = 5 µm. Bars represent the mean ± SD. ^#p < 0.01 versus Control group; *p < 0.05 versus OxyHb group. c Representative transmission electron microscopy images indicated that DHA improved the integrity of mitochondrial membrane and cristae, promoted mitochondrial fusion, and further reduced mitochondrial fission 24-h post-SAH. n = 3 for each group. Scale bars = 1 µm. Symbols indicate the following: asterisk = normal mitochondria; arrow = swelling and cristae vague; triangle = mitochondria under fission; square = mitochondria under fusion. Vehicle, 1% dimethylsulfoxide and sterile saline

Fig. 5

Expression of mitochondria dynamics-related proteins in neurons. Double immunofluorescence staining and quantification of a Drp1 (red) and b OPA1 (red) with neurons (NeuN, green). SAH induction increased the expression of Drp1 and decreased OPA1. n = 3 for each group. Scale bars = 50 µm. ^#p < 0.05 versus Sham group. DAPI (blue) was used as a nuclear marker. Vehicle, 1% dimethylsulfoxide and sterile saline

Fig. 6

DHA improved the balance of mitochondrial fission and fusion after SAH in vitro and in vivo. Representative western blot images showing the effect of DHA on the expressions of Drp1, p-Drp1 (Ser616), L-OPA1, and S-OPA1 a in vitro and b in vivo. Quantitative analysis of above proteins among all groups showed that DHA treatment decreased the expression of Drp1 and p-Drp1 (Ser616) and increased the expression of L-OPA1 and S-OPA1 c in vitro and d in vivo. n = 6 for each group. Bars represent the mean ± SD. ^#p < 0.05 versus Control or Sham group; *p < 0.05 versus OxyHb or SAH + vehicle group. Vehicle, 1% dimethylsulfoxide and sterile saline