ABSTRACT
Objective To investigate synergistic effects of extracellular signal regulated kinase (ERK1/2) and phosphatidylinositol 3 kinase (PI3-K) pathway inhibitors on autophagy in the hippocampus of rats with subarachnoid hemorrhage (SAH),for identification of therapeutic targets in SAH.Methods Totally,200 male SD rats were randomly divided into a sham operated group,SAH group,inhibitor U0126 group,inhibitor LY294002 group,and a U0126+ LY294002 group.Animal models were established by injecting autologous blood twice into the cisterna magna.Morphological changes in the hippocampus nerve cells were detected by HE staining;ERK1/2,PI3-K,beclin-1,and LC3 mRNA expression in the hippocampus were detected by real-time PCR,and phosphorylated ERK 1/2,PI3-K,beclin-1,and LC3-Ⅱ protein expression were detected by immunohistochemistry.Results Neuronal death rate and phosphorylated ERK1/2,PI3-K,beclin-1,and LC3-Ⅱ levels in the hippocampus in the SAH group were higher than in the sham group (all P ＜ 0.05).Neuronal death rate in U0126 or LY294002 group was higher than in SAH group,while ERK1/2,PI3-K,beclin-1,and LC3 mRNA and phosphorylated ERK 1/2,PI3-K,beclin-1,and LC3-Ⅱ protein levels were lower than in SAH group (all P ＜ 0.05).Neuronal death rate in U0126 +LY294002 group was higher than in U0126 or LY294002 group,while ERK1/2,PI3-K,beclin-1,LC3 mRNA and phosphorylated ERK 1/2,PI3-K,beclin-1,and LC3-Ⅱ protein levels in the hippocampus were lower than in U0126 or LY294002 group (all P ＜ 0.05).Conclusion Co-targeting the inhibition of ERK 1/2 and PI3-K pathways can significantly reduce neuronal cell autophagy and aggravate cells loss after SAH.
ABSTRACT
Objective Moderate autophagy helps improve the viability of neurocytes.This study aims to investigate the effect of SP600125 on the autophagy and loss of nerve cells in the hippocampus in rats with subarachnoid hemorrhage (SHA).Methods Forty healthy male SD rats were equally randomized into a sham operation, an DMSO group, an SAH model, and an SP600125 group.The SAH model was established by vascular puncture and the rats of the SP600125 group were injected with 10 μL of SP600125 (3 μg/μL) into the lateral cerebral ventricle at 30 minutes before modeling.Sham group and SAH group were injected with equal volume of normal saline, DMSO group was injected with the same amount of DMSO.The animals were sacrificed at 24 hours after modeling for observation of the changes in the morphology and the number of neurons in the hippocampus by HE staining and qualitative and quantitative determination of the expressions of the p-JNK protein and the autophagy markers beclin-1 and LC3-II by immunohistochemistry and Western blot.Results Compared with the sham operation group, the neurons exhibited a disordered arrangement and the cells were polygonal and decreased in number in the hippocampus of the SAH models, while milder neuronal injury and more cells were observed in the rats of the SP600125 group than in the SAH models.The mean optical density values of Beclin-1, LC3-II and p-JNK in the hippocampus were significantly higher in the SAH models (14.66±4.40, 12.62±3.46, and 12.82±3.68) and DMSO (13.85±3.85、11.59±4.52、13.03±3.53), and the SP600125 group (9.86±3.14, 6.78±2.56, and 5.60±2.42) than in the sham operation group (1.56±0.28, 1.60±0.30, and 1.58±0.32) (P<0.05), but markedly lower in the SP600125 than in the SAH model group (P<0.05).The expressions of Beclin-1, LC3-II and p-JNK were remarkably increased in the SAH models (0.474±0.122, 0.668±0.130, and 0.496±0.124) and DMSO (0.432±0.102、0.628±0.113、0.416±0.094) and the SP600125 group (0.264±0.106, 0.332±0.113, and 0.219±0.104) than in the sham operation group (1.56±0.28, 1.60±0.30, and 1.58±0.32) (P<0.05), but significantly decreased in the SP600125 group as compared with the SAH models (P<0.05).Conclusion SP600125 has a protective effect on the neurocytes in the hippocampus of SAH rats, which may be associated with SP600125 moderately activating neuronal autophagy by inhibiting the activity of the JNK signaling pathway.
ABSTRACT
Objective To investigate the therapeutic effect of edaravone(Ed)combined with rapamycin (RAP)on neural injury in rats with subarachnoid hemorrhage(SAH),and to provide the evidence for clinical medication. Methods 100 male Sprague-Dawley rats were randomly divided into the following 5 groups:sham operated(Sham)group,SAH group,RAP group,Ed group and RAP+Ed group. A rat model of SAH was estab-lished by using the 2 injection of the classic pillow.Learning and memory function assessment was performed by us-ing Morris water maze. The morphological changes of neurons in hippocampus were observed by using optical mi-croscopy. The MDA content was measured by the thiobarbituric acid method. The expressions of autophagy marker proteins(Beclin-1 and LC3-Ⅱ)were detected by immunohistochemistry assay.Results The number of dead neu-rons,MDA content,the expressions of Beclin-1 and LC3-Ⅱwere higher in SAH group than those in Sham group, but learning and memory function index was worse than that in Sham group(P<0.05).The number of dead neu-rons,MDA content in RAP or Ed group were lower than those in SAH group(P < 0.05,respectively),while the expressions of Beclin-1 and LC3-Ⅱwere higher and learning and memory function index was better in RAP or Ed group than those in SAH group(P<0.05,respectively).The number of dead neurons,MDA content in RAP+Ed group were lower than those in RAP or Ed group(P < 0.05,respectively),but the expressions of Beclin-1 and LC3-Ⅱwere higher and learning and memory function index was better in RAP+Ed group than those in RAP or Ed group(P < 0.05,respectively). Conclusions Edaravone combined with RAP had better therapeutic effect on neural injury in rats with SAH,which was associated with enhancing the autophagy of neural cells.