Studies On The Neuroprotective Effects And Mechanism Of Aceglutamide And Guhong Injection On Cerebral Ischemia-Reperfusion Injury

Cerebral ischemia is a leading global cause of death and adult permanent disability in the world. Ischemic survivors suffer from long-term disability and severe morbidity due to motor deficits. Currently, one of the clinical treatments for the ischemic stroke is the use of thrombolysis drugs in combination with the neuroprotective drugs. The main problem is how to protect reperfusion injury after thrombolysis treatment. So far, there is no effective and safe drug for the treatment of cerebral ischemia rcperfusion injury.In china, Aceglutamide injection exhibited the efficacy in improving neurological functions in clinical practice. There are also several clinical evidences confirmed the protective effect of Aceglutamide against cerebral ischemia in China. However, the underlying mechanism was unclear. Therefore, the objective of present study was to investigate the neuroprotective effect and mechanism of Aceglutamide on the cerebral ischemia-reperfusion injury in rats.Guhong injection is a compound containing safflower extract and aceglutamide. Clinical trials have demonstrated that its great clinical value in ameliorateing the neurological function in patients with cerebral infarction. However the related mechanism is unclear. The main purpose of this study is to reveal the mechanism of Guhong injection using the gene chip technology.Part Ⅰ:Study on the neuroprotective effects of aceglutamide on cerebral ischemia-reperfusion injury1. In MCAO rats, body weight loss was witnessed for all the MCAO animals (P<0.05) and neurological deficit scores were significantly increased (P<0.001) with the total time of bilateral forelimb completely removed the sticker significantly longer (P<0.001).Treatment with Aceglutamide signicantly increased the body weight, reduced neurological deficit score and shortened the time of removal-test (P<0.05).2. Bean-walking test was performed at 14 d after treatment with Aceglutamide.The passing time of model group was significantly longer than that sham group(P<0.01).Treatment with Aceglutamide significantly shortened the time of beam-walking with a dose-dependent manner(P<0.05).3. Results of Y-maze test showed that Aceglutamide significantly reduced the number of mistakes and improved working memory of ischemia-reperfusion treated rats (P<0.05).In the spontaneous movement test, Aceglutamide obviously improved mental disorder induced by cerebral ischemia-reperfusion.4. Results of TTC staining showed that infarct volume in model group was significantly increased (P<0.01).Treatment with medium and high dose of Aceglutamide significantly decreased the infarct volume (P<0.05).Part Ⅱ:Study on the mechanisms of Aceglutamide on cerebral ischemia-reperfusion injury1. Immunohistochemistry analsis indicated that MCAO decreased the number of TH positive cells and IOD in striatum area (P<0.01), and treatment with Aceglutamide obviously reversed the changes induced by MCAO injured (P<0.01).The results indicated that Aceglutamide significantly improved the secondary injury of the substantia nigra induced by cerebral ischemia-reperfusion.2. Ibal immunohistochemical analysis showed that the number of Iba-1-positive cells significantly increased in model group (P<0.01) in substantia nigra region, with activated microglia morphology.Aceglutamide significantly reduced the number of Iba-1 positive cells compare with the model group (P<0.01).3. TUNEL staining in the substantia nigra showed that, the ratio of TUNEL-positive cells significantly increased in the substantia nigra region of the model rats compared with sham-operated group (P<0.01) and after Aceglutamide treatment, middle and high dose group significantly redced the the ratio of TUNEL-positive cells compared with model group(P<0.01).4. The western-blotting results demonstrated that pre-apoptosis inducing factor TRAF1 expression was increased but P-Akt was decreased in mesencephalon of model group at 14 th day after ischemia reperfusion (P<0.05). Meanwhile, Aceglutamide treatment significantly decreased TRAF1 and elevated the phosphorylation level of Akt in the mesencephalon tissue (P<0.05). Aceglutamide treatment dose-dependently increased the Bcl-2/Bax level in the mesencephalon tissue (P<0.05).5. CD31 immunostaining and the expression of VEGF in cortex showed that, the number of microvessels significantly increased in model rats compared with sham group (P<0.05), with disorder and irregular morphology.Treatment with aceglutamide significantly increased the microvessels density compared with the model group (P<0.05).Meanwhile, the expression of VEGF significantly decreased compared with the sham group, and aceglutamide treatment elevated the expression of VEGF level compared with the model group (P<0.05).6. Nestin staining in SVZ area showed that the Nestin expression in model group cortex was significantly increased compared with sham group, while the Nestin expression in Aceglutamide group was also significantly increased compared with the model group, and there is a trend of migration to the stratum.7.1μM and10 μM Aceglutamide exhibited the neuroprotection against the damage induced by the Na2S2O4 in PC12 cells. The results showed that Aceglutamide potentiated cellular viability than model group (P<0.05).10μM Aceglutamide obviously inhibited PC12 cell apoptosis induced by Na2S2O4.8. Na2S2O4 resulted in a significant decline in the mitochondrial membrane potential (MMP) in the PC 12 cells and cultured mesencephalic neurons, and Aceglutamide reversed the above changes (P<0.05).9. In PC 12 cells, the TRAF1 expression was up-regulated in time-dependent manner after Na2S2O4 treatment.10μM Aceglutamide significantly reduced the expression level of TRAF1 evoked by Na2S2CM (P<0.05) and increased the expression level of P-Akt (P<0.01) and Bcl-2/Bax compared with model group (P<0.05).10. The expression of TRAF1 significantly increased after exposed to Na2S2O4 in PC12 cells.10 μM Aceglutamide-treatment obviously weakened the fluorescence intensity of TRAF1 under the identical conditions.11. Na2S2O4 exposure significantly decreased the level of intracellular glutathione (GSH) compared with the control group (P<0.01). While treatment with 10μM Aceglutamide elevated the level of glutathione (P<0.05).Part Ⅲ:Study on the effect and mechanisms of Guhong injection on cerebral ischemia-reperfusion injury using microarray analysis1. Guhong injection significantly improved motor dysfunction in MCAO rats and elevated the TH-positive cells in substantia nigra.2. Using cDNA microarray analysis,1303 genes were found to be differentially expressed in model group compared with sham group, where 985 genes were upregulated and 318 genes were down-regulated. Treatment with Guhong injection, 812 genes was influenced by Guhong treatment with 234 genes up-regulated,578 down-regulated.3. Gene set enrichment analysis showed that these genes were involved in various biological processes, such as receptor activity, transporters, cell growth, protein metabolism, systemic development, transmembrane receptor and cytoskeletal protein.4. Seven genes differentially expressed in gene chip were validated by real-time PCR. The results showed that the expression trend of these genes were in line with the results of gene chip.5. By functional analysis of differentially expressed genes and relevant literature, we selected 26 genes involved in the molecular mechanisms of cerebral ischemia reperfusion injury as a candidate verification gene.