| Background and purpose Many factors are involved in cerebral ischemia-reperfusion injury, among them inflammatory reaction and free radicals play an important role in early stage. Edaravone, a kind of free radical scavenger, has been used to treat cerebral infarction and verified effective, while the precise mechanisms are unclear. At present, there is no an effective medicine on restrain inflammatory reaction. The proteasome inhibitors can induce apoptosis on proliferating tumor cells and are permitted to use for the multiple myeloma. Recent research shows that the proteasome inhibitors can control inflammatory reaction on transcriptional level and are used in treating cerebral infarction. Now the mechanism is unclear.The study plans to apply the animal model of cerebral ischemia-reperfusion to observe the changes of inflammatory reaction, free radicals, mitochondria and cell apoptosis, and to treat with Edaravone and Velcade on the proper time. Comparing the changes pre- and post–treatment and observing the effects on the cerebral ischemia-reperfusion injury, the aims is to explore the protective mechanisms of Edaravone and Velcade on cerebral reperfusion inury.Methods1. Experimental animal and group 175 Wistar rats were randomly divided into the normal group(5), the sham operation group and cerebral ischemical reperfusion group (55 in each group), the physiological saline control group, Edaravone treatment group, Velcade treatment group and combined treatment group (15 in each group). The transient middle cerebral artery occlusion models were applied and reperfused after 2 hours. All the groups were divided into 5 subgroups according to different reperfusion time points, 0h, 6h, 12h, 24h, 48h (10 in each group). The others were used to measure infarct volume with TTC stain.2. Decapitation and detection methods Edaravone was given instantly and on 12h (3mg/kg), and Velcade was given instantly too (0.2mg/kg) after reperfusion via vena caudalis injection. Combined treatment method was given according to the above all. The physiological saline was used in control group. After 24h 5 rats were infused , decapitated and fixed with 4% paraformaldehyde after reperfusion 24h, alcoholic dehydration, dimethyl benzene transparence, paraffin imbedding and sect serial sections. Immunohistochemistry was used to detect the expression of Cyt C, Caspase-3, NF-κBp65 and IL-1βprotein, while cell apoptosis was detected by TUNEL. At the same time, the expression of Cyt C and Caspase-3 protein were detected by Western-blot. Another 5 rats were decapitated on ice plate immediately. The 2/3 brain tissue was used to make brain homogenate for detecting MDA and SOD, the others were preserved in -70℃refrigerator for Western-blot. After TTC stain, the images were measured and the cerebral infarct volume were calculated with Motic Images Advanced 3.2 image analysis system.Results1. Histological changes: The chromatospherites was clear, endochylema was profuse in normal group and sham operation group, while in reperfusion group karyopyknosis,工团chromoplasm condense. The interspace between cells became wide. brain tissue became loose. These changes were gradually obvious with time .2. There were seldom immune reaction positive cells and apoptosis cells in normal group and sham operation group. The difference between MDA and SOD was not significant (p>0.05) in them. MDA increased gradually after reperfusion and peaked at 12h, while SOD decreased obviously at that time. Compared with the physiological saline control group , the combined treatment group and single treatment groups MDA decreased and SOD activity restored, the differences were significant (p<0.05). There were lots of NF-κBp65, IL-1β, CytC and Caspase-3 positive cells in all the intervention groups. All kinds of positive cells increased gradually until 24h, compared with all the subgroups of the sham operation group, the differences were significant (p<0.05). Compared the single treatment group with the physiological saline control group and combined treatment group with all the other groups, NF-κBp65, IL-1β, CytC and Caspase-3 positive cells and apoptosis cells decreased obviously, the differences were significant (p<0.05). The expression of Cyt C, Caspase-3 by Western-blot in different time points and pre-and post-treatment were consistent with the results of immunohistochemistry methods.3. Cerebral infarct was found in all the operation groups except the sham operated group, and the infarct volumes with TTC stain in treatment group became smaller than the physiological saline group (p<0.05).Conclusion1. The change of inflammatory reaction, free radicals and mitochondria damage involved in cerebral ischemical reperfusion injury were dynamic . Cell apoptosis was coincident to the above changes.2. Edaravone can inhibit lipid peroxidation, scavenge free radicals, restrain inflammatory reaction, protect mitochondria and reduce cells apoptosis. So Edaravone maybe mitigate cerebral ischemical reperfusion injury through blocking mitochondria apoptosis pathway. On the other hand, Velcade can restrain inflammatory reaction and free radicals following that which can protect mitochondria and reduce cells apoptosis. So we assumed that Velcade mitigate cerebral ischemical reperfusion injury by blocking mitochondria apoptosis pathway too.3. Edaravone can scavenge free radicals and Velcade can inhibit inflammatory reaction. Combined treatment of them may deduce synergistic inhibition to mitochondria apoptosis pathway. The protection will be more effectively than single treatment.
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