| Objective: Using the dl-3n-butyphthalide(NBP), SP600125(specific inhibitor of JNK) to intervene the models of ischemia reperfusion animal, the experimental method were used,such as,Western blot, HE staining, TUNEL and transmission electron microscopy(sem) to discusse the influence of NBP on the autophagy,the mechanism in rats of ischemia-reperfusion injury and the change of autophagy related protein Beclin 1, LC3-Ⅱ.the research also tried to discuss whether the JNK3 signal pathways were involved in the regulation of autophagy and whether it was the JNK3 signaling pathways that the NBP played role in which.Methods:The one hundred male Sprague-Dawley(250-300 g,8-10 weeks old) were randomly divided into 5 groups: sham-operated group, model group, NBP intervention group, SP600125 intervention group, NBP+SP600125 intervention group.The group NBP and NBP+SP600125 were treated by liquid,which 8 mg NBP solution 1 m L,1ml/100 g of rat gavage seven days before. but the same volum ofsesame oil for other three groups. The group SP600125 and NBP +SP600125 were intraperitoneal injected by 1m L,100g-1.liquid, which 40% PEG400 as 1mg.m L-1solution seven days before,but the same volum of 40%PEG400 for other three groups. Each one was treated by 10% chloral hydrate(0.3ml/l00g)intraperitoneal injection of anesthesia.Using the rule of Longa line plug to make a rat MCAO model of ischemia reperfusion 2 hours.After cerebral ischemia 12 hours,make themdeath.Then,neurologiealbehavior evaluation was Performed by the method of Longa,5seoring.The Pathologiec hanges were observed by hematoxylinandeosin(HE).staining at 12 h after reperfusion in cerebral cortex of rats.To assess cerebral ischemia reperfusion injury in afterbrain function damage by NSE levels.Meanwhile,apoptotic cells were detected by TUNEL technique.Autophagy is observed of lysosome were observed in CA1 area of rats after cerebral ischemia reperfusion by using transmisson electron microscope.Using Western Blot testing CA1 area of Beclin 1,LC3-Ⅱrotein expression of rats autophagy related and the expression of JNK,p-JNK3 protein involved in signaling pathways.Results:1. Longa score:(1)The first grade after the success of the operation:Compared with sham-operated group,the model group(2.00 + 0.82) significantly increased reated the grade of the rat neurologieal defects(p<0.05);NBP treatment group(1.83±0.50), SP600125 group(1.880±0.52) and NBP +SP600125 group(1.85±0.41) three sets oftwo is no statistical significance(p>0.05), but in model group(p<0.05).(2) The grade after ischemia reperfusion 12h: model group(2.65 ±0.50), respectively, and the score for the first time and control group were significantly increased(p<0.05);NBP treatment group(1.53± 0.52), SP600125 group(1.55±0.43) and NBP+ SP600125 group(1.58±0.55) three sets of two is no statistical significance(p>0.05), but and for the first time score were significantly lower in the model group(p < 0.05). 2.TTC staining: Sham-operated group was not observed infarction.Cortical and subcortical visible white infarction were detected in model group、NBP intervention group 、SP600125 intervention group and NBP+SP600125 intervention group. The relative infarct volume of groups respectively:the relative infarct volume of model group 12 h is 25.56 ± 1.86,were significantly increased compared with the Sham-operated group(p<0.01).NBP intervention group(18.26±2.23), NBP+ SP600125 intervention group(17.86 ± 2.12), SP600125 intervention group(17.98 ± 2.52) for the comparison between two groups has no statistical significance(p>0.05), but in model group(p<0.05).. 3. HE staining showed the hippocampal CAl of cerebral pathology of 12 h after reperfusion in each group:the brain tissue slices of sham-operated group of 12 h is visible for nerve cells with normalmorphology,integrated and clears tructure,the nucleus of pyramidal cell is large and round,and nucleolus is obvious. The model group of 12 h showed ischemic changes,the structure of normal tissue disappeared in the infarct area,and the delineation of the nucleus and the cytoplasm is unclear,the volume of nerve cell is reduced,nucleus pyknosis, cytoplasm is eosinophilic,partial cells is necrosis.The ischemic changes compared with the model group was significantly reduced in intervention group(NBP,NBP+SP600125 group, SP600125 group)of 12 h,and the extent of necrosis is decreased,complete cell structure,less nucleus pyknosis,and interstitial edema of cells is light. 4. TUNEL staining showed the hippocampal CAl of neurons apoptosis expression of 12 h after reperfusion in each group: a few scattered TUNEL-positive cells can be observed in the sham-operated group. Compared with sham-operated group(0.05 ±0.02)、NBP group(0.31±0.03, SP600125 group(0.30 ± 0.02)and NBP + SP600125 group(0.32±0.04),the model group(0.52± 0.05)were increased significantly(p< 0.05).No significant chang at NBP group 、SP600125 group 、and NBP + SP600125 group(p>0.05). 5.NSE:the sham-operated group(1.89±0.13),NBP group(5.10±0.15),SP600125 group(5.21±0.11) and NBP+SP600125 group( 4.98±0.15) respectively in the model group, the results showed that the serum NSE levels were lower than the model group(p< 0.01), with significantstatistical significance.And no statistical significance of NBP group、SP600125 group and NBP+ SP600125 group(p>0.05). 6. Westem blot, the experimental results shows: P- JNK, JNK, Beclin 1, LC3-Ⅱlevels in model group than butyl phthalide, SP600125 group, butyl phthalide +SP600125 group, the control group(group A) increased significantly(p < 0.05), with statistical significance.And butyl phthalide, SP600125 group, butyl phthalide + SP600125 group between two comparison shows(p>0.05), no statistical significance. 7. Transmission electron microscopy(sem) : each CA1 area electron microscopy: basic normal control structure of nerve cells, mitochondria swelling, did not see autophagy body;Model group visible bubble autophagy-lysosome, neurons in the nucleus chromatin pyknosis;Butyl phthalide, SP600125 group, SP600125 +butyl phthalide group of double membrane structure is forming, surrounding the organelles autophagy body formation, a small circular lysosome, mostly located in the nucleus, and visible primary lysosome, secondary lysosome populations and mitochondrial swelling.Conclusion: 1. The model of ischemia-reperfusion injury in rats can induce the excessive activation of autophagy. 2. The autophagy can be regulated in ischemia-reperfusion injury of rats through JNK3 Signaling pathways. 3. The dl-3n-butyphthalide can attenuate brain ischemia reperfusion induced autophagy. The mechanism by which the can decrease the JNK and p-JNK levels at first, and then interfere with the functions of Beclin 1 during the execution of autophagy. |
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