| Objective:1.To observe the expression of phosphorylation c-Jun amino terminal kinase in brain tissue after cerebral ischemia-reperfusion injury in rats.2.To observe the changes of phosphorylation c-Jun amino terminal kinase after the inhibitor SP600125.3.To prepare for the JNK inhibitor as a treatment target for the ischemic cerebrovascular disease.Method:65 healthy adult male Wistar rats of clean grade, Weight 240 ~ 270g, Randomly divided into 5 groups:Normal control group (3), sham operation group (3), ischemia-reperfusion group (IR group,3), ischemia-reperfusion + DMSO group (DM group, 28) and ischemia reperfusion + DMSO + SP600125 group (SP group, 28). DM group and SP group was randomly divided into four subgroups according to reperfusion time 6h, 12h, 24h, 72h ,each subgroup has 7 rats. Preparate middle cerebral artery occlusion-reperfusion model by modified Longa intraluminal thread,reperfusion is after occlusion for 2h.1.To observe neurological behavioral changes by Longa neurological function standard.2.Observe Infarct volume by TTC staining.3.Observe ischemic neurons necrosis by HE staining.4.Detect the activation of c-Jun amino terminal kinase (p-JNK) by Immunohistochemistry and Western blot of rat brain tissue at different time . 5.Observe the effect of p-JNK inhibitor SP600125 on the expression of p-JNK.Results:1.The success rate of Intraluminal model of ischemia-reperfusion was 75.6%. The control group and sham group is asymptomatic.The neurological deficit symptoms of DM group is the most severe. The neurological deficit symptoms of 6h~12h subgroup is the most severe;the 24h subgroup is better;the 72h subgroup is the least severe.Compared with the control group and the sham group ,the neurological function scores of DM group is significantly different (P <0.01).Compared with the DM group,the neurological deficit scores of 6h～12h subgroup of SP group is significantly different (P <0.01);the neurological deficit scores of 24h～72h subgroup is no significant difference (P> 0.05).2. TTC staining showed infarction lesions in the middle cerebral artery feeding area in IR,DM and SP group,but no found in control group and sham group.Compared with the DM group, the infarction lesions of the SP group is decreased. HE staining showed ischemic infarct necrosis of the neurons.3. The immunohistochemistry study on p-JNK showed that brown stained of neurons and glial cells in rats of control group were negative; the neuronal and glial cellular nucleus of DM groups after 6h reperfusion were stained brown;after 12h reperfusion, Nuclear stained, positive cells increased; after 24h reperfusion the number of positive cells reduced; to 72h continued high level of expression. The expression of p-JNK was significantly decreased (P <0.05) in the rats of SP600125 intervention group compared with which of DM group.4. Western blot study found that expression of p-JNK was increased in brain tissue after 6 h reperfusion, peaked after 12 h reperfusion, and decreased after 24h, still expressed after 72 h reperfusion. Compared with DM group, the expression of p-JNK in SP600125 intervention group was significantly decreased (P <0.01), the peak was not obvious (P> 0.05).Conclusions:The results showed that the activity of p-JNK after cerebral ischemia-reperfusion injury in rats was time-dependent increase. Meanwhile, the effect was inhibited by SP600125.It showed that JNK play a positive regulatory role in ischemia-reperfusion induced apoptosis.SP600125 protected neurons by inhibiting the JNK activity. It may be used as treatment targets in ischemic brain injury. |
PDF Full Text Request