The Interaction Of C-Jun N-Terminal Kinase And Endoplasmic Reticulum Stress After Cerebral Ischemia-Reperfusion Injury In Rats

PART?THE FUNCTION OF C-JUN N-TERMINAL KINASE IN CEREBRAL INJURY AFTER ISCHEMIA-REPERFUSIONIN RATSObjective To Observe the changes of phosphorylation of c-Jun amino terminal kinase (p-JNK) in brain tissue after cerebral ischemia-reperfusion injury in rat,and the protection of the inhibitor SP600125 on ischemia-reperfusion injury in neurons.Methods1. Prepare middle cerebral artery occlusion-reperfusion model by modified Longa intraluminal thread. The rats were randomly divided into control, ischemia-reperfusion (IR) group, ischemia-reperfusion+dimethyl sulfoxide (IR+ DMSO) group, ischemia-reperfusion+DMSO+SP600125 (IR+SP) group. Rats of IR+DMSO group and IR+SP group were randomly divided into 2 h occlusion and 6,12,24,72 h reperfusion groups.2. Observe Infarct volume by TTC staining, and ischemic neurons necrosis by HE staining.3. Detect the activation of c-Jun amino terminal kinase (p-JNK) by Immunohistochemistry and Western blot, observe the changes of p-JNK in brain during the ischemic-reperfusion time courses.4. Observe the effect of p-JNK inhibitor SP600125 on the expression of p-JNK. Results1. The achievement rate of intraluminal model of ischemia-reperfusion was 75.6%; TTC staining showed infarction lesions in the middle cerebral artery feeding area; HE staining showed ischemic infarct necrosis of the neurons.2. 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 IR+DMSO 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 significantly decreased (P<0.05) in the rats of SP600125 intervention group compared with which of IR+DMSO group.3. Western blot study found that expression of p-JNK 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 IR group and IR+DMSO group, the expression of p-JNK in SP600125 intervention group significantly decreased (P<0.01), the peak was not obvious (P>0.05).Conclusions1. The morphological changes of the neurons manifested as necrosis after ischemia-reperfusion cerebral injury in rat induced by modified Longa intraluminal thread technique.2. The activity of p-JNK in the cerebral injury showed time-dependent increase, and the change can be suppressed by SP600125. 3. SP600125 can reduce the neuronal damage caused by ischemia and reperfusion.PART?THE ROLE OF ENDOPLASMIC RETICULUM STRESS IN CEREBRAL INJURY AFTER IS CHEMIA-REPERFUSION IN RATS AND THE EFFECT OF P-JNK INHIBITOR Objective Observed the changes of GADD153, caspase-12 ----the apoptosis-related proteins of endoplasmic netcom road, after cerebral ischemia-reperfusion injury, and explored the effect of p-JNK inhibitor SP600125 on the expression of the two.Methods1. Estabished modified Longa intraluminal middle cerebral artery occlusion model. The rats were randomly divided into control, ischemia-reperfusion (IR) group, ischemia-reperfusion+dimethyl sulfoxide (IR+DMSO) group, ischemia-reperfusion +DMSO+SP600125 (IR+SP) group. Rats of IR+DMSO group and IR+SP group were randomly divided into 2 h occlusion and 6,12,24,72 h reperfusion groups.2. Detected GADD153, caspase-12 changes at different time points by Immunohistochemistry, Western blot and Double-label immunofluorescence.Results1. Immunohistochemistry and double immunofluorescence analysis showed that the expression of GADD153 increased in cortex and striatum, and the positive cells of cytoplasm and nuclus staining in striatum were significantly more than in cortex (P<0.05); Western blot showed expression of GADD153 increased after 6h reperfusion, continued increasing on 72h reperfusion, P<0.05.2. Compared with that of rats without SP600125 treated, expression of GADD153 of SP600125 intervention group was not significantly different after 6h,12h reperfusion(P>0.05); but the expression decreased significantly after 24,72h reperfusion(P<0.05).3. Immunohistochemistry and double immunofluorescence analysis showed that expression of caspase-12 was increased in cortex and striatum, the expression of caspase-12 in striatum was significantly higher than in cortex (P<0.05); Western blot showed that expression of caspase-12 increased after 6h reperfusion, to the peak after 24h, and still maintained a high level after 72h.4. Expression of caspase-12 in SP600125 intervention rats was significantly lower than that in rats without SP600125 treated (P<0.05), the peak was not obvious (P>0.05).5. Double immunofluorescence staining of DMSO group showed that GADD153, caspase-12 single-positive and double-positive cells were visible mainly in striatum at 6h reperfusion; during 12?24h both single and double staining positive cells significantly increased (P<0.05), mainly in striatum, a small amoun in cortical areas also; to 72h, the number of GADD153 single-positive cells reduced, caspase-12 single-positive cells were still more marked, and the number of double-positive cells reduced (P<0.05).6. Compared with the DMSO group, GADD153 single-positive cells of SP group decreased slightly, caspase-12 single-positive cells and double-positive cells significantly reduced (P<0.05).Conclisions1. Expression of GADD153 and caspase-12 increased in brain tissue after ischemia-reperfusion, and endoplasmic reticulum stress involved in the pathological process of ischemic brain injury.2. During the course of ischemia-reperfusion brain injury, c-Jun amino terminal kinase pathway might involved in the process of endoplasmic reticulum stress by caspase-12 pathway.