| Cerebrovascular diseases which increase with the acceleration of population aging have become one of the main causes of human death and permanent disability,has brought heavy economic burden to the society and families.Stroke has highest morbidity among cerebrovascular diseases.Ischemic stroke is account for 80% of stroke.The main treatment of ischemic stroke is blood reperfusion.However,the recovery of blood perfusion inevitably leads to the second injury to the brain tissue in the ischemic region,which is called ischemia/reperfusion(I/R)injury.Because of the complex mechanisms of cerebral I/R injury,many researches has focused on the study of the mechanisms.It has been demonstrated that activation of epidermal growth factor receptor(EGFR)has protective effects against cerebral ischemic injury.However,the protective mechanisms of activating EGFR against I/R injury remain to be clarified.JAK2/STAT3 pathway is a signal transduction pathway which was found in recent years and can be stimulated by cytokines.With the further exploration of the JAK2/STAT3 pathway,more and more studies showed that phosphorylation of JAK2/STAT3 pathway promoted antiinflammatory,anti-apoptosis and angiogenesis,and played an important role against cerebral I/R injury.Researches showed that activating EGFR could phosphorylate JAK2/STAT3 pathway in anti-cancer effects.However,little research has explained whether activating JAK2/STAT3 pathway is benefit to the protective effects of activating EGFR against cerebral I/R injury.This study was carried out in vivo and vitro experiments to investigate the role and mechanism of JAK2/STAT3 pathway in the protective effects of EGFR activation against cerebral ischemia/reperfusion injury in rats and to provide a new theoretical basis for clinical treatment of cerebral I/R injury.Objective: 1.To investigate the relationship between EGFR and JAK2/STAT3 pathway against cerebral I/R injury in rats.2.To clarify the mechanism of JAK2/STAT3 pathway in the EGFR activation against cerebral I/R injury in rats.Methods: In vivo: 1.The cerebral I/R injury of rats were induced by middle cerebral artery occlusion(MCAO)for 2 h and reperfusion for 24 h.To investigate the relationship between EGFR and JAK2/STAT3 pathway against cerebral I/R injury in rats,sprague dawley(SD)rats were randomly divided into sham group,I/R + epidermal growth factor(I/R + EGF)group(EGF,1 μg/kg was given to rats by tail vein injection 90 min after ischemia),I/R + EGF + AG1478 group(AG1478,1 mg/kg,the antagonist of EGFR,and it was given to rats 30 min after ischemia by tail vein injection).To clarify the effects of JAK2/STAT3 pathway in the EGFR activation against cerebral I/R injury in rats,AG490,an inhibitor of JAK2/STAT3 pathway was used,and the SD rats were further divided into sham group,I/R group,I/ R + EGF group,I/R + EGF + AG490 group(3 mg/kg,105 min after ischemia by intraperitoneal injection).The operation of sham group was the same as the other groups except inserting the nylon monofilament into middle cerebral artery.Rats in the sham group and I/R group were given the same amount of saline.In each of the above groups,at least 10 rats were successfully established.After ischemia for 2 h and reperfusion for 24 h,the neurobehavioral scores of rats were detected by Longa score,the infarct volume of rats was measured by 2,3,5,triphenyltetrazolium chloride(TTC)staining,the apoptosis of neurons in different group was detected by terminal dexynucleotidyl transferase(Td T)-mediated d UTP nick end labeling(TUNEL)staining and the expressions of p-EGFR,p-STAT3,Bax and Bcl-2 were detected by Western blot.In vitro: 2.After the primary neurons cultured for 10 day,the hypoxia/reoxygenation(H/R)injury model was prepared for the following experiments.Neurons were divided into control group,H/R group,H/R + EGF group(EGF,50 ng/ml,was added to primary cultured neurons 1 h before hypoxia)and H/R + EGF + AG1478 group(AG1478,10 μmol/L,was added to primary cultured neurons after hypoxia),H/R + EGF + AG490 group(AG490,50μmol/L,was added to primary cultured neurons 45 min before hypoxia).The survival rate of neurons was detected by methyl thiazolyl tetrazolium(MTT)assay,the apoptosis of neurons was detected by Hoechst 33258,and the expression of p-EGFR,p-STAT3,Bax and Bcl-2 protein was detected by Western blot.Results: In vivo: 1.The neurobehavioral scores were increased when rats suffered from cerebral I/R injury compared with sham group.EGF pretreatment significantly decreased infarct volume compared with I/R group.However,the antagonist of EGFR,AG1478 attenuated the EGF-induced reduction of infarct volume compared with I/R + EGF group.Moreover,the inhibitor of JAK2/STAT3,AG490 undermined the protective effects stimulated by activating EGFR in rats with I/R injury and the neurobehavioral scores were increased compared with I/R + EGF group.2.The infarct volume in different group was detected by TTC staining and measured by Image J software.The infarct volume was increased of rats in I/R group compared with sham group.Pretreatment with EGF decreased the infarct volume compared with I/R group,however the protective effect was undermined by AG1478 and AG490,the infarct volume was increased compared with I/R + EGF group.3.After ischemia for 2 h and reperfusion for 24 h,the apoptosis of neurons in different group was detected by TUNEL staining.Results showed that,the apoptosis of neurons in I/R group was increased significantly compared with sham group.After activating EGFR,the apoptosis of neurons was decreased compared with I/R group.On the contrary,the apoptosis of neurons was strongly increased after rats were given EGF together with AG1478 or AG490 compared with I/R + EGF group.4.When rats suffered from I/R injury,the ratio of Bax/Bcl-2 was increased compared with sham group,EGF pretreatment increased the expression of Bcl-2 and reduced the expression of Bax compared with I/R group.The ratio of Bax/Bcl-2 was increased after rats were given EGF together with AG1478 or AG490 compared with I/R + EGF group.In vitro: 5.MTT results showed that the survival rate of neurons in H/R group was decreased compared with control group,EGF pretreatment significantly increased the survival rate of neurons compared with H/R group.However,the protective effect of EGFR activation was undermined by AG1478 and AG490,and the survival rate of neurons was decreased in H/R + EGF + AG1478 group and H/R + EGF + AG490 group compared with I/R + EGF group.6.The result of Hoechst 33258 showed that the apoptosis of neurons in H/R group was significantly increased compared with control group.After pretreatment with EGF, the apoptosis of neurons was decreased compared with H/R group.AG1478,an antagonist of EGFR and AG490,an inhibitor of JAK2/STAT3 pathway,undermined the protective effects which stimulated by activating EGFR and the apoptosis of neurons was increased compared with I/R + EGF group.7.The expression of p-EGFR,p-STAT3,Bax and Bcl-2 was detected by Western blot.When neurons suffered from H/R injury,the ratio of Bax/Bcl-2 was increased compared with control group.Pretreatment with EGF significantly increased the expression of p-STAT3 and the ratio of Bax/Bcl-2 was decreased compared with H/R group.On the basic of activating EFGR,using AG1478 antagonized EGFR and AG490 inhibited JAK2/STAT3 pathway activation,the expression of p-STAT3 the ratio of Bax/Bcl-2 was increased compared with I/R + EGF group.Conclusions: 1.Activating JAK2/STAT3 pathway is involved in the protective effects of EGFR activation against cerebral ischemia/reperfusion injury in rats.2.Reduction of cell apoptosis induced by cerebral ischemia/reperfusion injury may involve in JAK2/STAT3 pathway activation,which contributes to the protective effects of EGFR activation.3.Reduction of cell apoptosis is related to the reduction of Bax/Bcl-2 in activating JAK2/STAT3 signaling pathway,which contributes to the protective effects of EGFR activation against cerebral I/R injury in rats. |
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