Investigation of the Endoplasmic Reticulum Stress Associated Neuronal Apoptosis Following Cerebral Ischemia and Reperfusion in Rat and the Effects of Edaravone
|School||Central South University|
|Keywords||cerebral ischemia/reperfusion PERK ATF4 CHOP GADD34 ERS UPR neuronal apoptosis edaravone|
Background and objective: Many genes modulate the neuronal apoptosis following ischemia brain damage. There are mainly three pathways modulating cellular apoptosis. Compared with the pathways of chondrosome and death receptor, the studies on endoplasmic reticulum pathway was relative inadequate and focus on cell assay in vitro. The endoplasmic reticulum stress reaction resulted from calcium homeostasis unbalance and peroxidation damage after cerebral ischemia/reperfusion could down-regulate protein translation level and induce cellular apoptosis. PERK mediated UPR signal transduction pathway plays an important role in the processes. The eIF2αphosphorylation activated by PERK gene could inhibit directly protein synthesis, and induce postischemia target genes(ATF4, CHOP, GADD34, etc) expression. Previous studies suggested that PERK mediated protein synthesis inhibition correlated with cellular self-protection and cell survival promotion in short time, however, long-playing protein synthesis inhibition could regulate some pro-apoptosis genes(CHOP, caspase-12) expression and induce cellular apoptosis. Presently, few studies on PERK mediated UPR signal pathway in cerebral ischemia models had been reported. Furthermore, the more system studies on PERK, ATF4, CHOP and GADD34 genes expression development should be progressed. In this work, we detected the expression development of PERK, ATF4, CHOP, GADD34 genes, and explored the roles and association with brain damage after cerebral ischemia/reperfusion in rats. Furthermore, the effects of edaravone(a novel free radical scavenger) on related genes expression in PERK pathway were detected. Then the mechanism of neuroprotective effect produced by edaravone were eluciateded, which provide us with new theory and method for clinical treatment of cerebral ischemia. Methods: The 156 healthy male sprague-dawley rats were randomly divided into three groups: normal group(n=12), sham operation group(n=72) and cerebral ischemia model group(n=72) in part 1. But in Part 2, the 144 spraue-dawley rats were randomly enrolled in control group(n=72) and edaravone-treated group (n=72) after established the cerebral ischemia model successfully. Transient focal cerebral ischemia was induced by middle cerebral artery occlusion(MCAO) for 2 hours followed by reperfusion in the rats. Then, neurological behavior evaluation and cerebral infarction volume were evaluated by the methods of Longa’s scoring and TTC staining. The pathologic changes were observed by hematoxylin and eosin (HE) staining and Nissl staining at 1 h, 3 h, 6 h, 12 h and 24 h after reperfusion in cerebral cortex of rats, respectively. Meanwhile, the expression of p-PERK, ATF4, CHOP, GADD34 protein and/or mRNA were measured with methods of immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR).The neuronal apoptosis was detected by the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL).Results: 1. The expression of PERK、ATF4、CHOP、GADD34 were up-regulated in peri-infarction after cerebral ischemia/reperfusion in rats. Among these, the expression level of PERK mRNA and p-PERK protein reached a peak at 1 h after reperfusion. However, GADD34, ATF4, CHOP mRNA expression reached a peak at 3h,6h,12h after reperfusion, respectively, and the three different proteins expression simultaneously reached a peak at 24h under the same conditions.2. Cerebral infarction volume and the apoptosis cell counting were increasing gradually after cerebral ischemia/reperfusion, and reached a peak at 24h after reperfusion.3. Compared with the saline control group, treatment with edaravone could reduce the cerebral infarction volume, neuronal apoptosis and improve rat neurological deficits at 12h,24h,72h after reperfusion(P＜0.05).4. Compared with the saline control group, treatment with edaravone could decrease PERK mRNA and p-PERK protein expression in parietal lobe at 1h,3h,6h after reperfusion(P＜0.01). In this study, we have observed that injection of edaravone significantly down- regulated ATF4 mRNA expression level at 3h,6h,12h after reperfusion(P＜0.05),and the ATF4 protein expression level at 6h,12h,24h after reperfusion was also down-regulated as well(P＜0.05). Furthermore, edaravone treatment could decrease CHOP mRNA(P＜0.01) and protein(P＜0.05) expression at 3h,6h,12h,24h and 6h,12h,24h after reperfusion, compared with the saline control group, respectively. Meanwhile, GADD34 mRNA expression at 1h,3h,6h,12h after reperfusion(P＜0.05) was decreased in edaravone-treated group, and the down-regulation of GADD34 protein expression was also detected after 6h, 12h, 24h as well(P＜0.05).Conclusions: 1. Cerebral ischemia reperfusion may induce ERS, which initiate PERK mediated URP signal pathway.2. CHOP expression development paralleled with the changes of the apoptosis cell counting after cerebral ischemia/reperfusion in rats, which suggested that CHOP play a role in neuronal apoptosis.3. Edaravone could reduce neurological deficits and cerebral infarction volume after cerebral ischemia/reperfusion in rats.4. Edaravone could inhibit endoplasmic reticulum stress reaction and apoptosis signal pathway by the expressive down-regulation of PERK, ATF4, CHOP and GADD34 after cerebral ischemia/reperfusion in rats, which may result in decreases of neuronal apoptosis and ischemical reperfusion injury.