Protective Effect Of EFF1A1in Cerebral Ischemia

Stroke is a group of localized or wide range brain function deficit syndromecaused by acute cerebral circulatory disorders. It includes ischemic stroke andhemorrhagic stroke and has a high morbidity and disability in the world. With theaccelerating of aging in China, ischemic stroke has gradually become a heavy burdento the family and social. It is urgent to find effective drugs to preventing or healingischemic stroke. When brain tissue lack of blood and oxygen supply, it will finallylead to cell apoptosis through a variety of mechanisms such as excitotoxic damage,oxidative stress, inflammatory response and so on. Studies have shown thatinflammation plays an important role in the process of cerebral ischemia. Ischemiccells secrete IL-1β, TNF-α, E-selectin, P-selectin, intercellular adhesion molecule(ICAM-1) and chemokines, which promote the inflammatory cells infiltration to theischemic tissue and change blood-brain barrier permeability, and further aggravatevascular occlusion and finally a "no-reflow" phenomenon will appear. Thus, it isimportant to reduce the damage through inhibiting the inflammatory response. Ourprevious study found a anti-inflammatory activity peptide MLIF, which can reduceinfact area in rat and mice cerebral ischemia model, upregulate eNOS, anddownregulate ICAM-1and VCAM-1to reduce cell damage by interacting witheukaryotic elongation factor1A1(eEF1A1). Based on previous studies, this studyintends to further study the protective effects of eEF1A1in cerebral ischemia,provides new idea for cerebral ischemia.Methods and Results1、Use co-immunoprecipitation methods to find eEF1A1-interacted protein. Afteridentified with mass spectrometry and compared with protein databases, weconfirmed the interacting protein was HSC70.2、Use Western Blot method and immunofluorescence method to validate the resultsof co-immunoprecipitation.After hybridizing with HSC70antibodies in Western Blot, there was a clear bandin71kD, eEF1A1and HSC70were also well co-localization in the result ofimmunofluorescence. According to these results, we confirmed eEF1A1and HSC70had interaction in bEnd3cells.3、 Ue siRNA interference method to inhibit eEF1A1and HSC70expression separately, and observe the other protein expression. Detected by Western Blot, wefound that neither of the protein expression changed when the other protein’sexpression was inhibited. But the binding of these proteins increased firstly and thendecreased with the increasing of hypoxia time, suggest hypoxia may influence thebinding of eEF1A1and HSC70according to the result of immunoprecipitation.4、Use flow cytometry to identify apoptotic cells stained by Annexin V-FITC/PI.HSC70siRNA was transfected in bEnd3cells,48h later more than50%cellswere apoptotic. The apoptosis rate was significantly higher than the negative controlgroup (P＜0.01). Although apoptosis rate didn’t change in eEF1A1gene knock downgroup, the apoptosis rate of RNAi group became higher than negative control group(P＜0.01)after8h hypoxia. Up on these results, we could make the conclusion thateEF1A1and HSC70could protect bEnd3cells from apoptosis in cerebral ischemia.5、Detected by Western Blot, we found the expression of JNK signal pathwayassociated protein rising such as phosphorylated JNK, phosphorylated c-JUN (Ser63,Ser73), cleaved caspase-9and cleaved caspase-3when bEnd3cells was treated withhypoxia for different hours. This phenomenon could be inhibited by JNK inhibitorsp600125. This result demonstrated that hypoxia could lead apoptosis throughactivating JNK-related signaling pathway.6、After transfected with eEF1A1siRNA or HSC70siRNA in bEnd3cells for48h, wedetected JNK signaling pathway related protein expression by Western Blot. InHSC70RNAi group, phosphorylated JNK, phosphorylated c-JUN (Ser63, Ser73),cleaved caspase-9and cleaved caspase-3increased compared as negative controlgroup (P＜0.01); It also showed significant rise in the group of eEF1A1RNAi+hypoxia(P＜0.01). Up on these results, we could make the conclusion thateEF1A1and HSC70might play a protective effect by inhibiting the activation of JNKsignaling pathway in cerebral ischemia.7、Based on literatures, we detected LAMP-2A, a protein located on mitochondrialmembrane and related to chaperone-mediated autophagy, by Western Blot. Neitherhypoxia group nor eEF1A1RNAi+hypoxia group had significant difference comparedwith negative control group, indicating that the protection of eEF1A1and HSC70incerebral ischemia didn’t through chaperone-mediated autophagy.Conclusion: 1、 In bEnd3cells, eEF1A1interact withHSC70with the form of molecularchaperones.2、eEF1A1and HSC70can inhibit cell apoptosis through inhibiting the activation ofJNK signaling pathway, which play a protective role in ischemic stroke process.