The Neuroprotective Effect Of MLIF In Cerebral Ischemic Injury

Purposes Stroke is mainly composed of ischemic stroke and hemorrhagic apoplexy,87% of which is ischemic stroke.The human physical and psychological health is seriously threatened by stroke because of its high disability rate and fatality rate.Numerous studies have demonstrated that the pathogenesis of stroke is complicated involving in different factors,such as inflammation,apoptosis,cytotoxicity,oxidative stress and so on.Therefore,it is still lack of effective drugs in clinic.Inflammatory response and neuronal cell apoptosis induced by cerebral ischemia are key mechanisms inducing cerebral cell death and brain lesions and death.So,it has profound scientific value and social significance to find the multi-target drug with anti-inflammation and anti-apoptosis.Monocyte locomotion inhibitory factor,MLIF,is a heat-stable pentapeptide.In previous studies,we have revealed that MLIF could significantly shrink the cerebral infarction area in MCAO mice and rats models,and bind to the ribosomal protein eukaryotic translation elongation factor 1A1 in the cerebrovascular endothelium.MLIF could up-regulate the expression of e NOS by interacting with the 3'-UTR of e NOS and decrease the adhesion molecule expression of ICAM-1 and VCAM-1.To further study the cerebral protective mechanism of MLIF,the current research applies in SH-SY5Y cells and MCAO animal model to provide new idea for the prevention and treatment for ischemic cerebrovascular diseases.Methods and Results1.The ischemic model in vitro was established by oxygen and glucose deprivation(OGD)or oxygen and glucose deprivation/reperfusion(OGD/R)in SH-SY5Y cells.We evaluated the cell survival and cell apoptosis after OGD with MTT assay,LDH assay,flow cytometry and Hoechst 33258 staining.In addition,we established OGD/R model and evaluated the cell survival and cell apoptosis with MTT assay and LDH assay.Results showed that neuronal injury was induced by oxygen and glucose deprivation model and confirmed OGD for 6 h was the best OGD time and MLIF enhanced cell survival and inhibited apoptosis and necrosis induced by OGD or OGD/R(P<0.01).2.The effect of MLIF on the levels of apoptosis related proteins in SH-SY5Y cells exposed to OGD treatment was evaluated by western blot assay.We found that the expression levels of p-JNK,p-p38,p-ERK,p53,cleaved caspase3,cleaved caspase9 were significantly increased in OGD group compared with control group(P<0.01 or P<0.05)and the expression levels of p-JNK,p53,cleaved caspase3 and cleaved caspase9 were significantly decreased after treatment with MLIF compared with OGD group(P<0.01 or P<0.05).These results indicated that MLIF could inhibit neuronal cell apoptosis induced by OGD via inhibiting p-JNK/p53 apoptosis pathway.3.Pull-down assay was used to find the binding protein of MLIF in rat primary neuron and SH-SY5Y cells.Pull-down assay was carried out as followings: biotin-MLIF group and negative control group was interacted with two equivalent cell lysates respectively.After SDS-PAGE assay and coomassie brilliant blue staining,we took the specific band in gel for enzymolysis and found the specific binding protein of MLIF in neuronal cells with mass spectrum.Then we searched the database matching number,compared with the molecular weight,and finally confirmed the 50 k Da e EF1A2 as the binding protein in neuronal cells.To further confirm this result,we studied in neuron and SH-SY5Y cells by western blot.To further study the combination and distribution of the interaction between MLIF and e EF1A2 in SH-SY5Y cells,we used immunofluorescence technique and found FITC-MLIF and e EF1A2 were well co-localization.According to these results,we confirmed the specific binding protein of MLIF in neuronal cells was e EF1A2.4.SiRNA assay was used to detect the effect of MLIF in SH-SY5Y cells exposed to OGD after transfected with e EF1A2 siRNA.It was indicated that cell survival in e EF1A2 siRNA group was inhibited compared with negative control group in MTT assay(P<0.01),and cell apoptosis rate in e EF1A2 siRNA group was significantly increased and nucleus pycnosis was more serious compared with negative control group in flow cytometry assay and Hoechst 33258 staining(P<0.01),and the effect of MLIF inhibiting the expression of apoptotic protein p-JNK/p53 weaken(P<0.05).5.MCAO was used to make rat cerebral ischemic/reperfusion model,according to the experiment,which is divided into three groups: sham,model and MLIF group.To comprehensively measure the neuroprotection of MLIF on ischemic model in vivo,Nissl staining was used to observe the morphous and the structure of the cerebral cortex and the hippocampus nerve cell after MCAO for 7days,14 days and 28days;corresponding kits were used to detect the levels of SOD,MDA and the inflammatory factors IL-1? and TNF-? in serum after MCAO for 7 weeks;Morris water maze was used to evaluate the spatial learning and memory function after MCAO for 7 weeks.Results suggested MLIF could improve the morphous and the structure of the cerebral cortex and hippocampus nerve cell,increased the levels of SOD,and decreased the levels of MDA,IL-1? and TNF-?,and improved the spatial learning and memory function.Conclusion:1.MLIF increased cell survival and inhibited cell apoptosis and necrosis induced by OGD in SH-SY5Y cells.2.The effect of MLIF on inhibiting the OGD induced apoptosis was targeting eEF1A2 to inhibit p-JNK/p53 signaling pathway.3.The neuroprotection of MLIF was worked by decreasing neuronal damage of cerebral cortex and hippocampus after cerebral ischemia,ameliorating cerebral ischemia induced memory deficit,scavenging oxygen free radicals by increasing SOD and decreasing MDA,inhibiting the levels of inflammatory factor IL-1? and TNF-?.