Anti-inflammation Mechanism Of MLIF In Cerebral Ischemia

PurposeStroke is one of the three leading causes of death and also the most common reasons for adults’ disability world widely.Among all types of stroke,ischemic stroke,which was caused by blood vessel occlusion,accounts for the majority(over 80%).Mechanisms involved in the process of stroke are extremely complicated,including excitotoxicity,calcium overload,oxidative stress,inflammatory response and apoptosis.Previous studies have shown that inflammation plays an important role in neuron damage after ischemic stroke.Microglia are innate immune cells in central nervous system,they involved in antigen presentation,phagocytosis and secretion of pro-inflammatory/anti-inflammatory factors etc.Differential polarization of microglia could likely explain the biphasic role of microglia in ischemia.Microglia could generally polarize ―classically activated‖ pro-inflammatory M1 microglia and ―alternatively activated‖ anti-inflammatory M2 microglia.In the process of cerebral ischemia,the activation of microglia and its polarization play an very important role in nerve injury or repair.Hence,we should promote the M2 phenotype polarization instead of inhibiting the activation of microglia simply.Monocyte locomotion inhibitory factor(MLIF)is a heat-stable oentapeptide,which is produced by Entamoeba histolytica.Our study group previously found that MLIF could significantly decrease the cerebral infarction in the MCAO mice and rats model,and it could decrease the ICAM-1,VCAM-1 expression and increase the eNOS expression in oxygen and glucose deprivation(OGD)or ox-LDL induced bEnd3 cell by targeting eukaryotic elongation factor1A1(eEF1A1).Moreover,we also find MLIF could inhibit OGD induced apoptosis in SH-SY5 Y neuron cell by targeting eEF1A2.Thus,on the basis of our previous studies,the current research further study the regulation effects of MLIF on the microglia activation and polarization to illuminate the anti-inflammation mechanism of MLIF in ischemic stroke and provide the new thought for the prevention and treatment for ischemic stroke.Methods and Results1.The in vitro study model was esbilished by the oxygen and glucose deprivation(OGD)in BV2 cells and SH-SY5 Y cells.The conditioned medium transfer system was used to study the effects of microglia conditioned medium on the neuron in OGD model in vitro.The culture medium of normal or OGD of BV2 microglia was transfer to the normal or OGD insulted SH-SY5 Y neuron cells.MTT assay was used to evaluated the cell survival of SH-SY5 Y.Results showed that the conditioned medium of OGD insulted BV2 cells could aggravate SH-SY5 Y cells injury,and MLIF treatment conditioned medium enhanced cell survival.2.Real-time PCR and ELISA were used to evaluate the regulation effects of MLIF on BV2 microglia polarization suffered OGD treatment.We found that OGD induced the activation of BV2 microglia,and the expression of M1 markers including CD11 b,CD32,CD86,iNOS mRNA and IL-1β,TNF-a protein significantly increased compared with control group.MLIF treatment inhibited the expression of M1 markers.Compared with OGD group,M2 markers Arg-1,CD206,Fizz-1,Ym-1 mRNA and IL-4,IL-10 protein significantly enhanced in MLIF group.These results implied MLIF treatment could promote the transform of OGD induced BV2 microglia from M1 phenotype to M2 phenotype.3.We used the MCAO method to make rats or mice ischemic/reperfusion model.TUNEL staining was used to detect apoptotic cells in rats brain tissues;HE staining and transmission electron microscope wereutilized to observe the pathological damage in microstructure and ultra structure in the rats brain tissues.Real-time PCR and ELISA were used to detect the expression of M1 or M2 microglia markers in the rats brain tissues.We also used the immunofluorescence to detected the expression of M1(CD16/32)or M2(CD206)microglia markers in the mice brain tissues.It shown that MLIF could inhibit apoptotic cells,improve the ischemic pathological damage in ischemic rats brain tissues.And MLIF could regulate the microglia/macrophage polarization toward M2 phenotype after ischemic stroke.4.The NF-κB pathway and MAPK pathway which associated with MLIF were evaluated by Western blot assay.It found that MLIF could inhibit the OGD induced NF-κB pathway activation,but has no significant effect on MAPK pathway.5.Pull-down assay was used to find the binding protein of MLIF in BV2 cells.The biotin labeling MLIF group and negative group interacted with two equivalent cell lysates respectively.After pull-down assay,we used the SDS-PAGE assay and coomassie brilliant blue staining to find the specific band at 50 kDa.Then we used the western blot assay finally confirmed the 50 kDa eEF1A1 as the MLIF specific binding protein in BV2 microglia.6.We used the siRNA to knockdown the eEF1A1 protein,then study the regulation effects of MLIF on OGD induced BV2 microglia polarization.We found that M1 markers(CD11b,CD 32,iNOS,IL-1β,TNF-a)were significantly increased and the M2 markers(Arg-1,CD206,IL-4,IL-10)were significantly decrease in negative control group.MLIF treatment could inhibited the expression of M1 markers and increase M2 markers compared negative control group,which indicated MLIF may regulated BV2 microglia toward M2 phenotype transform.The regulation effects of MLIF on OGD induced BV2 microglia polarization was inhibited,and the inhibition of MLIF on NF-κB pathway was weaken.This indicated MLIF may regulated OGD induced microglia polarization by targeting eEF1A1.Moreover,we found that the expression of M1 markers in eEF1A1 siRNA group were more significantly up-regulate compared the negative control group;M2 markers in eEF1A1 siRNA group were more significantly down-regulate compared the negative control group.This indicated that eEF1A1 protein could regulate OGD induced microglia toward M2 phenotype transition.Conclusion1.MLIF exerted anti-inflammation effect by regulating actived microglia toward M2 phenotype polarization in vivo and in vitro brain ischemia model,thus exert neuroprotective effect in brain ischemia.2.MLIF promoted microglia toward M2 phenotype polarization by targeting eEF1 A 1 to inhibit NF-κB pathway,thus exert anti-inflammatory effect in brain ischemia.And eEF1A1 may be a new target macrophage/microglia polarization in brain ischemia.