| Background and purpose:Microglia are the innate immune cells of the nervous system,which play an important role in the occurrence and development of many diseases of the central nervous system.Different polarization phenotypes of microglia are critical to the progression of injury and the outcome of nerve recovery after ischemic stroke,which is influenced by the dynamic changes of the microenvironment at the ischemic site.Previous studies have found that artificial induction of M1-type microglia cells into M2-type microglia cells can reduce the infarct size and improve neurological function recovery of transient cerebral ischemia mouse models.The effect of different polarization phenotypes may explain the duality of microglia under different pathophysiological conditions of the same disease.Therefore,it is one of the important research directions in the treatment of ischemic stroke to seek research schemes to inhibit the polarization of M2-type cells to M1 type,or induce the transformation of M1 type to M2 type,or improve the ratio of M2 type /M1 type.IL-4 is mainly produced by activated T cells and has a regulatory effect on a variety of immune cells in the body.IL-4 stimulates the transformation of macrophages and microglia into an anti-inflammatory phenotype,thereby inhibiting the progression of inflammation,promoting tissue repair and playing a neuroprotective role.At present,IL-4 has been used in the study of cerebral hemorrhage,spinal cord injury,epilepsy,Alzheimer's disease,and many other neurological diseases.This study was designed to investigate the therapeutic value of IL-4 in ischemic stroke.Methods:1.Transient middle cerebral artery occlusion(t MCAO)rat model was established and divided into sham operation group and model group.Changes of inflammatory factors such as TNF-?,IFN-?,IL-4,TGF-?,and BDNF in peripheral blood of rats were detected by ELISA.The polarization phenotypes of microglia were detected by immunofluorescence.Peripheral blood samples of patients with acute cerebral infarction and healthy adults were also collected to detect the changes of inflammatory factors by ELISA.2.t MCAO rat model was established and divided into sham operation group,model group,IL-4 group,and IL-4+AS1517499 group.Neurological function score was performed 3 days later.Peripheral blood of rats was collected to detect the changes of inflammatory factors.The rats were sacrificed,TTC staining was used to calculate the infarct volume,microglia polarization phenotypes were detected by immunofluorescence,apoptosis was detected by TUNEL staining,and neuronal injury was detected by Nissl staining.3.HAPI microglia cell line was cultured and the microglia cell oxygen glucose deprivation(OGD)model was established and divided into normal culture group,OGD group,OGD+IL-4 group,and OGD+IL-4+ AS1517499 group.The changes of inflammatory factors such as IL-1?,IL-2,TNF-?,IL-10,TGF-?,and BDNF in supernatant of cell culture were detected by ELISA.The expression levels of JAK1,p-JAK1,STAT6,and p-STAT6 were detected by Western blot.The polarization phenotypes of microglia were detected by immunofluorescence.The endocytosis of microglia was detected by fluorescence microsphere method.4.The RN-C neuronal cell line was cultured,and the OGD model of neurons was established.The neurons were co-cultured with pretreated microglia cells and divided into RN-C normal culture group,RN-C+HAPI group,RN-C+HAPI IL-4 group,and RN-C+HAPI IL-4+AS1517499 group.Flow cytometry was used to detect neuronal apoptosis,JC-1 method was used to detect the changes of neuronal mitochondrial membrane potential,and Western blot was used to detect the expression of neuronal apoptosis proteins.5.Statistical analysis was performed using Graph Pad Prism9.Continuous variables were expressed as mean ± standard deviation,t-test was used for comparison between two groups,and one-way analysis of variance was used for comparison between multiple groups.Nonparametric test(Kruskal-Wallis test)was used for neurological function assessment,and Dunn's multi-group comparison was used for comparison between multiple groups.P<0.05 was considered statistically significant.Results:1.Compared with healthy controls,the levels of IFN-?,TNF-?,TGF-?,BDNF,and IL-4 in the blood of patients with cerebral infarction were significantly increased,among which the levels of IFN-?,TNF-?,BDNF and,IL-4 were most significantly increased on the first day after the infarction,and began to decrease on the third day after the infarction,but were still significantly higher than those of healthy controls.2.Compared with the sham operation group,the levels of IFN-?,TNF-?,and IL-4 in the blood of the model group were all increased.The level of BDNF decreased.After the administration of IL-4,the levels of IL-1?,IL-2,and TNF-? were decreased,while the levels of IL-10,TGF-?,and BDNF were increased.3.Compared with the sham operation group,the activation of microglia in the ischemic penumbra was increased in the model group,and both M1 and M2 phenotypes were increased.M2-phenotype microglia increased after administration of IL-4,and decreased after administration of IL-4+AS1517499.4.Compared with the sham operation group,there were obvious cerebral infarction in each model group.Compared with the model group,the cerebral infarct volume of rats was decreased after IL-4 administration,while the cerebral infarct volume of rats was increased after IL-4+AS1517499 administration.5.Cell apoptosis was detected by TUNEL staining.Compared with the sham operation group,the apoptosis of cells in the ischemic penumbra in the model group was significantly increased,and the apoptosis was significantly decreased after the administration of IL-4.Nissl staining was used to detect neuronal repair.Compared with the sham operation group,neuronal Nissl bodies were significantly reduced in the model group,increased after IL-4 administration,and decreased after IL-4+AS1517499 administration.6.Compared with the normal group and the OGD group,the expression of CD86 was down-regulated and the expression of CD163 was increased after the administration of IL-4;the expression of CD86 was up-regulated and the expression of CD163 was decreased after the administration of IL-4+AS1517499.Compared with the normal group and the OGD group,phosphorylation of JAK1(p-JAK1)and phosphorylation of STAT6(p-STAT6)were increased in microglia after IL-4administration,and phosphorylation of JAK1 was not changed and phosphorylation of STAT6 was decreased after administration of STAT6 phosphorylation inhibitor AS1517499.Compared with the normal group and the OGD group,the expression of IL-1?,IL-2,and TNF-? were significantly down-regulated,and the expression of IL-10,TGF-? and,BDNF were significantly increased after the administration of IL-4.After the administration of IL-4+AS1517499,the expressions of IL-1?,IL-2,and TNF-? were increased,while the expressions of IL-10,TGF-? and,BDNF were decreased.7.Compared with the normal group and the OGD group,the endocytosis of microglia was increased after the administration of IL-4,while the endocytosis was decreased after the administration of AS1517499.8.In the co-culture model of microglia and neurons,compared with the Control group,the RN-C cells in the OGD group were significantly increased,and the apoptosis of RN-C was significantly reduced and the expression of apoptotic proteins were down-regulated after IL-4 treatment.Compared with the IL-4 group,the apoptosis and expression of apoptotic proteins were increased in the IL-4+AS1517499 group.Conclusions:1.IL-4 promotes microglial polarization to M2 phenotype through JAK1-p JAK1-STAT6-p STAT6 signaling pathway.2.IL-4 can improve the neurofunctional prognosis of rats following ischemic stroke.After blocking the STAT6 phosphorylation pathway,the therapeutic effect of IL-4 is reversed.IL-4 can reduce the infarct volume,decrease neuronal apoptosis,increase the mitochondrial membrane potential,and down-regulate the expression of apoptotic protein in the ischemic stroke rat model.After blocking the phosphorylation pathway of STAT6,the effect of IL-4 is reversed,suggesting that IL-4 can play a neuroprotective role by promoting the polarization of microglia to M2 phenotype.3.After the administration of IL-4,microglial secretion of pro-inflammatory factors(IL-1?,IL-2,TNF-?)decreased and secretion of anti-inflammatory factors(IL-10,TGF-?,BDNF)increased.The microglial phagocytosis was enhanced.However,these effects were reversed by blocking the phosphorylation pathway of STAT6.These results suggest that microglia may play a neuroprotective role in ischemic stroke by inhibiting neuroinflammation and increasing endocytosis. |
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