| ObjectiveThe purpose of this study was to explore the potential mechanism of midbrain astrocyte derived neurotrophic factor in improving cognitive impairment in diabetes mice.MethodsForty healthy male C57BL/6 mice were divided into five groups: normal group(CON group,n=8),diabetes group(DM group,n=8),diabetes MANF overexpression group(DM+AAV-MANF group,n=8),MANF virus vector group(DM+AAV-NC group,n=8),diabetes MANF overexpression STAT3 activator group(DM+AAV-MANF+colivelin group,n=8).In addition to the normal control group,other mice were intraperitoneally injected with small dose of streptozotocin(100mg/kg)to establish diabetes model.After the establishment of the model,the DM+AAV-MANF group and DM+AAV-NC group were injected with MANF overexpression adeno-associated virus and MANF empty virus vector into the lateral ventricle by stereotactic brain positioning.After 12 weeks of modeling in each group,water maze test was conducted to monitor the learning and memory ability of mice.Nissl staining was used to observe the morphology of hippocampal neurons in mice.Detection of IL-17 and TGF-β in brain tissue of experimental mice by ELISA Protein expression level.The content of Th17 cells,Treg cells and the ratio of Th17/Treg in the peripheral blood of experimental mice were detected by flow cytometry.The expression of MANF,IL-17,TGF-β,FOXP3,STAT3 and p-STAT3 proteins in hippocampus were detected by Western blot.ResultsIn the diabetes model established by STZ,compared with CON group,the weight of DM group mice decreased,and the blood sugar increased significantly(P<0.05).The difference was statistically significant.In the water maze navigation test,the escape latency of DM group was significantly longer than that of CON group,and the escape latency of DM+AAV-MANF group was significantly shorter than that of DM group(P<0.05);In the space exploration test,compared with the CON group,the number of times the DM group crossed the platform and the percentage of time to stay in the target quadrant decreased significantly,compared with the DM group,the number of times the DM+AAV-MANF group crossed the platform and the percentage of time to stay in the target quadrant increased significantly(P<0.05),the difference was statistically significant.Nissl staining observation showed that the morphology of hippocampal neurons in DM group was sparse,loosely arranged,and the number decreased compared with that in CON group;Compared with DM group,the neurons in DM+AAV-MANF group arranged compactly and increased in number(P<0.05).Flow cytometry showed that compared with CON group,the percentage of Th17 cells in DM group increased and the percentage of Treg cells decreased(P<0.05).The results of ELISA test showed that the expression level of IL-17 in DM group was significantly higher than that in CON group,the expression level of TGF-β in DM group was significantly lower than that of CON group(P<0.05);the expression level of IL-17 in DM+AAV-MANF group was significantly lower than that in DM group,the expression level of TGF-β in DM+AAV-MANF group was significantly higher than that of DM group(P<0.05).Western blot showed that compared with CON group,the levels of IL-17,STAT3 and p-STAT3 in DM group were significantly increased,The expression of TGF-β and FOXP3 was significantly decreased(P<0.05);compared with DM group,the expression levels of IL-17,STAT3 and p-STAT3 in DM+AAV-MANF group decreased significantly,the expression of TGF-β and FOXP3 was significantly increased(P<0.05);compared with DM+AAV-MANF group,IL-17 in DM+AAV-MANF+colivelin group were significantly increased,the expression of TGF-β and FOXP3 was significantly decreased(P<0.05).ConclusionsMANF can improve the learning and memory ability of diabetes mice by regulating Th17/Treg immune imbalance,and its mechanism may be related to the down-regulation of STAT3 expression. |
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