| [Objective]Previous studies of our research group have found that Finglimod can play an anti-inflammatory role by promoting the activation of M2-type microglia cells.However,but the influence and mechanism of Finglimod on the neovascularization in cerebral cortex ischemia area and the function of cerebral microvascular endothelial cells are still unclear.Therefore,We will explore the effect of Finglimod on neovascularization in cerebral ischemic area and its possible mechanism on tubule formation,proliferation and apoptosis of cerebral microvascular endothelial cells.[Method](1)In vivo experiments: Male adult wild-type mice were given photochemically induced cerebral ischemia models,which were divided into blank control group,simple modeling group,and Finglimod group.After 24 hours of successful modeling,the mice were intraperitoneally injected with 0.3mg/kg fentanyl every day,and brain tissue were sacrificed at day 14.The immunofluorescence staining(Brdu+CD31)were used to observe the neovascularization in the marginal area of cerebral infarction in each group(corresponding area were taken from the blank control group).(2)In vitro experiments: Primary culture of microglial cells and primary culture of endothelial cells were performed in brain tissue of wildtype lactation mice.Western Blotting were used to detect the expression of Arg-1,CD206,CD16 and iNOS in microglia of each group,and the supernatant of each group were collected.Endothelial cells were placed in a hypoxic incubator to induce Oxygen and Glucose Deprivation model.The number and total length of tubules of endothelial cells in different groups were determined by Cell Tubule Formation Assay.Annexin VFITC/PI double staining flow cytometry were used to detect the apoptosis of different endothelial cells.The proliferation of endothelial cells in each group were observed by Brdu staining.[Result]1.Finglimod can increase the formation of new blood vessels at the cerebral cortex infarct margins.When the mouse model of cerebral ischemia induced by photochemical method were successfully constructed and intraperitoneal injection of Finglimod were given every day for 14 days,The immunofluorescence staining(CD31+Brdu)results were obtained blank control group,simple modeling group,:Compared with the blank control group,the number of neovascularization in the peripheral area of cortical infarction increased in the Finglimod group and the simple modeling group,and the number of neovascularization increased more significantly in the Finglimod group than in the simple modeling group.These results indicate that Finglimod can increase the formation of neovascularization in the cerebral cortex infarct margins.2.It were verified again that Finglimod could play an immunomodulatory role by promoting the transformation of M1-type microglia into M2-type microglia and ensure the inflammatory environment of supernatant of microglia in subsequent experiments.When microglia cells were treated with different drug interventions after 24 hours: Western Blotting results showed that the M2-type related protein expression of microglia such as CD206 and Arg-1 increased,while the M1-type related protein expression of microglia such as CD16 and iNOS decreased after the intervention of Finglimod.The above results further verified that Finglimod can promote the generation of M2-type microglia,inhibit the generation of M1-type microglia,improve the brain inflammatory environment,and ensure the reliability of microglia supernatant used in the next experiment.3.Finglimod can indirectly affects endothelial tubule formation,neovascularization and apoptosis.The supernatant obtained after drug intervention of microglia cells in different groups was further used to intervene the OGD model of endothelial cells for 24h:(1)The results of endothelial tubule formation experiment showed that indirect administration of Finglimod could increase the number and total length of tubule formation of endothelial cells,while direct administration showed no significant difference,indicating that Finglimod indirectly enhanced the tubule formation ability of endothelial cells by intervening in microglial cell typing;(2)The results of Brdu staining showed that indirect administration of fentanyl increased the number of Brdu-infected cells,while the direct administration showed no significant effect,indicating that fentanyl could indirectly promote the proliferation of endothelial cells by reducing inflammation in brain tissue;(3)Annexin V-FITC /PI double staining flow cytometry results showed that neither direct nor indirect administration of Finglimod could reduce the apoptosis of endothelial cells,indicating that Finglimod had no significant effect on the apoptosis process of endothelial cells.[Conclusion]1.Finglimod can increase the production of new blood vessels in the cerebral cortex at the edge of infarction.2.Finglimod can improve cerebral inflammatory environment by promoting the transformation of microglia type M1 to type M2,and then promote the proliferation and tubulogenesis of cerebral blood endothelial cells,but does not affect the apoptosis of endothelial cells. |
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