Effect And Mechanism Of Earthworm Extract On Promoting Angiogenesis Of Brain Microvascular Endothelial Cells By Regulating Microglia Polarization

Severe ischemic stroke can lead to disability and death.During treatment,the blood flow in the ischemic area can be restored,and the most effective way is to promote collateral circulation,which is strongly associated with the prognosis and can reduce the risk of hemorrhage.Angiogenesis belongs to the third-grade collateral circulation.Microglia cells can affect the formation of new blood vessels.Microglia is activated rapidly after ischemic stroke,and they can express pro-inflammatory cytokines in large quantities,trigger the rapid disintegration of blood vessels,and can interact with many other cells in the central nervous system to regulate the proliferation of brain endothelial cells and promote angiogenesis in a biphasic manner,and participate in tissue repair.Therefore,it may be a therapeutic strategy for stroke by promoting the transformation of microglia to repair phenotype.In modern pharmacological research,earthworms can exert anti-platelet aggregation and anti-inflammatory effects,and are usually widely used in the treatment of brain injury diseases caused by stroke and thrombosis.The purpose of this study is to explore the phenotypic transformation effect of Earthworm extract on microglia and its influence on angiogenesis,so as to provide experimental evidence for strengthening the rational use of earthworm in clinical medicine.Objective:1 Explore the role and mechanism of Earthworm extract in inhibiting microglia inflammation and promoting phenotypic transformation.2 Explore the angiogenesis effect and mechanism of Earthworm extract by stimulating the polarization of microglia.Method:1 BV-2 cells were cultured in vitro with LPS to establish an inflammation model,and the Earthworm extract was added for intervention.NO kit and CCK-8 kit to screen the optimal drug concentration;immunofluorescence method to detect the protein expression of CD16;Reactive oxygen species detection kit to detect the expression of ROS;ELISA method to detect the secretion of IL-1β,TNF-α and IL-6 in the cell culture medium,Western Blot method to detect IL-1β protein expression;RT-PCR method to detect IL-1β,TNF-α and i NOS m RNA expression;immunofluorescence method to detect CD206 protein expression;ELISA method to detect the secretion of IL-10 and VEGF in the cell culture medium;Western Blot method to detect IL-10 and TGF-β protein expression;RT-PCR method to detect IL-10 m RNA expression;Western Blot method to detect the phosphorylation level of AKT and NF-κB.2 Human brain microvascular endothelial cells HCMEC/D3 were cultured in vitro,hypoxia-reoxygenation model was established to simulate ischemiareperfusion injury,and microglia conditioned medium treated with Earthworm extract was added during reoxygenation.CCK-8 kit to detect brain microvascular endothelial cell proliferation;Scratch method to detect brain microvascular endothelial cell migration;Matrigel tube formation method to detect the formation of cerebral microvascular endothelial cells;Western Blot method to detect VEGF,MMP-9,Ang-1,Ang-2,p-Tie-2 protein expression.Result:1 Earthworm extract can significantly inhibit LPS-induced protein expression of M1 type microglia marker CD16(P<0.05),and reduce the release of inflammatory factors such as NO,ROS,IL-1β,TNF-α,IL-6(P<0.05),reduce the protein expression of IL-1β and m RNA expression levels of TNF-α and i NOS(P<0.05).Earthworm extract can significantly increase the protein expression of M2 type microglia marker CD206(P<0.05),and it can significantly increase the m RNA and protein expression levels of the anti-inflammatory factor IL-10(P<0.05).increase the release of IL-10 and VEGF(P<0.05);increase the protein expression level of TGF-β(P<0.05);significantly reduce the phosphorylation level of AKT and NF-κB(P<0.05).2 The conditioned medium of microglia treated with Earthworm extract can significantly promote the proliferation and migration of brain microvascular endothelial cells(P<0.05);increase the tube length and the number of branch points(P<0.05);upregulate the protein expression of VEGF,MMP-9,Ang-1,and p-Tie-2 in brain microvascular endothelial cells(P<0.05),and reduce the protein expression of Ang-2(P<0.05).Conclusion:1 Earthworm extract can inhibit LPS-induced M1 type microglia inflammatory response and promote the expression of M2 type microglia anti-inflammatory factors.The mechanism may be related to the inhibition of the AKT/NF-κB pathway.2 Earthworm extract can promote the proliferation,migration and tube formation of brain microvascular endothelial cells by modulating the polarization of microglia.The mechanism may be related to the activation of the Ang-1/Tie-2 pathway.