| Stroke is a disease with high morbidity,mortality and disability,which seriously endangers people's health.Neuronal death caused by insufficient blood supply is the main pathology of ischemic stroke.Therefore,it is an important therapeutic strategy to promote vascular repair and remodelling to restore cerebral blood flow in ischemic regions of brain tissue,and angiogenesis is an important way to increase blood flow after cerebral ischemia.Dl-3-N-butylphthalide(NBP)is a small molecule drug independently developed in China for the treatment of ischemic stroke.Previous studies have shown that NBP has a multi-target effect,which can inhibit apoptosis,platelet,thrombosis,antioxidant damage and so on.The role of NBP in promoting angiogenesis has been proved to increase the number of blood vessels and the expression of angiogenic growth factors,whereas the underlying mechanism remains unknown.In this study,we combined in vivo and in vitro experiments to explore the role of NBP in promoting angiogenesis and its possible mechanism.We simulated clinical ischemic stroke by using the rat model of transient middle cerebral artery occlusion with suture and evaluated the therapeutic effect of NBP by detecting the cerebral infarction volume,weight and neurobehavioral function scores.The number of CD31~+microvessels and CD31~+/BrdU~+endothelial cells were detected by immunofluorecent staining.The perfused vascular density was detected by synchrotron radiation angiography.The expression of angiogenic growth factors was detected by western blot.In addition,the effect of NBP treatment on the expression of sonic hedgehog was also investigated by Real-time PCR,western blot and immunofluorescence staining in brain tissue and in cultured astrocytes.In this study,siRNA was also used to interfere with the expression of sonic hedgehog in astrocytes in vitro to explore whether the expression of angiogenic growth factors and pro-angiogenesis effect after NBP treatment were regulated by astrocytic sonic hedgehog.The results showed that compared with oil control group,NBP could reduce the cerebral infarction volume(p<0.01),alleviate weight loss(p<0.05),promote the recovery of neurobehavioral function(p<0.05)and improve the prognosis of rats after transient middle cerebral artery occlusion.The number of CD31~+microvessels,the number of CD31~+/BrdU~+endothelial cells and the perfused vascular density increased after NBP treatment(p<0.05).The expression of angiogenic growth factors were also increased in NBP-treated group(p<0.05).NBP treatment could promote the expression of hedgehog protein in astrocytes in vivo and in vitro(p<0.05).Besides,NBP-treated group promoted human umbilical vein endothelial cells proliferation,migration and tube formation,which were reversed after sonic hedgehog knockdown with siRNA(p<0.05).NBP promoted angiogenesis through the upregulation of sonic hedgehog expression in astrocytes,which further increased the expressions of VEGF and Ang-1.With the further understanding of possible therapeutic mechanism,NBP may have a better clinical application and prognosis. |
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