The Effect Of PGRN On Angiogenesis After Cerebral Ischemia And Its Molecular Mechanism

Cerebral ischemia is a metabolic and dysfunctional disease caused by reduced cerebral blood flow.The ischemic lesions formed after cerebral ischemia is composed of the ischemic core and the ischemic penumbra at the edge of the core region,can protect and rescue the reversible tissue cell damage in the ischemic penumbra.These lesions also can promote the recovery of blood flow in the ischemic injury and play an important role to improve the recovery of cerebral ischemic disease.Cerebral ischemia and hypoxia can induce the angiogenesis of damaged tissues in the central nervous system(CNS),and can be disposed as a compensatory mechanism to repair the blood flow recovery after cerebral ischemia injury.However,some studies have shown that new blood vessels are insufficient to improve the recovery of nerve function after ischemia.Therefore,promoting angiogenesis after cerebral ischemia is vital on the recovery of the disease.Progranulin(PGRN)is a secreted N-linked glycoprotein,it involved in many different biological processes,ranging from embryonic development to organization structure maintenance and adult tissues repair.It is considered to take effect in maintaining normal physiological function and neuronal survival in the CNS,and can protect hypoxic-ischemic brain injury by three mechanisms,inhibiting the destruction of blood brain barrier(BBB),inhibiting neuroinflammation and promoting cell proliferation and differentiation.Studies have shown that PGRN can be expressed in capillary endothelial cells of wound granulation tissue,and it facilitates mitosis and migration of adult skin microvascular cells with tumor angiogenesis.In addition,PGRN can also directly affect endothelial cells cultured in vitro,enhancing cell proliferation,migration,and the capacity of tubes formation.Therefore,we speculated that PGRN might protect ischemic brain injury by promoting vascular regeneration after cerebral ischemia.In this study,we first established a model of permanent middle cerebral artery occlusion(pMCAO)in C57BL/6 mice with the method of line occlusion,and compared endogenous PGRN and neovascularization expression in the ischemic penumbra of mice by immunofluorescence staining.The expression of VEGF and the changes of Erk1/2 and AKT signaling pathways were detected by western blot.Then,HUVEC and bEnd.3 hypoxia models were established in vitro to explore the effects of PGRN on endothelial cell proliferation,migration and tubes formation.Finally,the molecular mechanism through which PGRN promotes vascular regeneration of endothelial cells after hypoxia was further verified by western blot.Research results:1.The endogenous PGRN expression of penumbra in mice increased after cerebral ischemia.Compared with mice in the sham operation group,the levels of endogenous PGRN expression in the ischemic penumbra at 3 d,7 d and 14 days after ischemia were significantly increased(P<0.001;P<0.001;P<0.001).2.PGRN promoted the expression of CD31-positive cells and BrdU+/CD31+cells in the ischemic penumbra after cerebral ischemia in mice.After injecting 1ng of RhPGRN into the lateral ventricle,the expression of CD31-positive cells in the ischemic penumbra was significantly increased after 3 d,7 d and 14 d of ischemia in the PGRN group compared with the mice in the PBS group(P<0.05;P<0.05;P<0.05);the expression of BrdU+/CD31+cells also increased significantly(P<0.001;P<0.001;P<0.001).3.PGRN promotes the increase of Erk1/2 and AKT phosphorylation in ischemic penumbra after cerebral ischemia in mice.After injecting 1ng of RhPGRN into the lateral ventricle,the expression of VEGF protein in the PGRN group was not significantly different from that of ischemic mice after 7 days of ischemia,but the expression levels of Erk1/2 and AKT phosphorylation were significantly higher than those of ischemic mice after 7 days of ischemia(P<0.001;P<0.001).4.PGRN improves endothelial cell proliferation,migration,and tube formation after hypoxia.After HUVEC and bEnd.3 were cultured for 24 h and 12 h after hypoxia,respectively;the result showed that PGRN could significantly improve the cell survival rate after hypoxia(P<0.01;P<0.05);After 24 h of HUVEC hypoxia,PGRN could significantly improve the cell migration ability(P<0.001).Moreover,after 24 h of HUVEC hypoxia,PGRN could significantly improve the cell tube-forming ability,and the number of nodes,cross-over points,and total tube length of tube formation increased significantly compared with the simple hypoxia group(P0.001;P<0.001;P<0.001).5.PGRN promotes Erk1/2 and AKT phosphorylation in HUVEC cells after hypoxia.After 24 h of HUVEC hypoxia,the CD31 protein expression in the PGRN group was significantly higher than that in the control group(P<0.05);the expression of VEGF protein was almost unchanged compared to the simple hypoxia group;the phosphorylation levels of Erk1/2 and AKT were significantly increased compared with the control group(P<0.01;P<0.01).Research conclusions:1.PGRN can promote angiogenesis after cerebral ischemia.2.PGRN may promote angiogenesis by activating Erk1/2 and AKT signaling pathways.This study not only proved the protective effect of PGRN on cerebral ischemic injury,but also laid a theoretical foundation for the study and treatment of the molecular mechanism of the disease.