| Background and ObjectiveIschemic cerebrovascular disease remains the leading cause of death and long-term disability.Timely vascular recanalization is the most effective treatment approach at present,but most patients cannot receive vascular recanalization treatment for various reasons,while promoting angiogenesis and neuroprotection is an effective way to promote functional recovery.Vascular endothelial growth factor(VEGF)is an effective proangiogenic factor and has a neuroprotective effect.Recently,a short peptide(PR1P)derived from the extracellular VEGF-binding domain of the glycoprotein Prominin-1 is reported to bind specifically to VEGF and promote the action of VEGF.In order to realize the sustained release of VEGF,combining with the targeting function of collagen hydrogel by collagen binding domain(CBD),we constructed a bi-functional peptide-CBD-PR1 P.Through specific binding of CBD and PR1 P with collagen and VEGF respectively,VEGF was targeted into collagen hydrogel to limit the diffusion of VEGF and prolong the release of VEGF.The collagen hydrogel containing CBD-PR1 P and VEGF was injected into the cerebral ischemic cortex of rats,and the therapeutic effect of CBD-PR1P+VEGF/collagen targeted delivery system on cerebral ischemic injury of rats was observed.Methods1.The binding capacity of CBD-PR1P/PR1 P peptide and collagen hydrogel,CBDPR1P/PR1 P peptide and VEGF,and the sustained release effect of VEGF in collagen hydrogel were detected by Elisa.2.MTT assay was used to detect the proliferation of HUVECs,and Matrigel assay was used to detect in vitro tubule formation,to evaluate the effect of CBD-PR1 P peptide on the biological activity of VEGF.3.The right middle cerebral artery occlusion(MCAO)in rats was induced by Nylon thread,and cerebral ischemia cortex was injected for treatment,which were divided into Model group,VEGF/collagen group,CBD-PR1P+VEGF/collagen group and Sham group.4.Functional recovery of rats was evaluated by behavioral tests.5.HE staining and Nissl staining were used to detect the histopathological changes in the ischemic area,and TTC staining was used to evaluate and compare the changes of infarct volume.6.Anti-CD68 antibody was used to detect inflammatory response;Anti-Integrinβ-1antibody was used to detect cell adhesion;Anti-VEGFR-2 antibody was used to label VEGF receptor 2;Anti-v WF antibody was used to detect cerebral angiogenesis on the ischemic side;Tunel staining was used to evaluate the apoptosis of cells around infarction;Anti-Neu N antibody was used to label the survival of neurons around infarction.Results:1.It was confirmed by Elisa that CBD-PR1 P peptide could bind more PR1 P peptide in collagen hydrogel through specific binding of CBD to collagen,and more VEGF could be retained in collagen hydrogel through specific binding of PR1 P peptide to VEGF,further prolonging the release of VEGF.2.Proliferation of HUVECs was detected by MTT assay,and cells were grown in medium containing VEGF and VEGF+CBD-PR1 P peptide for 2d,3d,4d.The results showed that CBD-PR1 P peptide did not affect the biological activity of VEGF,and the proliferation of HUVECs in the medium containing CBD-PR1 P peptide was slightly faster than that in the medium VEGF only.And the results of in vitro angiogenesis showed that CBD-PR1 P peptide did not affect the angiogenesis activity of VEGF in vitro.3.After the preparation of MCAO model,Zea-Longa scoring was performed after the rats were fully awake,and scores of 1-3 were included in subsequent experiments.4.Behavioral tests were performed 1 and 2 weeks after surgery,including open field test and claw strength test,and the results showed that the rats in the CBDPR1P+VEGF/collagen group recovered better than the VEGF/collagen group and Model group.5.2 weeks later,paraffin sections of brain tissue were histologically stained,HE staining and Nissl’s staining showed that the degree of cell necrosis in the infarction area in the CBD-PR1P+VEGF/collagen group and VEGF/collagen group was lighter than the Model group,and the cells around the infarction area were more orderly arranged.And the results of TTC staining showed the infarct volume of CBD-PR1P+VEGF/collagen group and VEGF/collagen group was significantly smaller than that of Model group.6.2 weeks later,CD68,Integrinβ-1,VEGFR-2,v WF and Neu N immunofluorescence staining and Tunel staining were performed on paraffin sections of brain tissue.The results showed that compared with VEGF/collagen group and Model group,CBDPR1P+VEGF/collagen group could better reduce inflammation,promote angiogenesis and neuron survival,reduce cell apoptosis,thus protecting brain tissue.ConclusionsIn summary,CBD-PR1 P peptide can target VEGF to injectable collagen hydrogel and control the release of VEGF.CBD-PR1P+VEGF/collagen delivery system could reduce inflammation,promote angiogenesis and neuron survival,reduce cell apoptosis,and improve muscle strength and motor function in rats with cerebral ischemia injury,thus providing a promising therapeutic strategy for cerebral ischemia. |
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