| Background and ObjectiveAs a serious threat to human health,cerebral ischemia has a high mortality and disability rate.Timely recanalization is the most effective therapy for ischemic stroke.However,the time window for this treatment is only 3-4 hours,so many patients do not receive timely and effective treatment.Basic fibroblast growth factor(bFGF)has been proved to play a neuroprotective and angiogenic role in ischemic brain injury,but its targeted delivery to ischemic brain remains difficult.Continuous up-regulation of connective tissue growth factor(CTGF)after the occurrence of ischemic injury is a potential target for the treatment and administration of cerebral ischemia.Subsequently,targeted peptide CFBP screened by phage display technology was shown to specifically bind to CTGF.In this study,recombinant CFBP-bFGF protein was designed,constructed and prepared,and CFBP-Bf GF could specifically guide the delivery of bFGF to ischemic brain by binding to upregulated CTGF.Finally,we further explored the role of CFBPbFGF in the therapy of cerebral ischemia in rats.Methods1.CFBP-bFGF recombinant proteins were constructed by genetic engineering and purified by nickel column,and the purity of recombinant proteins was detected by Western blot and SDS-page.2.The proliferative ability of human skin fibroblasts(HSFs)was detected by MTT assay to detect the activity of CFBP-bFGF recombinant protein.The OGD/R model was constructed with PC12 cells to verify that CFBP-bFGF could target neurons producing CTGF from hypoxia and play a protective role.3.The back of rats was subcutaneously embedded with a gel sponge and divided into PBS group,bFGF group and CFBP-bFGF group.14 days later,the tissue blocks were removed for HE and α-SMA staining to verify the in vivo angiogenesis promoting by CFBP-bFGF.4.The model of right middle cerebral artery occlusion(MCAO)was prepared by thread embolization method and divided into PBS group,bFGF group and CFBPbFGF group.The expression of CTGF was detected by Elisa,Western blot and in vivo fluorescence imaging.The immunofluorescence double-staining of bFGF and CTGF in ischemic brain tissue was detected the co-location of CFBP-bFGF and CTGF.5.The recovery of motor function of rats was evaluated by open field and rotating rod.6.H&E and Nissl staining were used to detect pathological changes of cerebral ischemia sites.Immunofluorescence was used to detect the inflammatory response,angiogenesis,apoptosis and neuronal survival at the site of cerebral ischemia injury.NeuN antibody was used to mark the survival of neurons.vWF antibody was used to detect the angiogenesis of ischemic brain tissue.CD68 antibody was used to detect macrophage inflammatory response.TUNEL staining was used to evaluate apoptosis in ischemic injury sites.7.Tissue protein was extracted and Western blot was used to detect apoptosis and the downstream pathway of regulation.BCL-2 and Caspase-3 antibodies were used to detect apoptosis,and ATK/p-AKT was used to detect the regulation of CFBP-bFGF on the downstream pathwayResults:1.The molecular weight of CFBP-bFGF recombinant protein was about 21 KD,which was slightly larger than bFGF.2.In the in vitro proliferation experiment of HSFs detected by MTT method,cells were cultured in medium containing the same gradient concentration of bFGF and CFBPbFGF fusion proteins for 2 days.bFGF and CFBP-BFGF fusion proteins had similar ability to promote the proliferation of HSFs cells in vitro,indicating that after the fusion of CFBP peptide,the biological activity of CFBP-bFGF was not affected.In addition,it has been reported that the expression of CTGF in PC12 cells is upregulated after hypoxia.Subsequent PC12 cells were cultured in the medium containing the same gradient concentration of bFGF and CFBP-bFGF fusion protein,and were reoxygenated for 18 h after 6h of hypoxia.The results showed that low concentration of CFBP-bFGF and bFGF had similar protective effects on hypoxic PC12 cells.However,the protective effect of high concentration of CFBP-bFGF was superior to that of bFGF.We speculated that CFBP-bFGF could better play its protective effect by targeting and binding to upregulated CTGF after hypoxic injury.3.Western blot detected the up-regulated expression of CTGF in rats after skin injury.As CTGF was upregulated,more CFBP-bFGF could be detected in the injured site.After 14 days of subcutaneous embedding in the back of rats,the results of section staining showed that CFBP-bFGF exhibited better angiogenesis function than bFGF.Combined with the targeting of CTGF by CFBP-bFGF,it was proved that CFBPbFGF could better exert its biological activity.4.The MCAO model was prepared by the suture-occluded method.Zea-Longa score was used after the rats were fully awake,and the rats with scores of 1-3 were included in the follow-up experiment.Protein was extracted to detect CTGF expression,and the results showed high expression at 3 days after operation.The fluorescent dye labeled protein was injected into the caudal vein and then in vivo fluorescence imaging was performed.The results showed that CTF-BFGF accumulated in the brain.This result was validated by the fluorescence observation of frozen section,that the injured right brain had higher fluorescent intensity.Consistent with the fluorescence,Elisa assays showed at 6h and 24 h after recombinant protein injection,significant more bFGF was detected in the ischemic brain of CFBP-bFGF group compared with bFGF and PBS group.Finally,the co-location of bFGF and CTGF fluorescence staining showed most of bFGF in CFBP-bFGF group was colocalized with CTGF but there was less colocalization of bFGF and CTGF in bFGF group and PBS group,with significant difference.These results revealed CFBP-bFGF could target to the ischemic brain through interacting with the upregulated CTGF.5.Behavioral tests were performed 1 and 2 weeks after surgery,including open field test and rod rotation,and the results showed that the recovery of rats in CFBP-bFGF group was better than that in bFGF group and PBS group.6.Two weeks later,the brain was taken for paraffin section.HE staining and Nissl staining showed that the necrosis of infarct cells in the CFBP-bFGF group was lighter than that in the bFGF and PBS groups.Immunofluorescence of CD68,vWF and NeuN showed that the CFBP-bFGF group could reduce inflammation,promote the formation of blood vessels and protect nerve cells,thus protect brain tissue.7.TUNEL staining of paraffin sections of brain tissue 2 weeks later showed that CFBPbFGF could effectively inhibit cell apoptosis.2 weeks later,protein was extracted from brain tissue for Western blot,and BCL-2,Caspase-3 and ATK/p-AKT antibody were used for detection.The results showed that the expression of apoptosis was inhibited in the CFBP-bFGF group,and ATK/p-AKT pathway might involve in the regulation of CFBP-bFGF.ConclusionsIn present study,a specific peptide was used to modify bFGF to construct recombinant CFBP-bFGF,and CFBP-bFGF could specifically deliver to ischemic brain through binding with the upregulated protein-connective tissue growth factor(CTGF).When CFBP-bFGF was used in rats with cerebral ischemia by intravenous injection,local concentration of the bFGF in ischemic brain was significantly increased.In addition,enhanced neurons survival,increased angiogenesis,decreased neuroinflammation were observed,that improved the motor functional recovery of cerebral ischemic injury.These results demonstrated that the targeting delivery of CFBP-bFGF would be a potential therapeutic approach for cerebral ischemia. |
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