Research Of Absorption Characteristics And Molecular Mechanism Of TAT-HaFGF Via Nose-to-brain Transport Pathways

Objective:The aim of the dissertation were focused on the evaluation of the safety andmucosal absorption characteristics of TAT-HaFGF via intranasal administration.Even more important, to study the cellular and molecular mechanism of TAT-HaFGFacross the blood-brain barrier (BBB).Methods:1、Nasal ciliotoxicity studies were carried out using an in situ palate model.After the rats treated withTAT-HaFGF (100,300,600μg/kg) through nose for onetime or lasting5weeks， olfactory nerve marker protein (OMP)immunohistochemical staining and hematoxylin and eosin (HE) staining were testedto evaluate the safety of the drug.2、After rats were administered125I-TAT-HaFGF intranasally, the blood and othertissures were collected after15,30and60min following the start of administration.Drug concentrations in tissues were calculated using a γ counter. Confocalmicroscopy was used to localize the TAT-HaFGF (anti-TAT) and OMP (anti-OMP) ofnasal mucosa.3、The BBB model was build by mouse brain microvascular endothelial cell line(bEnd.3). Elisa, SEM and Western blotting (WB) were used to study the mechanismof TAT-HaFGF through BBB.Result:1、Rats were administrated with TAT-HaFGF (100,300,600μg/kg) intranasallyfor5weeks. The results of HEstaining showed that there were no pathologies in anyof the organs investigated, such as brain, heart, olfactory bulbs liver, spleen, lung,kidney, and noses. OMP immunohistochemical staining demonstrated that there wasno significant difference in the number of olfactory sensory neurons (OSNs) of allintranasal treatment groups. The results of an in situ palate model suggested that thenasal ciliotoxicity of TAT-HaFGF is negligible. 2、Fifteen minutes after intranasal administration of125I-TAT-HaFGF to rats,different concentrations of radioactivity were observed in the hypophysis, olfactorybulb, brainstem and cerebellum. At30min, stronger radioactive signals wereobserved in the brainstem, cerebrum confocal microscopy confirmed that labeledTAT-HaFGF in the olfactory mucosa was mainly localized to the olfactory epithelium,Bowman’s glands, and bundles of olfactory nerves.3、TAT-HaFGF has an obvious advantage compared with haFGF in the ability ofacrossing BBB model. TAT-HaFGF can activate Rho signaling to enlarge the gap ofcells resulting from the reduction of the ZO-1protein.Conclusion:1、The nasal ciliotoxicity of TAT-HaFGF is negligible. The results of OMPimmunohistochemical staining demonstrated that intranasal TAT-HaFGF does notcause damage to tissue structure or the numbers of OSNs.2、The present study demonstrates that TAT-HaFGF can bypass the blood-brainbarrier to reach multiple sites within the brain and spinal cord likely by olfactory andtrigeminal pathways.3、Rho signaling is related to TAT-HaFGF in the molecular mechanism to enlargethe gap of cells resulting from the reduction of the ZO-1protein.