| Objective:(1) To investigate the expressions of endothelin A receptor and calcitonin gene-related peptide in brain vessel after SAH and their relationship with the delayed cerebral vasospasm (DCVS). (2) To explore new methods designing and synthesizing the novel selective polypeptide ETRA antagonists and compare the committed steps and parameters in the course of synthesis and purification, thus provide a simple and feasible way of synthesizing selective polypeptide ETRA antagonists using the solid-phase peptide synthesis (SPPS) technology, moreover, the polypeptides' bioactivity was observed in experimental SAH in vivo model.Methods:(1) According to the structure-function relationship of endothein-1 (ET-1), five novel ETRA agonist hexapeptides were screened by application of phage random peptide library, computer aided design such as Lasergene software and the systematical structural analysis of target sequences. The five novel peptides were isolated and purified by solid-phase synthesis, mass spectrometric detection and reversed-phase high-performance liquid chromatography purified strategy and observed their antagonism in brain vessels in rabbit experimental SAH models in vivo at last. (2) Two hundred and ninety Japanese rabbits were randomized into: blank group (n=35), sham group (n=35), saline group (n=35), SAH group (n=35), the novel ETRA antagonist hexapeptides group (n=150). Each group were divided into five subgroups by different time periods, they were 1h, 3d, 5d, 7d, 10d after SAH. Five rabbits in each subtype were applied to immunohistochemical studies (CGRP and ETRA) while two of them were assessed cerebral vessel ultramicro-structure. There were six subgroups in ETRA antagonists group: typical antagonist (BQ485), SC-01, SC-02, SC-03, SC-04, and SC-05 group (five rabbits in each subgroup). (3) Basilar arteries were obtained from double cisternal blood injection model after animals were sacrificed by perfusion. SABC immunohistochemistry and HE were used to stain the tissue. The expressions of ETRA and CGRP in the basilar artery and its histopathological changes were assessed with light and electron microscopy, and the circumference of the vessels was measured by the image analysis. Furthermore, we studied the antagonistic effect of the novel ETRA antagonists on cerebral vessels and the statistical analysis was performed by the analysis of variance.Results:(1) The basilar artery contracted significantly after SAH especially in 5d and SAH could cause ultrastructure changes which presented dynamic variations in the course of disease. (2) ETRA was expressed a little in the basilar artery in blank, sham and saline groups; it was high in the SAH group especially in 3d subgroup and on vascular endothelial cell. Smooth muscle cell also expressed ETRA. The expression was still high in the 10d group. (3) Meanwhile, scattered CGRP was expressed irregularly on basilar artery in blank, sham and saline groups. Its regularity was as follows: significantly high in lh and decreased in 3d and 5d in turn, became lowest in 5d, then increased from 7d gradually, and was relatively high in 10d but couldn't achieve the normal level yet. (4) In the synthesized five novel selective antagonists, the effect of SC-01 antagonist was obvious compared with the typical antagonist (BQ-485) and other antagonists (P<0.05), and the effect of SC-05 was second only to SC-01(p<0.05). SC-02, SC-03, SC-04 antagonists had no conspicuous distinction with typical antagonist (P>0.05). SC-01 antagonist and SC-05 antagonist attenuated the DCVS obviously after SAH.Conclusions:(1) The expression of CGRP and ETRA on cerebral vessels present dynamic variations after SAH, but their change trend is derangment; The CGRP and ETRA play an important role in DCVS. (2) These results demonstrate that the novel ETRA antagonist hexapeptides can attenuate SAH-induced delayed cerebral vasospasm efficaciously.
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