| Objective: To screen the combination peptide of ouabain from Ph.D.-7 phage display peptide library, and analyze its biological activity, and study the interaction between the ouabain and sodium pump and it's role in essential hypertension which is correlated with endogenous ouabain.Methods: (1) Ouabain combination peptides were screened from Ph.D.-7 peptide library by biopanning.①Affinity screening: Ouabain-ovalbumin compounds were adsorbed in enzyme-labeled plates. After incubation overnight, the plates were blocked with 5% BSA, and incubated with peptide library samples diluted 1:10 in blocking buffer. Non-combination peptides were abandoned and special combination peptides were added into ER2738 enriched culture, the enriched phages were obtained.②Adsorption experiment: Ovalbumin was adsorbed in enzyme-labeled plates and then the plates were cultured with the third round phages, non-combination peptides were collected.③Tite measurement: Each round's infected phages were spreaded on IPTG-Xgal cultivate plates, and cultured 12h and the locus cinereus were counted.④ELISA identification: Enzyme- labeled plates were adsorbed by ouabain-ovalbumin comp- ounds,then the third round's phages, anti-M13 antibody and H2O2 were added by turns. The phages'activity were valuated by A450.⑤The ssDNA of phage positive clone were extracted, then identified by electrophoresis. The DNA sequences of each selected phage was determined,the sequences of amino acid were deduced and analyzed through internet NCBI/BLAST.⑥Homology analysis: The sequences and the homology of the deduced amino acid were analyzed with gene bank.(2)Biological activity of ouabain combination peptide.①Combin- ant activity of 3H-ouabain and OCP was detected by radioligand binding assay.②Growth inhibition effect of ouabain and OCP on endothelial cell line EAhy926 was analyzed by MTT assay.③Microscopic structure changes. The feature of cell death was studied by Hoechst 33342/PI staining, nuclear morphological assessment of dead cells was observed by fluorescence micro- scopy.④The mRNA expression of Na+-K+-ATPaseα1-subunit,β1-subunit, VE-cadherin and Snail was studied by reverse transcription PCR(RT-PCR).Results: (1) Efficiency of screening phage display peptide library for ouabain combination peptides with OVA-OUA combination was 2.0×10-3 % in the first round, 2.1×10-3% in the second round, and 3.8% in the third round. Through three times'biopanning, the specific combination peptide was highly enriched with OVA-OUA combination. The phage peptide library(2×1013 pfu/ml) was absorbed by OVA for three times, and more specific peptide library(1.8×102 pfu/ml) was obtained. Three kinds of peptide were selected. Peptide A(Arg-Cys-Met -Thr-Ser-Arg-Ser) was occupied in 64.3% (9/14) , its homologous protein was a kind of multiprotein complex which participates in cell signal transduction, located in 643-648 site. Peptide B(Leu-Ala-Thr-Thr-Val-Pro-His) was occupied in 16.7% (4/14), homologous protein: carbamyl aspartate, located in 26-32 site. Peptide C(Thr-Ala-Thr-Thr-Llr-Pro-Thr) was occupied in 7.14% (1/14),homologous protein: imagination protein, located in 248-252 site. (2) There was some bonding ability between 3H-ouabain and Ouabain combination peptide (OCP). Regression Equation: B/F=-0.743B+94.5276 (r=-0.768). K=0.836nmol/L, Bmax=127.7fmol/mg protein. Equilibrium constant: 0.836 nmol/L. Receptor density: 127.7 fmol/mg. (3) 10μmol/L ouabain treating for 24h could stimulate the necrosis of EAhy926 cells. The median inhibitive concentration (IC50) of ouabain for 24, 48, 72h on EAhy926 cells was 0.507, 0.126, 0.038μmol/L respectively. Ouabain could inhibit EAhy926 cells growth in a dose and time-dependment manner. (4) OCP could antegia the inhibition action of ouabain on EAhy926 cells. 1μmol/L OCP has stronger protection action on EAhy926 cells. The median inhibitive concentration (IC50) of ouabain for 24, 48, 72h on EAhy926 cells was 0.612, 0.364, 0.174μmol/L respectively. OCP could antegia ouabain in a dose and time-dependent manner. (5) When EAhy926 cells were treated with 0.1μmol/L ouabain for 24～48 hours, the cells became sphericalin shape, detached, obviously defluxion. Apoptotic cells shew nuclear chromatin condensation, chromatin margination and the loss of cell-cell adhesion. When EAhy926 cells were treated with 0.1μmol/L ouabain combined with 0.1 and 1μmol/L OCP respectively for 24～48 hours, more cells had better shape and adhesiveness than ouabain group, and the number of apoptotic cells had decreased. (6) OCP could inhibit the up-regulated expression of Na+,K+-ATPaseα1-Subunit and down-regulated expression of Na+,K+-ATPaseβ1-Subunit induced by ouabain in EAhy926 cells. (7) OCP could inhibit the up-regulated expression of Snail and down-regulated expression of VE-cadherin induced by ouabain in EAhy926 cells.Conclusion: (1) A specific ouabain combination peptide was obtained, it's base sequence was CAACGTACTGGACG T CCACT, and it's amino acid sequence was RCMTSRS. (2) OCP could antegia the growth inhibition and death induction of ouabain in EAhy926 cells in a dose and time-dependent maner. (3) OCP could inhibit the up-regulated expression of Na+, K+-ATPaseα1-Subunit and Snail, and down-regulated expres- sion of Na+,K+-ATPaseβ1-Subunit and VE-cadherin induced by ouabain in EAhy926 cells. These molecular changes may play important role in signal transduction and cell-cell adhesion.
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