Urechis unicinctus derived peptide attenuates erectile dysfunction via up-regulating NO/cGMP pathway in in vitro co-culture system of EA.hy926 and HNPrSM cells

Abstract

According to the recent research, men suffering from erectile dysfunction (ED) are more than 30%. ED is known to be a common phenomenon modulated by multifactorial disorder that is associated with several factors including psychogenic or organic, neurologic, and endocrinologic. Among others, related to the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is one of the important cause that directly influence to ED. Moreover, NO plays important roles in physiological processes, including smooth muscle relaxation. Therefore, this study was designed to investigate the regulatory effect of active peptide extracted from marine natural organism, U. unicinctus on NO/cGMP pathway related to ED in vitro. U. unicinctus has high content of protein (78.5 ± 1.7%) which can be readily hydrolyzed into bioactive peptides. U. unicinctus protein was hydrolyzed by high pressure/temperature and ultra-sonication methods and purified using. fast protein liquid chromatography (FPLC) and reverse phase high performance liquid chromatography (HPLC) to obtain bioactive peptides. The accurate molecular mass and amino acid sequences of the purified peptides was determined using a hybrid quadruple-TOF LC/MS/MS mass spectroscopy as DDL- 362 Da, DGL- 303 Da and DLF- 393 Da. To determine the effect of the peptides on NO/cGMP pathway, endothelial cells and smooth muscle cells were used. Endothelial cells are in contact with the underlying smooth muscle cells and the interaction between endothelial cell and smooth muscle cells in vessel wall are considered to be related with smooth muscle relaxation through NO/cGMP pathway. As a study model a co-culture system of human umbilical vein endothelial cells (EA.hy926) and human normal prostate smooth muscle cells (HNPrSM cells) was used. The results show that the peptide DDL markedly induced the production of NO than other candidate peptides via up-regulating the protein and gene expression levels of endothelial nitric oxide synthases enzyme (eNOS). This was resulted due to DDL mediated induction of intracellular inositol triphosphate (IP3) and Ca2+ levels. In co-culture system the peptide mediated induced NO secretion in endothelial cells up-regulated the cGMP concentrations in HNPrSM cells through NO/cGMP pathway and finally lead to decrease in Ca2+ levels. The decrease in intracellular concentration of Ca2+ in HNPrSM cells induces the smooth muscle cell relaxation. According to these results the peptide DDL could be effectively used to attenuate erectile dysfunction.