Studies On The Protective Effects On Sensory Hair Cells Of Bioactive Peptides From Rapana Venosa

Gentamicin is one of the few thermally stable antibiotics which can effectively treat gram-negative bacterial infection.The use of gentamicin is limited by its ototoxicity.Gentamicin-induced ototoxicity is thought to be related with apoptosis induced by reactive oxygen specie.Therefore,in the field of research and development of ototoxicity preventive drugs,researchers are gradually focusing on active oxygen scavengers.Due to their unique living environment,marine organisms have formed many active substances with novel structures and diverse functions in the process of evolution.Marine bioactive peptides are a kind of antioxidant active substances produced by marine organisms.However,it has not been widely reported whether marine bioactive peptides with antioxidant activity can be used in the prevention and treatment of gentamicin ototoxicity,the mechanism of action.This paper was based on previous research.We selected the Rapana venosa as the research object and extracted polypeptides by semi-bionic enzymatic hydrolysis.After analyzing and identifying the components of the mixed polypeptide,we found that the mixed polypeptide was composed of six active peptides(1-6).These 6 peptides were composed of different amino acids.In vitro DPPH scavenging assay and in vivo antioxidant assay both confirmed that the mixed polypeptide had strong antioxidant activity.Based on the close association between antioxidant and auditory hair cell protection,we used zebrafish as the experimental animals to study the protective activity and mechanism of auditory hair cell of the active peptide derived from Rapana venosa.We counted the mortality rate,hatchability ratio and heart rate.And we used many experimental methods including fluorescent labeling of hair cells,detection of mechano-electrical transduction channel on hair cells,analysis of gentamicin uptake,evaluation of reactive oxygen species accumulation in hair cells,detection of apoptosis,Real-time q PCR and western blot.In this study we found that: 1)The optimal concentration of gentamicin-induced ototoxicity model of zebrafish was 125 μΜ,and the duration was 1 h.2)The mixed peptides exhibited certain protective activity against gentamicin-induced hair cells injury at concentrations of 120,200 and 280μg/m L.3)At the concentration of 50 μg/m L,the 1,2,3 and 6 peptides had certain protective activities against gentamicin-induced hair cells injury.4)At the concentration of 50 μg/m L,the 1,2,4 and 5 peptides showed certain developmental toxicity,while peptides 3 and 6 did not show obvious developmental toxicity.5)After comprehensive analysis of the safety and hair cell protective activity of the six peptides,we chosen 3-peptide as the main object.We found that when the concentration was 10,30 or 50 μg/m L,3-peptide did not affect mechanical-electrotransduction channels on hair cells.But it could reduce the the gentamicin uptake by hair cells,decrease the accumulation of ROS,reduce the occurrence of hair cells apoptosis,and increase the expression of Nrf2,Mn-SOD and Cu/Zn-SOD.In conclusion,a variety of novel polypeptides with antioxidant activity had been discovered from Rapana venosa.The active peptide from Rapana venosa could reduce the uptake of gentamicin by hair cells without damaging the function of mechanical-electrotransduction channels.At the same time,it could also activate the Nrf2-Keap1-ARE signaling pathway by up-regulating the expression of Nrf2 gene.Accordingly,the expressions of target genes such as Mn-SOD and Cu/Zn-SOD which were related to antioxidant were increased.Through these mechanisms mentioned above,the active peptide from Rapana Venosa finally reduced the excessive accumulation of ROS in hair cells and decreased the occurrence of hair cells apoptosis.The hair cell injury caused by gentamicin was weakeded.This study started from the active peptides derived from marine organisms and confirmed that they had a certain application potential in the field of hearing protection.This discovery can provide a new direction for the prevention and treatment of gentamicin-induced deafness and contribute to the realization of high-value utilization of marine biological resources.