| Objective(s): Stroke is the second largest cause of death in the world,and also the main cause of death and disability in adults in China.The incidence rate of stroke has increased rapidly in recent years,which has brought a huge burden to the global medical security and individual families.Cerebral ischemia reperfusion(CI/R)is the main cause of nerve injury after stroke and is one of the factors affecting the prognosis of the disease.However,the existing treatment methods for CI/R injury cannot meet the growing needs of clinical and patient diagnosis and treatment,and its related drugs still need further research and development.In recent years,peptide molecules have attracted wide attention due to their low toxicity,high selectivity,high activity and other characteristics,and gradually become the focus and new focus of drug development.Among the natural sources of peptides,amphibians(especially Ranidae),as the resource pool of bioactive peptides,have attracted more and more attention.However,reports on neuroprotective peptides from amphibians are still extremely scarce.The purpose of this study is to explore a new amphibian-derived neuroprotective peptide that can alleviate CI/R injury,and explore its neuroprotective mechanism from the perspective of cell-tissue-molecular;At the same time,the advantages of peptide molecules as molecular probes are used to explore possible drug targets and endogenous disease pathogenesis with exogenous polypeptide molecules.It provides more possibilities for the treatment of related central nervous system diseases,especially CI/R.Methods: By constructing a c DNA library of the Odorrana livida(O.livida),the peptide molecule OL-FS13 derived from O.livida was obtained.Its stability was explored using reverse phase high-performance liquid chromatography at 4 ℃,25 ℃,37 ℃,and 60 ℃,as well as in plasma.The neuroprotective activity of OL-FS13 in vitro was determined by the oxygen glucose deprivation/re-oxygenation(OGD/R)model and cell proliferation assay(MTS method)of renal pheochromocytoma cells(PC12 cells),and the degree of cell damage was determined by cell survival rate.The in vivo neuroprotective activity of OL-FS13 was evaluated using a rat model of middle cerebral artery occlusion using modified nerve function severity scale(m NSS),triphenyltetrazolium chloride staining,hematoxylin-eosin staining,and Nissl staining.The degree of oxidative stress injury in vivo and in vitro was assessed by the detection of superoxide dismutase(SOD),malondialdehyde(MDA),catalase(CAT)and lactate dehydrogenase(LDH).Detect the expression of proteins in related signaling pathways using protein immunoblotting.Multigroup sequencing of m RNA and mi RNA was used to evaluate the anti stroke mechanism of OL-FS13 from the perspective of non-coding RNA.The targeting relationship between mi R-21-3p and CAMKK2 was detected by double Luciferase method.Detect the expression levels of mi R-21-3p and CAMKK2 m RNA through real-time fluorescence quantitative PCR.Results: OL-FS13 was derived from the skin of O.livida,and the primary structure was FSLLLTWWRRRVC(Molecular weight: 1736.09 Da);The half-life of OL-FS13 in plasma is 19.9 min;OL-FS13 can be maintained stably at 4 ℃ and 25 ℃environment.OL-FS13 could alleviate the cell damage caused by OGD/R(the optimal concentration was 100 p M);OL-FS13 reduced the brain injury of rats induced by CI/R(the optimal effective dose was 10 μg/kg),which was manifested as improved m NSS score,reduced cerebral infarction area and reduced tissue injury;OL-FS13 could increase SOD and CAT levels and decrease MDA and LDH levels in vivo and in vitro;OL-FS13 alleviated oxidative stress by activating the Nrf-2/HO-1 axis,and alleviated endoplasmic reticulum(ER)stress by inhibiting the IRE1α/JNK axis.OL-FS13 enhanced the antioxidant stress system by activating the Nrf-2 pathway,and then reduced the damage of ER stress;Mi R-21-3p/CAMKK2 axis plays a key role in the neuroprotective effect of OL-FS13;OL-FS13 alleviated energy stress by activating CAMKK2/AMPK axis;OL-FS13 activated CAMKK2/AMPK signaling pathway by inhibiting mi R-21-3p,thus achieving neuroprotective effects.Conclusion(s): In this study,a novel neuroprotective peptide(OL-FS13)from the skin of O.livida was reported.OL-FS13 could antagonize the OGD/R damage to PC12 cells,and have a significant easing effect on the nerve damage of caused by CI/R.These neuroprotective effects may be achieved through activation of Nrf-2/HO-1 and inhibition of IRE1α/JNK signaling pathway,thus realizing the inhibition of oxidative stress and ER stress damage.In the anti-stroke process of OL-FS13,Nrf-2 plays a key role in its inhibition of oxidative stress and apoptosis induced by ER stress injury.Further studies showed that OL-FS13 alleviated OGD/R and CI/R injury induced damage by inhibiting mi R-21-3p and activating CAMKK2/AMPK/Nrf-2 axis.This study highlights the potential application value of OL-FS13 in the field of anti-stroke,and provides new possibilities for the research and application of peptides as molecular probes while increasing the number of research subjects for amphibian-derived neuroprotective peptides. |
