Isolation and Purification of Active Peptides from Monkfish Skin and Their Protective Effect on HK-2 Cell Injury
[Objective]To investigate the role of monkfish skin peptides(MSP)on palmitic acid-induced damage to human renal tubular epithelial(HK-2)cells.[Methods]Fresh Lophius litulon skin was selected as test object,the enzyme dosage,extraction time,pH,extraction temperature,and the liguid-to-material ratio were selected as the key factors,and the clearance rate of DPPH·as response value,to optimize the extraction process of MSP.The MSP was separated and purified using ultrafiltration,Sephadex G-25,and a fully automated high pressure preparative purification system.To perform the structural analysis,peptides with stronger antioxidant activity were screened using LC-MS/MS and de-novo sequencing.A PA-induced HK-2 cell damage model was created to assess the effects of MSP on oxidative stress,lipid metabolism,and inflammatory factor levels in HK-2 cells.[Results]The optimum conditions for the extraction of MSP were as follows:the mass ratio of acid protease to pepsin was 2∶1,enzyme dosage was 3 800 U/g,the material-liquid ratio was 1 g∶10 mL,pH 3,and the hydrolysis was carried out at 50℃for 4 h.Under these circumstances,the highest MSP clearance rate on DPPH·was 78.84%.The best antioxidant activity was found in the MSP with a molecular weight of less than 3 ku following ultrafiltration.This component was gathered and separated using Sephadex G-25 to produce three peaks,F1,F2,and F3,among which the F1 fraction had the highest DPPH radical scavenging activity.Following the HPLC separation and purification of the F1 fraction,six absorption peaks were obtained.The F1 fraction was separated and purified by high performance liquid chromatography to obtain six absorption peaks,among which the H2 fraction had the highest intensity,from which three oligopeptides were screened out by amino acid sequence identification,namely Phe-Glu-Ala-Thr-Ser-Gln-Glu-Leu(FEATSQEL,FL-8),Leu-Val-Ser-Cys-Ser-Ser-Leu(LVSCSSL,LL-7)and Pro-Leu-Glu-Asn-Tyr(PLENY,PY-5).The three peptides had different restorative effects in the HK-2 cell model of lipid damage:FL-8 and PY-5 increased the antioxidant capacity of the HK-2 cell model,which resulted in a significant increase in the levels of intracellular glutathione peroxidase(GSH-Px)activity and total antioxidant capacity(T-AOC),a decrease in the fluorescence intensity of reactive oxygen species(ROS)and a significant decrease in the level of malondialdehyde(MDA),whereas LL-7 reduced the levels of GSH-Px and T-AOC,and significantly reduced the level of MDA;as for regulating lipid metabolism,FL-8 and PY-5 reduced the levels of total cholesterol,triglycerides,and LDL-cholesterol to varying degrees,and significantly increased the level of HDL-cholesterol,respectively,whereas on the contrary,LL-7 exacerbated the intracellular accumulation of lipids;FL-8 and PY-5 significantly decreased the level of interleukin-18(IL-18),whereas LL-7 significantly upregulated IL-18(α=0.05).[Conclusion]MSP might reduce HK-2 cell damage caused by PA,which may offer a theoretical and experimental foundation for the use of active peptides from monkfish skin and the development of novel therapeutic medications for the mitigation of lipid nephrotoxicity.
monkfish skinactive peptideisolation and purificationHK-2 cellsprotection
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