罗非鱼鱼皮肽的体外ACE抑制活性、消化性及分子对接对比

Study on in Vitro ACE Inhibitory Activity, Digestibility and Molecular Docking Comparison of Tilapia Fish Skin Peptide

【目的】分析罗非鱼鱼皮多肽LSGYGP的ACE抑制活性、胃肠道消化稳定性以及与降压药卡托普利在分子对接的对比。【方法】用HHL法测定多肽的IC50值和抑制模式,测定多肽在模拟胃肠道消化中的稳定性,MTT法检测细胞活力,Western blot法检测诱导型一氧化氮合酶(i NOS)、环氧合酶-2(COX-2)、内皮素-1(ET-1)等蛋白表达情况,将多肽、卡托普利分别与ACE进行分子对接对比分析。【结果】LSGYGP的半抑制率(IC50)值为2.57μmol/L,表现为混合非竞争性抑制,4 h内模拟胃肠道消化较为稳定;MTT法验证多肽LSGYGP对HUVEC细胞无毒性作用(P> 0.05),且能明显提高Ang Ⅱ诱导组的HUVEC细胞活力(P<0.01);与对照组相比,随实验组多肽浓度增加,i NOS、COX-2和ET-1的表达明显逐渐减少(P<0.001)。对接结果表明,氢键是LSGYGP与ACE结合结构中的支撑力,而卡托普利通常与ACE的Zn2+离子形成紧密结合的相互作用。【结论】消化稳定的罗非鱼鱼皮多肽可明显抑制血管收缩因子和炎症因子的表达,展现了较好ACE抑制活性,LSGYGP与卡托普利的活性差异可能是由于ACE分子对接的作用力不同,这些可作为研发少副作用ACE抑制剂的药理基础。

【Objective】The ACE inhibitory activity of tilapia skin peptide LSGYGP, gastrointestinal digestive stability and molecular docking comparison with antihypertensive drug captopril were analyzed.【Methods】The IC50 value and inhibition mode of peptide were determined by HHL stability, Stability of peptide in the simulated gastrointestinal digestion was measured.Cell viability was detected by MTT method, protein expression of inducible nitric oxide synthase(iNOS), cyclooxygenase-2(COX-2) and endothelin-1(ET-1) were detected by western blot.Comparison of peptide and captopril with ACE in molecular docking was analyzed.【Results】The IC50 of LSGYGP was 2.57 μmol/L, which showed mixed non-competitive inhibition;Simulated gastrointestinal digestion was stable within 4 h;MTT assay indicated that the peptide LSGYGP had no toxic effect on HUVEC cells(P> 0.05), and viability of HUVEC in Ang Ⅱ-induced group was significantly increased(P<0.01).Compared with the control group, LSGYGP treatment could decreased the expression of i NOS, COX-2 and ET-1 in a dose-dependent manner(P<0.001).Docking results indicated that hydrogen bond is the supporting force in the binding structure of LSGYGP and ACE, while captopril usually forms a tight binding interaction with the Zn2+ ion of ACE.【Conclusion】The digestive-stabilized tilapia fish skin peptide LSGYGP significantly inhibited the expression of vasoconstrictor and inflammatory factors, and exhibited a good ACE inhibitory activity.The difference in activity between LSGYGP and captopril might be due to the different forces of ACE molecular docking, which might provide the pharmacological basis to develop ACE inhibitors with fewer side effects.