Studies in anti-inflammatory effect of spinorphin, an endogenous factor

已知Spinorphin对中枢神经系统中的多种脑啡肽降解酶如中性内肽酶、氨肽酶和二肽基肽酶III（DPPIII）具有抑制活性。本研究采用特异性底物（Arg-Arg-β-naphthylamide）和抑制剂，研究了从牛脊髓中纯化的内源性多肽Spinorphin对人中性粒细胞（PMN）的抗炎作用，并对Spinorphin对人PMN的致敏作用和人血清中Spinorphin的定量方法进行了研究。(1)刺激物诱导的PMNsSpinorphin（1μM）单独使用时不诱导超氧化物的产生。然而，通过与FMLP，一种炎症刺激剂的二次刺激，活动显着增强相比，未处理的控制。流式细胞仪分析表明，与对照组相比，Spinorphin诱导的细胞表面粘附分子CD 11b和CD 18的表达明显增加，提示Spinorphin诱导的细胞表面粘附分子的表达增加可能是其致敏作用的部分原因。(2)阿匹诺啡对二肽基肽酶Ⅲ酶活性的影响（3）酶联免疫吸附法测定棘啡的含量用匙孔血蓝蛋白（KLH）标记的棘啡免疫家兔，制备抗棘啡抗体。建立了棘啡肽的ELISA定量方法。抗spinorphin抗体在此方法中没有与其类似物完成。人血清中Spinorphin的基础水平为0.23±0.25 pmol/ml。从这些结果来看，Spinorphin可能是炎症中的肽调节剂。

It has been known that spinorphin has the inhibitory activity toward various enkephalin-degrading enzymes, such as neutral endopeptidase, aminopeptidase and dipeptidyl peptidase III (DPPIII) in central nerve system. In this study, the inhibitory activity toward DPPIII on human PMNs was found using a specific substrate (Arg-Arg-β-naphtylamide) and inhibitors.To characterize anti-inflammatory effect of spinorphin, an endogenous peptide purified from bovine spinal cord, priming of spinorphin on human neutrophils (PMNs) and quantitative method of spinorphin in human serum were investigates.(1) Priming effect of superoxide generation by stimulant-induced PMNsSpinorphin (1μM) alone did not induce superoxide generation. However, by secondary stimulation with FMLP, an inflammatory stimulant, the activity was markedly augmented compared to that of nontreated control. From FACS analysis, expression of CD11b and CD18, adhesion molecules was significantly increased compared with that of control, suggesting that the priming effect of spinorphin is in part due to increase of expression of adhesion molecules.(2) Effect of apinorphin on dipeptidyl peptidase III enzymatic activity(3) Quantitative method of spinorphin by ELISAAnti-spinorphin antibody was produced in rabbits immunized with keyhole limpet hemocyanin (KLH)-conjugated spinorphin. Quantitative method of spinorphin by ELISA was established. Anti-spinorphin antibody did not complete with its analogues in this method. Base levels of spinorphin in human serum were 0.23±0.25 pmol/ml.From these results, spinorphin may be a peptide regulator in inflammation.