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

已知Spin对中枢神经系统中的中性内肽酶、氨基肽酶和二肽基多肽酶III等脑啡肽降解酶具有抑制活性。在本研究中，利用特定底物(Arg-Arg-β-naphtylamine)和抑制剂对人中性粒细胞(PMN)产生DPPIII的抑制活性进行了研究。为了研究从牛脊髓中提纯的内源性多肽Spin的抗炎作用，研究了Spin对人中性粒细胞(PMN)的启动作用和人血清中性粒细胞中Spin的定量方法。(1)刺激剂诱导PMN产生超氧化物的启动效应Spinogin(1μM)单独作用不能诱导超氧化物的产生。然而，在炎症刺激剂FMLP的二次刺激下，与未经处理的对照组相比，该活性明显增强。流式细胞仪分析显示，CD11b、CD18等黏附分子的表达明显高于对照组，提示Spinogin的启动作用部分是通过增加黏附分子的表达来实现的。(2)Apinogin对二肽基肽酶III酶活性的影响。(3)建立了锁孔血蓝蛋白(KLH)标记的Spin免疫兔体内抗Spin抗体的ELISA法。建立了双抗体夹心酶联免疫吸附测定法(EL ISA)测定Spin含量的方法。在这种方法中，抗Spin抗体没有与其类似物一起完成。人血清中Spin的基础浓度为0.23±0.25pmol/ml，提示Spin可能是炎症中的一种多肽调节剂。

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.