Hao J L, Nagano T, Nakamura M, Kumagai N, Mishima H, Nishida T
Department of Ophthalmology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan.
Exp Eye Res. 1999 Dec;69(6):595-601. doi: 10.1006/exer.1999.0755.
The authors examined the effect of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase, Galardin, on collagen degradation by Pseudomonas aeruginosa (P. aeruginosa) in the presence or absence of keratocytes. Type I collagen gels, with or without suspended keratocytes, were incubated under medium containing sterile P. aeruginosa culture broth and/or Galardin for 24 hr. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline. The conditioned media were also subjected to gelatin zymography and Western blotting to analyse the activation, by P. aeruginosa factor(s), of matrix metalloproteinases (MMPs) released by keratocytes. The effects of protease inhibitors, aprotinin, leupeptin and pepstatin, on collagen degradation by P. aeruginosa were also examined. P. aeruginosa broth by itself induced collegen gel degradation. When keratocytes were present, P. aeruginosa broth increased the amount of degraded collagen even further. Galardin significantly reduced the amounts of collagen degraded by P. aeruginosa culture broth, whether keratocytes were present or absent in the gel. However, the protease inhibitors had no inhibitory effects on collagen degradation. Gelatin zymography and Western blotting revealed that inactive proMMP-1, -2 and -3, released by keratocytes, were converted to active forms in the presence of P. aeruginosa broth. Galardin decreased the amounts of active MMPs and increased those of inactive proMMPs, suggesting that Galardin inhibited the activation of proMMPs by P. aeruginosa. The present results suggest that Galardin inhibits the keratocyte-mediated collagen degradation by P. aeruginosa culture broth, resulting from preventing the conversion of proMMPs to active MMPs.
作者研究了基质金属蛋白酶的合成肽基异羟肟酸酯抑制剂加拉迪恩（Galardin）在有或没有角膜细胞存在的情况下，对铜绿假单胞菌（P. aeruginosa）胶原降解的影响。将含有或不含有悬浮角膜细胞的I型胶原凝胶在含有无菌铜绿假单胞菌培养液和/或加拉迪恩的培养基中孵育24小时。通过羟脯氨酸的释放来测量培养过程中胶原纤维的降解。条件培养基还进行了明胶酶谱分析和蛋白质印迹分析，以分析铜绿假单胞菌因子对角膜细胞释放的基质金属蛋白酶（MMPs）的激活作用。还研究了蛋白酶抑制剂抑肽酶、亮抑肽酶和胃蛋白酶抑制剂对铜绿假单胞菌胶原降解的影响。铜绿假单胞菌培养液本身可诱导胶原凝胶降解。当存在角膜细胞时，铜绿假单胞菌培养液进一步增加了降解胶原的量。加拉迪恩显著降低了铜绿假单胞菌培养液降解的胶原量，无论凝胶中是否存在角膜细胞。然而，蛋白酶抑制剂对胶原降解没有抑制作用。明胶酶谱分析和蛋白质印迹分析显示，角膜细胞释放的无活性前MMP-1、-2和-3在铜绿假单胞菌培养液存在的情况下转化为活性形式。加拉迪恩减少了活性MMPs的量，增加了无活性前MMPs的量，表明加拉迪恩抑制了铜绿假单胞菌对前MMPs的激活。目前的结果表明，加拉迪恩抑制铜绿假单胞菌培养液介导的角膜细胞胶原降解，这是由于阻止了前MMPs转化为活性MMPs。
