Biroc S L, Gay S, Hummel K, Magill C, Palmer J T, Spencer D R, Sa S, Klaus J L, Michel B A, Rasnick D, Gay R E
Berlex Biosciences, Richmond, California, USA.
Arthritis Rheum. 2001 Mar;44(3):703-11. doi: 10.1002/1529-0131(200103)44:3<703::AID-ANR120>3.0.CO;2-2.
Cysteine proteases are postulated to play a role in tissue destruction in the joints of animals with arthritis. The purpose of the present study was to confirm the concept that cysteine proteases are enzymes involved in the pathology of rheumatoid arthritis (RA).
Arthritis was induced in Lewis rats by adjuvant injection (adjuvant-induced arthritis [AIA] model) and scored for inflammation. At necropsy, the rear paws were either fixed in formalin and assigned a histologic score (based on synovial cell proliferation, cartilage erosion, bone erosion, and fibroproliferative pannus) or frozen, cryosectioned, and assayed for enzyme activity either by in situ cytochemical staining with a post-azo-coupling method using a chromogenic substrate (Z-arg-arg-MNA) or by a novel assay placing the tissue section directly in a cuvette using the fluorogenic substrate Z-arg-arg-AMC.
Enzymatic activity, measured either in frozen sections in situ or in the cuvette assay, was positively correlated with joint destruction (r = 0.7) and inflammation (r = 0.8). Activity was not inhibited significantly by Pefabloc (a serine protease inhibitor), EDTA (a metalloprotease inhibitor), or pepstatin A (an aspartyl protease inhibitor) but was inhibited by E-64 and vinyl sulfone irreversible inhibitors of cysteine proteases. The effect of one of the vinyl sulfone cysteine protease inhibitors, Mu-Leu-HomoPhe-vinylsulfone, was tested in vivo by dietary administration at 2.2 mg/kg/day in the AIA model; this resulted in a significant decrease in inflammation and in the amount of cysteine protease activity measured in the joint tissue.
Cysteine protease activity levels increase in the diseased state and may be an important target for designing small molecule inhibitors to reduce the inflammation and tissue destruction associated with RA.
据推测，半胱氨酸蛋白酶在患有关节炎的动物关节组织破坏中起作用。本研究的目的是证实半胱氨酸蛋白酶是参与类风湿性关节炎（RA）病理过程的酶这一概念。
通过佐剂注射在Lewis大鼠中诱导关节炎（佐剂诱导的关节炎[AIA]模型），并对炎症进行评分。尸检时，后爪要么用福尔马林固定并给予组织学评分（基于滑膜细胞增殖、软骨侵蚀、骨侵蚀和纤维增生性血管翳），要么冷冻、切片，然后使用发色底物（Z-精氨酸-精氨酸-4-甲氧基-β-萘胺）通过后偶氮偶联法进行原位细胞化学染色或使用荧光底物Z-精氨酸-精氨酸-7-氨基-4-甲基香豆素将组织切片直接放入比色皿中通过一种新方法测定酶活性。
在原位冷冻切片或比色皿测定中测得的酶活性与关节破坏（r = 0.7）和炎症（r = 0.8）呈正相关。活性不受Pefabloc（一种丝氨酸蛋白酶抑制剂）、EDTA（一种金属蛋白酶抑制剂）或胃蛋白酶抑制剂A（一种天冬氨酸蛋白酶抑制剂）的显著抑制，但受半胱氨酸蛋白酶的不可逆抑制剂E-64和乙烯砜抑制。在AIA模型中，通过每天以2.2 mg/kg的剂量饮食给药测试了一种乙烯砜半胱氨酸蛋白酶抑制剂Mu-Leu-HomoPhe-乙烯砜的体内作用；这导致炎症和关节组织中测得的半胱氨酸蛋白酶活性量显著降低。
在疾病状态下半胱氨酸蛋白酶活性水平升高，可能是设计小分子抑制剂以减少与RA相关的炎症和组织破坏的重要靶点。
