Buhimschi I A, Kramer W B, Buhimschi C S, Thompson L P, Weiner C P
Division of Perinatal Research, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Baltimore 21201-1559, USA.
Am J Obstet Gynecol. 2000 Feb;182(2):458-64. doi: 10.1016/s0002-9378(00)70239-0.
The mechanisms underlying membrane rupture at term and preterm are obscure. Collagenolytic activity of matrix metalloproteinases in amniochorionic membranes increases during spontaneous term and preterm labor associated with intra-amniotic infection. We sought to test the hypothesis that reduction-oxidation homeostasis, which is altered in inflammatory states, directly regulates amniochorionic matrix metalloproteinases.
Membranes were collected from 7 patients undergoing elective cesarean delivery at term, rinsed thoroughly, and immediately incubated in phosphate-buffered sodium chloride solution at 37 degrees C for 24 hours. Matrix metalloproteinase activity in the culture medium was assayed by substrate-gel electrophoresis and normalized against the dry weight of the tissue incubated. Superoxide anions were generated in the presence of membranes by a xanthine (2 mmol/L) and xanthine oxidase (20 mU/mL) mixture and monitored by reduction of ferri-cytochrome c to ferro-cytochrome c. Incubations were performed in the presence of xanthine alone, a xanthine-xanthine oxidase mixture, superoxide dismutase (500 U/mL), a xanthine-xanthine oxidase-superoxide dismutase mixture, nitro-L-arginine (a nitric oxide synthase inhibitor, 1 mmol/L), xanthine-xanthine oxidase-nitro-L-arginine, S-nitroso-N -acetylpenicillamine (a nitric oxide donor, 10 mmol/L), xanthine-xanthine oxidase-S-nitroso-N -acetylpenicillamine, N -acetylcysteine (a thiol-containing antioxidant, 0.1, 1, or 10 mmol/L), lipopolysaccharide (100 ng/mL), or lipopolysaccharide-N -acetylcysteine. Intracellular generation of superoxide anions was monitored by the reduction of nitroblue tetrazolium to formazan.
Basal matrix metalloproteinase 9 and matrix metalloproteinase 2 levels were detected in all samples. Superoxide anions significantly increased matrix metalloproteinase 9 activity but did not increase matrix metalloproteinase 2 activity, which effect was reversed by the addition of superoxide dismutase. N-acetylcysteine reduced basal activity of both matrix metalloproteinase 9 and matrix metalloproteinase 2 to 20%. Importantly, N-acetylcysteine completely inhibited intracellular formazan formation in cultured membranes both in the absence and in the presence of lipopolysaccharide. Neither nitric oxide synthase inhibition nor the nitric oxide donor S-nitroso-N -acetylpenicillamine had any effect on fetal membrane matrix metalloproteinase activity.
Matrix metalloproteinase activity in human fetal membranes is reduction-oxidation (redox)-regulated. Matrix metalloproteinase 9 activity in human fetal membranes is directly increased by superoxide anion, a byproduct of macrophages and neutrophils. Neither nitric oxide donors nor nitric oxide synthase inhibitors significantly affect matrix metalloproteinase activity in human fetal membranes. The glutathione precursor N-acetylcysteine dramatically inhibits amniochorionic matrix metalloproteinase activity in addition to inhibiting intrinsic superoxide generation within the tissue. Thus thiol-reducing agents, such as N-acetylcysteine, may be beneficial in preventing preterm premature rupture of the membranes.
足月和早产时胎膜破裂的潜在机制尚不清楚。在与羊膜腔内感染相关的足月和早产自然分娩过程中，羊膜绒毛膜中基质金属蛋白酶的胶原olytic活性增加。我们试图验证以下假设，即在炎症状态下发生改变的氧化还原稳态直接调节羊膜绒毛膜基质金属蛋白酶。
从7名足月行择期剖宫产的患者收集胎膜，彻底冲洗，然后立即在37℃的磷酸盐缓冲氯化钠溶液中孵育24小时。通过底物-凝胶电泳测定培养基中的基质金属蛋白酶活性，并根据孵育组织的干重进行标准化。在有胎膜存在的情况下，通过黄嘌呤（2 mmol/L）和黄嘌呤氧化酶（20 mU/mL）混合物产生超氧阴离子，并通过将高铁细胞色素c还原为亚铁细胞色素c进行监测。在单独存在黄嘌呤、黄嘌呤-黄嘌呤氧化酶混合物、超氧化物歧化酶（500 U/mL）、黄嘌呤-黄嘌呤氧化酶-超氧化物歧化酶混合物、硝基-L-精氨酸（一种一氧化氮合酶抑制剂，1 mmol/L）、黄嘌呤-黄嘌呤氧化酶-硝基-L-精氨酸、S-亚硝基-N-乙酰青霉胺（一种一氧化氮供体，10 mmol/L）、黄嘌呤-黄嘌呤氧化酶-S-亚硝基-N-乙酰青霉胺、N-乙酰半胱氨酸（一种含硫醇的抗氧化剂，0.1、1或10 mmol/L）、脂多糖（100 ng/mL）或脂多糖-N-乙酰半胱氨酸的情况下进行孵育。通过将硝基蓝四氮唑还原为甲臜来监测细胞内超氧阴离子的产生。
在所有样本中均检测到基础基质金属蛋白酶9和基质金属蛋白酶2水平。超氧阴离子显著增加基质金属蛋白酶9的活性，但不增加基质金属蛋白酶2的活性，添加超氧化物歧化酶可逆转该效应。N-乙酰半胱氨酸将基质金属蛋白酶9和基质金属蛋白酶2的基础活性均降低至20%。重要的是，N-乙酰半胱氨酸在不存在和存在脂多糖时均完全抑制培养胎膜中细胞内甲臜的形成。一氧化氮合酶抑制或一氧化氮供体S-亚硝基-N-乙酰青霉胺对胎膜基质金属蛋白酶活性均无任何影响。
人胎膜中的基质金属蛋白酶活性受氧化还原调节。人胎膜中的基质金属蛋白酶9活性直接由巨噬细胞和中性粒细胞的副产物超氧阴离子增加。一氧化氮供体和一氧化氮合酶抑制剂均未显著影响人胎膜中的基质金属蛋白酶活性。谷胱甘肽前体N-乙酰半胱氨酸除了抑制组织内源性超氧阴离子的产生外，还显著抑制羊膜绒毛膜基质金属蛋白酶活性。因此，硫醇还原剂，如N-乙酰半胱氨酸，可能有助于预防早产胎膜早破。
