Mitochondrial respiratory chain dysfunction modulates metalloproteases -1, -3 and -13 in human normal chondrocytes in culture.
Mitochondrial respiratory chain dysfunction modulates metalloproteases -1, -3 and -13 in human normal chondrocytes in culture.
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DOI：
10.1186/1471-2474-14-235
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发表时间：
2013-08-09
影响因子：
2.3
通讯作者：
Blanco FJ
中科院分区：
文献类型：
--
作者：
Cillero-Pastor B;Rego-Pérez I;Oreiro N;Fernandez-Lopez C;Blanco FJ
线粒体在骨关节炎(OA)的病理过程中起着重要作用。我们先前已经证明，线粒体呼吸链(MRC)的改变有助于软骨细胞的炎症反应。然而，它在软骨破坏过程中的作用还不是很清楚。在本研究中，我们探讨了培养的人正常软骨细胞中金属蛋白酶(MMPs)调控与MRC功能障碍的关系。人的正常软骨细胞是从没有风湿病病史的捐献者的尸检中分离出来的。分别用鱼藤酮、3-硝基丙酸(NPA)、抗霉素A(AA)、叠氮化钠和寡霉素抑制线粒体复合体I、II、III、IV和V的活性。实时荧光定量聚合酶链式反应检测MMPs-1、-3和-13mRNA的表达。免疫印迹法检测细胞内是否存在基质金属蛋白酶蛋白。用酶联免疫吸附试验检测这些蛋白释放到胞外培养上清液中的情况。Tolouidin蓝和藏红花/快速绿检测组织中蛋白多糖的存在。免疫组织化学方法检测组织中MMPs的表达。首先，用MRC抑制剂处理细胞24小时，检测其mRNA的表达。寡霉素5和100μg/ml(复合体V的抑制剂)作用24小时后，MMP1和MMP3mRNA表达上调。AA20和60μg/ml(复合体III抑制剂)与寡霉素共同孵育后，MMP13mRNA的表达明显降低。结果在蛋白水平上证实了寡霉素25μg/ml刺激后细胞内MMP1和MMP3水平的升高[分别为(15.20±8.46)和(4.59±1.83)vs基础值1(n=4；*P<；0.05)]。然而，AA和寡霉素降低了细胞内基质金属蛋白酶-13蛋白的表达水平(分别为0.70±0.16和0.3±0.24，与基础值相比分别为1)。为了了解MRC功能障碍是否对MMPs的释放有影响，对培养上清液中MMPs的水平进行了评估。刺激36h后，寡霉素25μg/mlvs基础值1，MMP1=18.06±10.35，寡霉素5μg/mlvs基础值1，MMP3=8.49±4.32(n=5，P<0.05)。AA60μg/ml组和寡霉素25μg/ml组上清液中MMP13水平分别为(0.5±0.13)和(0.41±0.14)g/ml(n=5)。外植体经寡霉素处理后，基质金属蛋白酶-1和-3的阳性表达增加。外植体经AA或寡霉素刺激后，基质金属蛋白酶-13的表达降低。蛋白多糖染色显示，寡霉素处理的组织中蛋白多糖水平降低。这些结果表明，MRC功能障碍调节了人正常软骨细胞MMPs的表达，表明MRC功能障碍在软骨破坏的调节中起着重要作用。
Mitochondrion has an important role in the osteoarthritis (OA) pathology. We have previously demonstrated that the alteration of the mitochondrial respiratory chain (MRC) contributes to the inflammatory response of the chondrocyte. However its implication in the process of cartilage destruction is not well understood yet. In this study we have investigated the relationship between the MRC dysfunction and the regulation of metalloproteases (MMPs) in human normal chondrocytes in culture. Human normal chondrocytes were isolated from human knees obtained form autopsies of donors without previous history of rheumatic disease. Rotenone, 3-Nitropropionic acid (NPA), Antimycin A (AA), Sodium azide and Oligomycin were used to inhibit the activity of the mitochondrial complexes I, II, III, IV and V respectively. The mRNA expression of MMPs -1, -3 and -13 was studied by real time PCR. The intracellular presence of MMP proteins was evaluated by western blot. The liberation of these proteins to the extracellular media was evaluated by ELISA. The presence of proteoglycans in tissue was performed with tolouidin blue and safranin/fast green. Immunohistochemistry was used for evaluating MMPs on tissue. Firstly, cells were treated with the inhibitors of the MRC for 24 hours and mRNA expression was evaluated. An up regulation of MMP-1 and -3 mRNA levels was observed after the treatment with Oligomycin 5 and 100 μg/ml (inhibitor of the complex V) for 24 hours. MMP-13 mRNA expression was reduced after the incubation with AA 20 and 60 μg/ml (inhibitor of complex III) and Oligomycin. Results were validated at protein level observing an increase in the intracellular levels of MMP-1 and -3 after Oligomycin 25 μg/ml stimulation [(15.20±8.46 and 4.59±1.83 vs. basal=1, respectively (n=4; *P<0.05)]. However, AA and Oligomycin reduced the intracellular levels of the MMP-13 protein (0.70±0.16 and 0.3±0.24, respectively vs. basal=1). In order to know whether the MRC dysfunction had an effect on the liberation of MMPs, their levels were evaluated in the supernatants. After 36 hours of stimulation, values were: MMP-1=18.06±10.35 with Oligomycin 25 μg/ml vs. basal=1, and MMP-3=8.49±4.32 with Oligomycin 5 μg/ml vs. basal=1 (n=5; *P<0.05). MMP-13 levels in the supernatants were reduced after AA 60 μg/ml treatment (0.50±0.13 vs. basal=1) and Oligomycin 25 μg/ml (0.41±0.14 vs. basal=1); (n=5; *P<0.05). The treatment of explants with Oligomycin, showed an increase in the positivity of MMP-1 and -3. Explants stimulated with AA or Oligomycin revealed a decrease in MMP-13 expression. Proteoglycan staining demonstrated a reduction of proteoglycan levels in the tissues treated with Oligomycin. These results reveal that MRC dysfunction modulates the MMPs expression in human normal chondrocytes demonstrating its role in the regulation of the cartilage destruction.
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影响因子：
0.7
作者：
Li, Heng;Li, Liqin;Wang, Dan
通讯作者：
Wang, Dan
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影响因子：
4.4
作者：
Blanco, FJ;López-Armada, MJ;Maneiro, E
通讯作者：
Maneiro, E