Down-regulation of collagen I biosynthesis in intestinal epithelial cells exposed to indomethacin: a comparative proteome analysis.
Down-regulation of collagen I biosynthesis in intestinal epithelial cells exposed to indomethacin: a comparative proteome analysis.
复制标题
暴露于吲哚美辛的肠上皮细胞中胶原蛋白 I 生物合成的下调：比较蛋白质组分析。
DOI：
10.1016/j.jprot.2014.03.022
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发表时间：
2014
影响因子：
3.3
通讯作者：
Sakai A et al
中科院分区：
文献类型：
--
作者：
Edogawa S;Sakai A et al
与与吲哚美辛等非甾体抗炎药（NSAID）相关的胃十二指肠损伤的累积知识相比，小肠粘膜损伤最近才被注意到，其确切机制仍有待阐明。为了阐明这一机制，我们对用吲哚美辛（200μM，24小时）或载体对照处理的IEC-6大鼠正常肠细胞进行了2-DE，并通过MALDI-TOF/TOF质谱鉴定了18个上调和8个下调的蛋白质。在这些蛋白质中，I型胶原蛋白和参与I型胶原生物合成和成熟的蛋白质，包括脯氨酰4-羟化酶亚基α1、蛋白质二硫键异构酶A3（PDIA3）、钙网蛋白和内纤蛋白，均被吲哚美辛下调。给予吲哚美辛的大鼠肠粘膜的免疫组织化学染色显示，肠细胞顶端表面的 I 型胶原蛋白减少。当 IEC-6 细胞在胶原蛋白 I 包被的平板上生长时，吲哚美辛诱导的细胞死亡被显着抑制。cis-4-Hydroxy-l-proline（一种抑制胶原蛋白合成的脯氨酸类似物）以浓度依赖性方式降低 IEC-6 细胞活力。 IEC-6 细胞中内源性 I 型胶原蛋白的短干扰 RNA 敲低也可诱导细胞死亡。总之，通过比较蛋白质组分析，我们确定 I 型胶原蛋白的下调是 NSAID 引起的肠道损伤的重要机制。生物学意义 NSAID 引起的小肠损伤在临床中备受关注。对于这些炎症反应的起源提出了各种假设，例如血流量减少、肠道过度运动、肠粘膜通透性异常、线粒体功能障碍和活性氧，其中许多与前列腺素合成的抑制有关。然而，确切的机制尚不清楚。病变的细胞过程必然涉及大量蛋白质的上调和下调以及它们之间复杂的相互作用。为了阐明这一点，对肠道细胞中受非甾体抗炎药影响的蛋白质进行全面、系统的鉴定至关重要。我们发现，吲哚美辛治疗后表现出表达减少的蛋白质是 I 型胶原蛋白以及参与 I 型胶原合成和成熟的蛋白质。与此相一致的是，免疫组织化学分析表明，吲哚美辛处理的大鼠肠粘膜细胞在其顶端表面表现出 I 型胶原表达减少。此外，通过使用胶原蛋白合成抑制剂、内源性胶原蛋白 I 的短干扰 RNA (siRNA) 敲低以及在胶原蛋白 I 包被的板上与未包被的板上进行细胞培养，证明了胶原蛋白对肠粘膜细胞的细胞保护作用。这些结果为肠粘膜胶原合成在 NSAID 诱导的小肠病变机制中的作用提供了重要信息。
In contrast to accumulated knowledge about gastroduodenal injury associated with nonsteroidal antiinflammatory drugs (NSAIDs) such as indomethacin, small intestinal mucosal injuries have been noticed only recently, and the precise mechanism remains to be elucidated. To clarify the mechanism, we performed 2-DE on IEC-6 rat normal intestinal cells that were treated with indomethacin (200 μΜ, 24 h) or a vehicle control and identified 18 up-regulated and 8 down-regulated proteins through MALDI-TOF/TOF mass spectrometry. Among these proteins, collagen I and proteins involved in collagen I biosynthesis and maturation, including prolyl 4-hydroxylase subunit α1, protein disulfide isomerase A3 (PDIA3), calreticulin, and endoplasmin, were all down-regulated by indomethacin. Immunohistochemical staining of the intestinal mucosa of indomethacin-administered rats showed a decrease of collagen I on the apical surface of intestinal cells. Cell death induced by indomethacin was prominently suppressed when IEC-6 cells were grown on collagen I-coated plates.cis-4-Hydroxy-l-proline, a proline analog that inhibits collagen synthesis, depressed IEC-6 cell viability in a concentration-dependent manner. Cell death was also induced by short interfering RNA knockdown of endogenous collagen I in IEC-6 cells. In conclusion, by comparative proteome analysis, we identified down-regulation of collagen I as an important mechanism in NSAID-induced intestinal injury.Biological significanceSmall intestinal lesions induced by NSAIDs are of great concern in clinical settings. Various hypotheses have been proposed for the origin of these inflammatory responses, such as reduction in the blood flow, intestinal hypermotility, abnormal intestinal mucosal permeability, mitochondrial dysfunction, and reactive oxygen species, many of which are related to the inhibition of prostaglandin synthesis. However, the precise mechanism is yet to be known. The cellular process of the lesions must involve up- and down-regulations of a large number of proteins and complex interactions between them. To elucidate it, global and systematic identification of the proteins in intestinal cells affected by NSAIDs is essential. We found that the proteins exhibiting reduced expression by indomethacin treatment are collagen I and the proteins involved in collagen I synthesis and maturation. Consistent with this, immunohistochemical analysis showed that the indomethacin-treated rat intestinal mucosal cells exhibits decreased collagen I expression on its apical surface. Furthermore, the cell-protective effect of collagen on intestinal mucosal cells was demonstrated by the use of a collagen-synthesis inhibitor, short interfering RNA (siRNA) knockdown of endogenous collagen I, and cell cultivation on collagen I-coated plates versus uncoated plates. These results give important information on the role of the collagen synthesis in intestinal mucosa in the mechanism of NSAID-induced small intestinal lesions.