摘要
目的探讨α-玉米赤霉醇对肿瘤坏死因子仅（TNF-α）刺激的人脐静脉内皮细胞（HUVEC）血红素氧化酶1（HO-1）表达及胞质游离钙水平的影响及其作用机制。方法用小分子RNA干扰（siRNh）技术消除HUVEC的NADPH氧化酶p47p^hox亚基；用分子探针2，7-DCF测定细胞内活性氧（ROS）的产生量；用RT-PCR测定HO-1 mRNA的表达以及用细胞免疫组化方法测定HO-1蛋白的表达；以Fluo-3／AM为探针，用激光共聚焦显微镜测定胞质游离钙整体水平的变化。结果TNF-α刺激使细胞内ROS的产生量较对照组增加了155％（228.3±50.6比89.4±23.7，P〈0.05）；α-玉米赤霉醇预处理呈剂量依赖地抑制TNF-α对ROS的诱导效应；p47p^hox的siRNA完全阻断TNF-α诱导ROS的产生。TNF-α刺激使细胞内HO-1的mRNA表达水平较对照组增加了145％（0.88±0.10比0.36±0.11，P〈0.01），蛋白的表达也明显增加；α-玉米赤霉醇预处理呈剂量依赖地抑制TNF-α对HO-1 mRNA表达的诱导效应；α-玉米赤霉醇预处理及转染p47p^hox的siRNA均明显降低TNF-α诱导的HO-1蛋白的表达。TNF-α刺激使内皮细胞胞质游离钙整体水平比对照组增加179％（107.3±4.9比38.5±0.6，P〈0.01）；α-玉米赤霉醇预处理及转染p47p^hox的siRNA分别使TNF-α诱导的胞质游离钙水平降低46％（58.5±0.3比107.3±4.9，P〈0.01）及57％（46.3±2.1比107.3±4.9，P〈0.01）。结论ROS仅部分介导TNF-α对HUVEC中HO-1表达的诱导作用，α-玉米赤霉醇主要通过抑制NADPH氧化酶途径产生的ROS而降低TNF-α诱导的HO-1表达及胞质游离钙水平增加。
Objective To investigate the effects of α-zearalanol on the expression of heme oxygenase-1 （HO-1） gene and cytosolic free calcium level in the tumor necrosis factor α（TNF-α）-stimulated human umbilical vein endothelial cell （HUVEC） and the mechanisms involved. Methods The siRNA expression vector for p47p^hox was constructed and used to block the NADPH oxidase in the HUVEC. The intracellular ROS production was detected by using 2, 7-dichlorofluorescin diacetate as probe. The mRNA expression of the HO-1 was determined by semiquantitative RT-PCR and the protein expression was measured by immunocytochemistery analysis. The level of cytosolic free calcium was determined by using Fluo-3/AM as probe with laser confocal microscope. Results TNF-α stimulation caused ROS output increased by 155% of control（228±51 vs 89±24, P 〈0.05） ; α-zearalanol was able to reduce the production of ROS in a dose-dependent manner. Knock down of the p47p^hox subunit for NADPH oxidase by siRNA abolished the production of ROS. TNF-α stimulation caused HO-1 mRNA increased by 145% of control （0.88±0.10 vs 0. 36±0. 11, P 〈0. 01 ）, and also obviously increased HO-1 protein expression; α-zearalanol inhibited the expression of HO-1 mRNA in a dose-dependent manner; Pretreatment with α-ZAL and p47P^hox siRNA both attenuated TNFα-induced HO-1 proein expression. The treatment of TNF-α for 24 hours up-regulated cytosolic free calcium level by 179% （ 107.3±4.9 vs 38.5 ±0.6, P 〈 0. 01 ） ; Pretreatment with α-ZAL and p47p^hox siRNA depressed TNFα-induced cytosolic free calcium level by 46% （ 58. 5±0. 3 vs 107.3±4.9, P 〈0. O1 ） and 57% （46. 3±2. 1 vs 107.3±4. 9, P 〈 0. 01 ） respectively. Conclusion ROS only partly mediated HO-1 ; expresson in the TNF-α- stimulated HUVEC ; α-ZAL has a potent inhibitory effect on the HO-1 expresson and cytosolic free calcium level in the TNF-α- stimulated HUVEC, mainly through the inhibition of ROS generation derived from NADPH oxidase.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2009年第46期3280-3284,共5页
National Medical Journal of China
基金
国家自然科学基金（30470769）
