Proteomic screening of antioxidant effects exhibited by Radix Salvia miltiorrhiza aqueous extract in cultured rat aortic smooth muscle cells under homocysteine treatment
Proteomic screening of antioxidant effects exhibited by Radix Salvia miltiorrhiza aqueous extract in cultured rat aortic smooth muscle cells under homocysteine treatment
复制标题
DOI：
10.1016/j.jep.2009.05.020
复制
发表时间：
2009-07-30
影响因子：
5.4
通讯作者：
Leu, Yann-Lii
中科院分区：
文献类型：
--
作者：
Hung, Yu-Chiang;Wang, Pei-Wen;Leu, Yann-Lii
研究目的：在氧化应激因子（如同型半胱氨酸（Hcy））的影响下，导致主动脉平滑肌细胞增殖减弱的细胞机制仍然知之甚少。因此，我们的目的是评估丹参是否会调节这种Hcy促进模型动物主动脉细胞系统生长的作用，丹参是一种广泛用于民间医学治疗各种心血管疾病的中草药。材料与方法：采用恒浓度双梯度洗脱窄孔高效液相色谱分析证实含有3,4-二羟基苯甲酸、3,4-二羟基苯基乳酸和丹酚酸B的丹参根水提物（SMAE），用于Hcy刺激下大鼠主动脉平滑肌A10细胞的治疗。采用二维电泳（2-DE）联用MALDI-TOF质谱法分析大鼠A10细胞对hcy诱导的氧化应激反应的蛋白变化。结果：本研究显示，低剂量（0.015 mg/mL） SMAE对Hcy刺激的大鼠A10细胞的生长有显著抑制作用（bbb60 %, p < 0.05）。此外，SMAE孵育后大鼠A10细胞胞内活性氧（ROS）浓度明显降低，过氧化氢酶活性升高。接下来，蛋白激酶C β -1 （PKC β -1）和磷酸化丝裂原活化蛋白激酶（p-MAPK）表达的显著下调表明，观察到的富含多酚的SMAE对hhy诱导的大鼠A10细胞生长的抑制作用是通过PKC/p44/42 mapk依赖途径实现的。检测大鼠A10细胞对hcy诱导的氧化损伤反应中α -4-原肌球蛋白、波形蛋白、F1F0-ATP合成酶（β亚基）、葡萄糖调节蛋白75 （GRP75）、肌动蛋白（片段）、禁止蛋白、capping蛋白、血小板红蛋白、内质网蛋白（ERp29）、肽基脯氨酸异构酶A （PPlase A）等10个蛋白点的强度变化，差异均有统计学意义（p < 0.05）。同时，我们发现SMAE可以分别抑制vimentin、α -4-原肌球蛋白和GRP75等特定细胞骨架蛋白和伴蛋白的羰基化，从而导致大鼠A10细胞的表型转化。结论：本实验提示，SMAE通过抑制大鼠主动脉平滑肌A10细胞循环ROS及随后调节蛋白羰基化发挥保护作用。本研究强调的氧化还原-蛋白质组学方案可能适用于促进评估潜在的新型分子治疗靶点，以降低各种人群中与Hcy水平升高相关的心血管风险，并阐明通过天然抗氧化剂氧化还原状态调节蛋白质功能的新机制。2009爱思唯尔爱尔兰有限公司版权所有。
Aim of the study: Still little is known about the cellular mechanisms that contribute to the attenuated proliferation of aortic smooth muscle cells under the influence of the oxidative stress factors such as homocysteine (Hcy). Thus, we aimed to evaluate whether Salvia miltiorrhiza Bunge (Labiatae), a Chinese medicinal herb widely used in folk medicine for therapy of variety of human cardiovascular disorders would modulate this Hcy promoted growth effect in model animal aortic cells system.Materials and methods: The Salvia miltiorrhiza roots aqueous extract (SMAE) containing 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenyl lactic acid and salvianolic acid B, as confirmed by narrow-bore H PLC analyses with binary gradient elution was used invariable concentrations for the treatment of the rat aortic smooth muscle A10 cells under Hcy stimulation. Two-dimensional electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry was applied for the elucidation of protein changes characterizing the response of the rat A10 cells into the Hcy-induced oxidative stress.Results: This study showed that a low dose (0.015 mg/mL) of the SMAE significantly inhibited growth (>60%, p < 0.05) of the Hcy stimulated rat A10 cells. in addition, concentration of intracellular reactive oxygen species (ROS) obviously decreased in the rat A10 cells after its incubation with SMAE in terms of catalase increasing activity. Next, marked down-regulation of protein kinase C beta-1 (PKC beta-1) and phosphorylated mitogen-activated protein kinase (p-MAPK) expression suggest that observed inhibitory effect of the polyphenol-rich SMAE on the Hcy-induced growth of rat A10 cells was realized via the PKC/p44/42 MAPK-dependent pathway. The intensity changes of 10 protein spots in response of the rat A10 cells to the Hcy-induced oxidative damage as alpha-4-tropomyosin, vimentin, F1F0-ATP synthase (beta subunit), glucose regulated protein 75 (GRP75), actin (fragment), prohibitin, capping protein, plakoglobin, endoplasmic reticulum protein (ERp29), and peptidylprolyl isomerase A (PPlase A), were detected with statistical significance (p < 0.05). Meanwhile, it was showed that used here SMAE resist carbonylation of specific cytoskeleton and chaperone proteins as vimentin, alpha-4-tropomyosin and GRP75, respectively, leading to phenotype transformations in the rat A10 cells.Conclusion: These data suggest that applied here SMAE exerts its protective effect through circulating ROS suppression and subsequent modulation of protein carbonylation in rat aortic smooth muscle A10 cells. Redox-proteomics protocol highlighted in this study may be applicable in facilitating the assessing potential novel molecular therapeutic targets to reduce cardiovascular risk related with elevated Hcy levels in various human populations and elucidating new mechanisms through which protein functions can be regulated by the redox status with the use of naturally occurring antioxidants. (C) 2009 Elsevier Ireland Ltd. All rights reserved.