Proteomic screening of antioxidant effects exhibited by radix Salvia miltiorrhiza aqueous extract in cultured rat aortic smooth muscle cells under homocysteine treatment.
OBJECTIVE
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.


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 HPLC analyses with binary gradient elution was used in variable 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 (PPIase 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.


CONCLUSIONS
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.
Hung
Yu-Chiang
YC
Graduate Institute of Clinical Medical Sciences, Kaohsiung Division, Chang Gung University, Kaohsiung, Taiwan.
Wang
Pei-Wen
PW
Pan
Tai-Long
TL
Bazylak
Grzegorz
G
Leu
Yann-Lii
YL
D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000494:pharmacology*; D001013:Aorta, Thoracic / Q000166:cytology / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006710:Homocysteine / Q000494:pharmacology*; D013058:Mass Spectrometry; D008938:Mitosis / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000187:drug effects*; D018384:Oxidative Stress / Q000187:drug effects; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology; D018517:Plant Roots / Q000737:chemistry; D050050:Protein Carbonylation / Q000187:drug effects; D040901:Proteomics; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D027561:Salvia miltiorrhiza / Q000737:chemistry*; D013778:Tetrazolium Salts; D013844:Thiazoles
D000975:Antioxidants; D010936:Plant Extracts; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D006710:Homocysteine; C022616:thiazolyl blue