Salvia Miltiorrhiza Bunge (also known as herb Danshen in Chinese) is a widely used Chinese herbal medicine. Tanshinone IIA (TSN IIA) is considered to be the most important bioactive ingredient in Danshen and exhibits an anti-atherosclerotic activity.
To evaluate the protective effect of TSN IIA on the human endothelial EA.hy926 cells injured by hydrogen peroxide in vitro and its possible mechanism.
The EA.hy926 cells were incubated for 24h with different concentrations of TSN IIA (5, 10 and 20 μg/μL ) or DMEM. Subsequently, cells were treated with 300 μmol/L H(2)O(2) for another 4h. Then, the percentage of cell viability was evaluated by 3-(4, 5-di-methylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The apoptosis of EA.hy926 cells was detected by flow cytometry with AnnexinV-FITC/PI double staining and laser scanning spectral confocal technique. The generation of intracellular reactive oxygen species (ROS) generation was analyzed by flow cytometry. The mRNA expressions of caspase-3, Bcl-2 and Bax were tested by real time-reverse transcription polymerase chain reaction (real time RT-PCR). The protein expression of Bcl-2 and Bax was determined by Western blotting. MDA levels, NO production, LDH leakage, and SOD as well as caspase-3 activities were also measured using standard methods.
Loss of cell viability and excessive cell apoptosis were observed in EA.hy926 cells after 4h of challenge with H(2)O(2) (300 μmol/L). However, cell apoptosis was attenuated in different concentrations of TSN IIA (5, 10 and 20 μg/μL) pretreated cells. Furthermore, TSN IIA markedly inhibited the elevation of ROS evoked by H(2)O(2). Real time RT-PCR and Western blotting analysis showed that TSN IIA significantly decreased the expressions of pro-apoptotic proteins (Bax and caspase-3) while significantly increased the expression of anti-apoptotic protein Bcl-2, and resulted in obvious reduction of Bax/Bcl-2 ratio in EA.hy926 cells induced by H(2)O(2).
These observations provide preliminary evidence that TSN IIA protects EA.hy926 cells against H(2)O(2) damage, which is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-3 activation leading to apoptosis.
"Labeled cells were observed under a LSM 780 Laser Scanning Confocal Microscope (Carl Zeiss SAS, Jena, Germany). In order to further discriminate the early and late cell apoptosis and perform a quantitative analysis, flow cytometry with Annexin V-FITC/PI double staining was employed as previously described . Briefly, PC12 cells were detached with 0.125% trypsin, centrifuged at 1,000 rpm for 5 min, and then washed twice with PBS. "
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is characterized by the accumulation of β-amyloid peptide (Aβ) and loss of neurons. Recently, a growing body of evidences have indicated that as a herbal compound naturally derived from grapes, resveratrol modulates the pathophysiology of AD, however, with a largely unclear mechanism. Therefore, we aimed to investigate the protection of resveratrol against the neurotoxicity of β-amyloid peptide 25-35 (Aβ25-35) and further explore its underlying mechanism in the present study. PC12 cells were injuried by Aβ25-35, and resveratrol at different concentrations was added into the culture medium. We observed that resveratrol increased cell viability through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) colorimetric assays. Flow cytometry indicated the reduction of cell apoptosis by resveratrol. Moreover, resveratrol also stabilized the intercellular Ca(2+) homeostasis and attenuated Aβ25-35 neurotoxicity. Additionally, Aβ25-35-suppressed silent information regulator 1 (SIRT1) activity was significantly reversed by resveratrol, resulting in the downregulation of Rho-associated kinase 1 (ROCK1). Our results clearly revealed that resveratrol significantly protected PC12 cells and inhibited the β-amyloid-induced cell apoptosis through the upregulation of SIRT1. Moreover, as a downstream signal molecule, ROCK1 was negatively regulated by SIRT1. Taken together, our study demonstrated that SIRT1-ROCK1 pathway played a critical role in the pathomechanism of AD.
PLoS ONE 03/2013; 8(3):e59888. DOI:10.1371/journal.pone.0059888 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atherosclerosis is considered a chronic inflammatory disease and injury to vascular endothelial cells is an important event at the onset of its pathogenesis. Oxidative stress is regarded as a pivotal pathogenic factor in the development of endothelial cell injury and apoptosis. Tanshinone IIA is considered the main active lipophilic component of the traditional Chinese medicine Danshen. It has been demonstrated that Tanshinone IIA protects endothelial cells from oxidative stress-triggered injury and apoptosis, but many of its anti-oxidant and anti-apoptotic mechanisms still remain to be elucidated. In this paper, our results show that HUVECs incubated with 1.1mM H2O2 for 12h had significantly decreased the viability of endothelial cells, which was accompanied with obviously cell apoptosis. However, treatment with Tanshinone IIA (8 mM, 24h) resulted in a significant resistance to H2O2-induced apoptosis. We used 2D-DIGE (Difference in Gel Electrophoresis) analysis followed by MALDI-TOF-TOF-MS to identify the proteins differentially expressed after human umbilical vein endothelial cells (HUVECs) were protected with Tanshinone IIA against oxidative damage by hydrogen peroxide. There were various differently expressed proteins, we picked out 32 proteins to analysis, and discovered that Tanshinone IIA could protect endothelial cells by its anti-inflammatory or antioxidant effects, regulating proliferation and differentiation, maintaining the cytoskeleton, and maintaining intracellular calcium homeostasis.
Current Proteomics 01/2013; 10(4). DOI:10.2174/1570164610999131125102832 · 0.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelium plays an important role in the physiological homeostasis of blood vessels. Endothelial injury is considered to be implicated in the pathogenesis of many cardiovascular diseases, including atherosclerosis. Farrerol, a flavonoid considered to be the major bioactive component in a traditional Chinese herb, "Man-shan-hong", which is the dried leaves of Rhododendron dauricum L., displays many bioactive properties, including antibechic, antibacterial, anti-inflammatory, and the inhibition of vascular smooth muscle cell (VSMC) proliferation. In this study, the protective effects of farrerol on hydrogen peroxide (H2O2)-induced apoptosis in human endothelium-derived EA.hy926 cells were investigated. The results showed that farrerol significantly inhibited the loss of cell viability and enhanced superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in H2O2-induced EA.hy926 cells. Meanwhile, farrerol inhibited H2O2-induced elevation in the levels of intracellular malondialdehyde and reactive oxygen species, as well as cell apoptosis. Furthermore, real time RT-PCR and Western blot analysis showed that farrerol significantly decreased the expression of Bax mRNA, Bax, cleaved caspase-3, and phosph-p38 MAPK, while increasing the exporession of Bcl-2 mRNA and Bcl-2 in H2O2-induced EA.hy926 cells. These results are the first demonstration that farrerol has protective effects against H2O2-induced apoptosis in EA.hy926 cells, and suggests that farrerol is a potential candidate for the intervention of endothelial-injury-associated cardiovascular diseases.
Canadian Journal of Physiology and Pharmacology 09/2013; 91(9):733-40. DOI:10.1139/cjpp-2013-0008 · 1.77 Impact Factor
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