Echinacoside Retards Cellular Senescence Of Human Fibroblastic Cells MRC-5

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The active ingredient of Cistanche: How does echinacoside anti-aging?

Hong Xie 1, Hui Zhu1,2, Cong Cheng1, Yu Liang1, Zhao Wang1

In this study, the effects of echinacoside, one of the phenylethanoids isolated from the stems of Cistanches salsa, a Chinese traditional herbal medicine, on human embryo lung fibroblastic MRC-5 cells, was investigated. The activity of cell proliferation was evaluated with Alamar Blue, showing that treatment with echinacoside could retard senescence. Flow cytometry results show that echinacoside could trigger cells in the G1 phase to enter the S phase and G2 phase, and could improve ROS degradation. The results from the comet assay indicate that echinacoside could protect cells from DNA damage, partly elucidating the mechanism of its effects. All of the above results suggest that echinacoside has potential anti-senescence activity.

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1. Introduction

Normal cells cultured in vitro proliferate for a limited number of population doublings (the ‘Hayflflick limit’) and enter a stage of replicative senescence. Senescent cells undergo cell growth arrest in the G1 phase and a change in morphology and metabolism, including cellular enlargement, increased lysosome biogenesis, and expression of higher -galactosidase activity at pH 6 (senescence-associated -galactosidase, SA- - -Gal) (Hayflflick and Moorhead 1961; Dimri 1995). The accumulation of senescent cells prognosticates the age-related decline in tissue/organ functions. To access the way of delaying or reversing aging, efforts to discover active anti-aging substances have never ceased. Echinacoside is one of the phenylethanoids isolated from stems of Cistanches salsa, a Chinese traditional herbal medicine, which has potential biological activities, both as an antiserum and antifatigue agent (Deng et al. 2004; Xiong et al. 1996). It has also been reported to behave in vitro as a potent free radical scavenger (Facino et al. 1995) and exhibits neuroprotective activities in vitro and in vivo (Koo et al. 2005; Geng et al. 2007). Echinacoside may be a candidate for an anti-aging substance, and an anti-aging activity might be one of its biological properties. Thus, we treated a senescent human embryo lung fibroblastic cell line MRC-5 with echinacoside, expecting it could retard or reverse the senescence.

2. Investigations and results

Firstly, we added echinacoside into the culture medium to check its influence on cell growth and cell cycles. It was observed that the viability of the cells with 48 h exposure to echinacoside increased evidently relative to the control, as the concentration increased (Fig. 1A), indicating that echinacoside helped senescent cells proliferate. Result of SA- Gal staining showed that the level of SA-  -Gal decreased obviously after treatment of echinacoside (data not shown), suggesting that a part of senescent cells were reversed from senescence. To confirm whether senescent cells were indeed propelled to proliferate by echinacoside, fellow cytometry was introduced to check the cell cycle phase alteration of the senescent MRC-5 cells after treatment with echinacoside. In our study, we observed that with the concentration of Echinacoside increased, the ratio of cells in the G1 period was reduced gradually. On the contrary, more cells were driven to enter the S phase and G2 phase (Fig. 1B). The accumulation of senescence-associated heterochromatin foci (SAHF) is another specific biomarker of senescent cells (Shay and Wright 2001). Senescent cells displayed punctuated DNA foci which were visualized by Hoechst staining (Fig. 1C). In contrast, MRC-5 cells treated with echinacoside appeared to have less SAHF.

The level of reactive oxygen species (ROS) increases as cells approach the Hayflflick limit (Hutter et al. 2002). Continuous treatment of cells with sub-lethal levels of ROS not only increases the level of oxidative damage products such as lipofuscin (Sitte et al. 2001) but also causes permanent growth arrest accompanied by the activation of the p53-growth inhibitory pathway (Toussaint et al. 2000). Therefore, at the cellular level, ROS plays a key role in inducing senescence. The FL fluorescent dye DCFH-DA was used to measure ROS contents with fellow cytometry. It was shown that compared with the control, the ROS contents were decreased evidently in cells incubated with echinacoside (Fig. 2A). These results indicate that echinacoside might act as an anti-oxidant, lowering ROS production or accelerating ROS removal.

Accumulated reactive oxygen species (ROS) cause the remarkable impairment of macromolecules in cells, including proteins, DNA, and telomeres (Kovtun et al. 2007). Oxidative DNA damages trigger p53-dependent G1 growth arrest, following the expression of p21Waf-1/SDI-1/Cip1 (Chen et al. 1998) and cell senescence in normal humans fibroblasts. We used a comet assay to measure the DNA damage and its repair. The size of DNA fragments and the number of breaks determine the migration and pattern of the comet seen. Tail DNA content (product of tail length and tail DNA content) increases with damage. Our results show that pretreatment with echinacoside can partly promote the repair of DNA damage, at least protect the cells from DNA lesions caused by H2O2 (Fig. 2B). Based on these facts, we conclude that echinacoside has anti-senescence activity at least in MRC-5 cells. The results of our study show that echinacoside can reduce ROS accumulation, protect cells from DNA damage, promote MRC-5 cells from the G1 phase into the S and G2 phases, and thus improve the proliferation of the cells. Generally speaking, the effects of echinacoside on MRC-5 cells could be divided into two aspects: to promote cell proliferation and to reduce ROS accumulation. Although the precise mechanism of cell senescence is still unknown and the network of cell cycle regulation remains controversial, we have demonstrated that echinacoside can protect cells from oxidative damage. We, therefore, speculate that echinacoside might be a good candidate for regulating senescence, even though the precise underlying mechanism remains an open question and further studies are in progress.

3. Experimental

3.1. Cell culture and treatment

Human embryo lung fibroblastic cell line MRC-5 (Institute of Biochemistry and Cell Biology, SIBS, CAS) was maintained in modified minimum essential medium (Gibco/BRL) and supplemented with 10% fetal bovine serum. MRC-5 cells undergo replicative senescence after multiple cell passages, assessed by the slowing of metabolism and cessation of division within 7 days. All cultures were seeded at a cell density of 104/cm2 unless otherwise noted and were allowed to proliferate, undisturbed, for 7 days. Various concentrations of echinacoside were added 48 h before examination with solvent as control unless otherwise stated. The concentration of DMSO in cultures was less than 0.3% (v/v). For H2O2 treatment, cells were induced with 250M H2O2 for 12 h.

3.2. Survival rate assay

Cells were cultured and treated with echinacoside on 96 well plates. Before detection, 10% Alamar BlueTM was added to the medium and incubated for 4 h. Absorbance was measured with a spectrophotometer.

3.3. Flow cytometry analysis

Flow cytometry was introduced to measure the DNA contents and intracellular ROS levels using propidium iodide and DCFH-DA respectively. Cells were plated at a density of 105cells/10 ml cell solution in 100 mm diameter dishes and supplemented with echinacoside for 48 h after cell seeding. After 48 h treatment, cells were harvested and washed in PBS after centrifugation, then resuspended in PBS solution with 0.1% RNase and 50 g/ml propidium iodide or culture medium containing 10 M DCFH-DA for 30 min. DNA or ROS contents were then determined by FL fluorescence-activated cell sorting on a Beckman Coulter Flow Cytometry System. The data were analyzed by MultiCycle software.

3.4. Hoechst staining

Cells were cultured and treated with echinacoside for 48 h on a 96 well plate. The medium was removed, Cells were washed with PBS, then fixed with 4% formaldehyde in PBS for 20 min, followed with washing with PBS once, and stained with Hoechst dye for 10 min. Pictures were captured.

3.5. Single-cell gel electrophoresis (comet assay)

Comet assay was conducted as reported (Olive and Banath 2006). Individual comet images were analyzed with CometScore software. Acknowledgment: This work was financially supported by the National Basic Research Program (973 Project) of China (No. 2007CB507406), the National Natural Science Foundation of China (No. 30572341), and the Tsinghua-Yue-Yuen Medical Sciences Fund (THYY20070008).