Echinacoside rescues the SHSY5Y neuronal cells from TNFα-induced apoptosis

doi:10.1016/j.ejphar.2004.09.059

European Journal of Pharmacology

Volume 505, Issues 1–3, 28 November 2004, Pages 11-18

https://doi.org/10.1016/j.ejphar.2004.09.059 Get rights and content

Abstract

We investigated the neuroprotective effect of echinacoside, one of the phenylethanoids isolated from the stems of Cistanches salsa, a Chinese herbal medicine, on tumor necrosis factor-alpha (TNFα)-induced apoptosis in human neuroblastoma (SHSY5Y) cells. Treatment of cultured SHSY5Y cells with TNFα 100 ng ml⁻¹ for 36 h stimulated apoptosis, as demonstrated by typical morphological changes, cell viability, DNA laddering, annexin-V binding, intracellular reactive oxygen species, mitochondrial membrane potential and caspase-3 activity. However, simultaneous treatment with echinacoside (1, 10 or 100 μg ml⁻¹) attenuated the TNFα-mediated apoptosis. The antiapoptotic action of echinacoside was partially dependent on antioxidative stress effects, maintenance of mitochondria function, inhibition of caspase-3 activity and was also associated with increasing the expression of the antiapoptotic protein Bcl2. Thus, echinacoside has the neuroprotective capacity to antagonize TNFα-induced apoptosis in SHSY5Y cells and may be useful in treating some neurodegenerative diseases.

Introduction

Apoptosis is a genetically determined mechanism of programmed cell death that can be triggered by various internal and external stimuli. During nervous system development, apoptosis is normal, but its reemergence appears to contribute to neurodegenerative disorders such as Parkinson and Alzheimer diseases and delayed encephalopathy after acute carbon monoxide poisoning (Honig and Ronsenberg, 2000, Martin, 2001, Claude et al., 1997). Several lines of evidence have strongly suggested that oxidative stress, a cellular imbalance between the production and elimination of reactive oxygen species, leads to neuronal apoptosis and necrosis (Kelso et al., 2001, Brown et al., 1997, Freyer, 1998). Therefore, it is valuable to identify compounds that can antagonize the deleterious action of reactive oxygen species and act as an antioxidant to protect neurons from apoptosis.

Echinacoside is one of the phenylethanoids isolated from the stems of Cistanches salsa, a Chinese herbal medicine, which is an important crude drug used both as an antisenium and antifatigue agent (Xiong et al., 1999). Several phenylethanoids have been shown to possess free radical scavenging properties and protect oxidative-stress-induced toxic injuries (Xiong et al., 1996, Xiong et al., 2000, Gao et al., 1999). Tumor necrosis factor-alpha (TNFα) is a toxic-interfering agent because of its important role in neurodegenerative diseases and being a well-characterized model of programmed cell death. Thus, we studied whether echinacoside can protect against TNFα-induced apoptosis in cultured SHSY5Y neuronal cells.

Section snippets

Reagents

Modified Eagle's medium (MEM) and fetal bovine serum (FBS) were purchased from GIBCO/BRL. Chromatin dye bisbenzimide (Hoechst 33342), poly-l-lysine, 3-(4,5-dimethylthiazal-z-yl)-2,5-diphenylterazolium (MTT), TNFα and 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA) were obtained from Sigma (St. Louis, MD, USA). Primary mouse monoclonal antibodies to human Bcl-2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The annexin-V fluorescein isothcocyanate (FITC) apoptosis

Effect of echinacoside on cell nuclear morphology

The Hoechst 33342 staining that is sensitive to DNA was used to assess changes in nuclear morphology following echinacoside and TNFα treatment. The nuclei in normal cells were larger and exhibited diffused staining of the chromatin (Fig. 1A). However, after exposure to 100 ng ml⁻¹ TNFα for 36 h, SHSY5Y cells underwent morphologic changes of apoptosis (Fig. 1B). A marked decrease was observed in the cells treated with echinacoside (Fig. 1C–E).

Effect of echinacoside on cell viability

After incubation with TNFα, approximately 42.9% of

Discussion

C. salsa (C.A. Mey) G. Beck, one species of Cistanches which belongs to Orobanchaceae family, is a parasitic plant native in the northwest of China. The stem of this plant is an important traditional Chinese medicine and used for kidney deficiency, female infertility, morbid leucorrhea, neurasthenia and senile constipation due to colonic inertia. The major active constituents of this herb are phenylethanoid glycosides (Lei et al., 2001). Several phenylethanoids have been shown to possess free

Acknowledgements

The project was supported by the National Nature Science Foundation of China (NO. 30070651). We are grateful to Dr. Lan Yuan and Lin Liu (Department of the Center for Cell Analysis, Peking University) for helpful assistance in the experiments of confocal and flow cytometry, respectively.

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