Milk thistle seed extract protects rat C6 astroglial cells from acute cocaine toxicity

Abstract

Cocaine is a powerful addictive drug, widely abused in most Western countries. It easily reaches various domains within and outside of the central nervous system (CNS), and triggers varying levels of cellular toxicity. No pharmacological treatment is available to alleviate cocaine-induced toxicity in the cells without side-effects. Here, we discerned the role of milk thistle (MT) seed extract against cocaine toxicity. First, we investigated acute cytotoxicity induced by treatment with 2, 3 and 4 mM cocaine for 1 h in astroglial, liver and kidney cells in vitro, and then in living shrimp larvae in vivo. We showed that astroglial cells are more sensitive to cocaine than liver, kidney cells or larvae. Cocaine exposure disrupted the general architecture of astroglial cells, induced vacuolation, decreased cell viability, and depleted the glutathione (GSH) level. These changes may represent the underlying pathology of cocaine in the astrocytes. By contrast, MT pretreatment (200 µg/ml) for 30 min sustained the cell morphological features and increased both cell viability and the GSH level. Besides its protective effects, the MT extract was revealed to be non-toxic to astroglial cells, and displayed high free-radical scavenging activity. The results from this study suggest that enhanced GSH level underlies cell protection, and indicate that compounds that promote GSH synthesis in the cells may be beneficial against cocaine toxicity.

Figure 1

Effect of cocaine on viability. (A) C6 astroglial, (B) liver and (C) kidney cells were treated with various concentrations of cocaine for 1 h in 96-well plates, and the viability was assessed by the crystal violet dye uptake method. (D) Living shrimp larvae were counted in each vial at the end of a 1-h cocaine treatment. Data are expressed as mean ± standard error of the mean (SEM), ^*P<0.05 or ^**P<0.01, compared to the control (0 μM cocaine).

Figure 2

Measurement of antioxidant and cytotoxic activities of milk thistle (MT) extract. (A) Various concentrations of MT dissolved in ethanol in 1-ml eppendorf tubes without cells, were incubated in 0.1 mM of the free radical 2,2-diphenyl-1-picrylhydrazyl for 30 min. (B) C6 astroglial cells were treated with different concentrations of MT for 1 h, and viability was assessed using the crystal violet dye (0.1%) uptake method. Data are expressed as means ± standard error of the mean (SEM), ^**P<0.01, compared to the control (0 μM MT).

Figure 3

Attenuation of cocaine-induced toxicity by milk thistle (MT) extract in C6 astroglial cells. Cells were pretreated with 200 μg/ml MT for 30 min, followed by co-treatment with 2, 3 or 4 mM cocaine for 1 h. (A) Cell viability was evaluated with the crystal violet (0.1%) dye uptake assay and (B) images of control (i), MT (ii), 4 mM cocaine (iii) and MT-pretreated and 4 mM cocaine co-treated cells (iv) were acquired under an inverted phase contrast 1X-70 Olympus microscope with a ×40 objective. Scale bar, 50 μm. (C) Vacuolation in control and 4 mM cocaine-treated cells was quantified with the neutral red dye (0.05%) uptake assay. Data are expressed as mean ± standard error of the mean (SEM), ^*P<0.05 or ^**P<0.01, compared to the respective controls; ^##P<0.01, cocaine treatment alone vs. MT-pretreated and cocaine co-treated group.

Figure 4

Effect of milk thistle (MT) extract on the total glutathione level. C6 astroglial cells were pretreated with 200 μg/ml MT for 30 min, followed by co-treatment with cocaine for 1 h. Data are expressed as mean ± standard error of the mean (SEM). ^*P<0.05 or ^**P<0.01, compared to the respective controls; ^##P<0.01, cocaine treatment alone vs. MT-pretreated and cocaine co-treated group. Abs, absorbance.