Baicalein attenuates 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells

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Abstract

It has been suggested that baicalein, a flavonoid obtained from the Scutellaria root, exerts a protective role on neurons against several neuronal insults. However, the protective mechanisms underlying this protective effect remain largely unknown. Our results indicate that baicalein protects SH-SY5Y cells, a dopaminergic neuronal cell line, from 6-hydroxydopamine (6-OHDA)-induced damage by the attenuation of reactive oxygen species (ROS). In order to determine the effects of baicalein on mitochondrial events, mitochondrial membrane potentialsΨm) and caspase cascades downstream of mitochondria were assessed. Baicalein inhibited the collapse of ΔΨm, suggesting that baicalein reduces the mitochondrial dysfunction associated with 6-OHDA treatment. Baicalein also inhibited caspase-9 and caspase-3 activation, which can be triggered by mitochondrial malfunctions. Furthermore, baicalein induced a significant reduction in the level of phospho-JNK, which is known as an apoptotic mediator in 6-OHDA-induced neuronal cell death. Our results indicate that baicalein protects neurons from the deleterious effects of 6-OHDA via the attenuation of oxidative stress, mitochondrial dysfunction, caspase activity, and JNK activation.

Introduction

Parkinson's disease (PD) is a typical neurodegenerative disorder, characterized by symptoms including rest tremors, postural instability, gait abnormality, bradykinesia and rigidity. Although genetically susceptible individuals exhibit early onset of PD, in most cases PD occurs sporadically with age (Alonso et al., 1986). PD is mainly characterized by the selective loss of the dopaminergic neurons in the substantia nigra. The mechanisms by which this neuronal loss occurs remain unclear (Moghal et al., 1994). The presence of apoptotic nigral neurons was demonstrated in post-mortem brains of PD patients (Anglade et al., 1997).

Insight into the potential mechanisms which contribute to PD-associated neurodegeneration has been obtained from animal PD models. One of the neurotoxins which can induce PD in this model is 6-hydroxydopamine (6-OHDA). 6-OHDA is a dopamine analog, which readily undergoes non-enzymatic oxidation, resulting in the production of hydrogen peroxide (H2O2), superoxide, and the hydroxyl radical, under physiological pH conditions (Cohen and Heikkila, 1974). Intrastriatal 6-OHDA injections result in a Parkinsonian pattern, together with neuronal loss in the substantia nigra of the rat brain (Ungerstedt, 1968). Although used as an exogenous neurotoxin in this model, some evidence suggests that 6-OHDA can be formed in vivo from dopamine. In PD, the neurons within the substantia nigra pars compacta (SNpc) contain an increased amount of iron (Hirsch et al., 1991). In addition, the effects of PD reduce the activities of catalase and glutathione peroxidase in the brain (Ambani et al., 1975; Kish et al., 1985). In the presence of free ferric iron and H2O2, the principal product of dopamine oxidation has been reported to be 6-OHDA (Jameson and Linert, 1999). Andrew et al. (1993) reported that the urine of PD patients treated with levodopa contained an increased amount of 6-OHDA. The fact that 6-OHDA is found in the brain and urine samples of PD patients indicates that 6-OHDA may constitute an important endogenous cause of PD pathogenesis (Andrew et al., 1993; Curtius et al., 1974; Jellinger et al., 1995).

Several studies have suggested that baicalein exerts an anti-oxidant effect (Gao et al., 1995; Hanasaki et al., 1994). Also, Hamada et al. (1993) reported that baicalein is capable of scavenging reactive oxygen species (ROS), including superoxide (O2−), H2O2, and hydroxyl radicals. Baicalein has also been shown to strongly inhibit iron-dependent lipid peroxidation in microsomes (Gao et al., 1995) and mitochondria (Miyahara et al., 1993). Gao et al. (1998) reported that baicalein prevented ROS-mediated damage of human dermal fibroblast cells better than the iron chelator deferoxamine or hydroxyl radical scavengers (including α-tocopherol and allopurinol).

Dopamine agonists including levodopa and ropinirole have been used in the treatment of PD (Rascol et al., 2000) with significantly greater benefit for patients treated with levodopa than with any other substance. However, patients treated with levodopa develop wearing off, dyskinesias, or on–off motor fluctuation (Parkinson Study Group, 2000). Moreover, levodopa cannot be administered for a long time, as the effectiveness of levodopa may be diminished, due to levodopa resistance (Sandyk et al., 1987). Thus, it is clearly important to search for a fundamental mix of therapeutics for the improvement of PD symptoms. Oxidative stress, one of the major causes of PD, may be a target for treatment.

Therefore, in the present study, we have examined the effects of baicalein on 6-OHDA-induced dopaminergic neuronal cell death in SH-SY5Y cells.

Section snippets

Cell culture

SH-SY5Y human neuroblastoma cells were cultivated at 37 °C in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL) in a 95% humidified air and 5% CO2 atmosphere. Before treating the cells with 6-OHDA (20 μM) they were cultured in MEM, containing 1% FBS for 2 h to assure the neuronal survival and the morphological integrity of the cells.

Pharmacological treatments

6-OHDA (Sigma Chemical Co.) was dissolved in 2% ascorbic acid and used at a final concentration of 20 μM, which

Baicalein attenuates 6-OHDA-induced SH-SY5Y cell death

Baicalein is one of the principal components of Scutellaria baicalensis, and its structure is shown in Fig. 1A. Baicalein per se hardly revealed any effect on SH-SY5Y cell viability (Fig. 1B; AlamarBlue assay). Treatment of SH-SY5Y cells with 6-OHDA alone resulted in an approximately 40% reduction in cell survival within 12 h, whereas the samples pre-treated with 6.25 and 12.5 μM baicalein showed a reduction of 6-OHDA-mediated cytotoxicity (Fig. 2A). We also observed morphological changes using

Discussions

The selective loss of dopaminergic neurons in the substantia nigra appears to be the direct cause of neurodegeneration in cases of PD. Also, 6-OHDA, which is commonly used for the induction of PD in experimental animals, is believed to cause dopaminergic cell death via a free radical mechanism. Our results indicate that 6-OHDA induced oxidative stress, and that its blockage resulted in attenuated cell death. These results indicate that ROS play a primary role in the induction of neuronal damage

Acknowledgments

This research was supported by grants from the Korea Health 21 R&D Project funded by the Ministry of Health and Welfare of the Korean government (01-PJ8-PG1-01 CN2-0003) and Rural Development Administration (02-N-I-02).

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