Keywords

1 Introduction

Arsenic (As), being a kind of toxic metalloid, is widely distributed around the world. Industrial and geological pollution is the major source of As which was found in contaminated water resource, food, ambient air and dust (Carlin et al. 2016). Humans are exposed to the toxin mainly through skin contact, digestive tract, and inhalation. The toxin presents as an organic compound or inorganic compound which involved with a myriad of disease, including cancer, neurologic deficits, psychiatric problems, kidney disease, diabetes, cardiovascular disease, respiratory outcomes, and reproductive abnormalities (Kuo et al. 2013; Navas-Acien et al. 2005; Peters et al. 2015). When arsenic was absorbed into the body, it would distribute into several tissues and organs. Our group previously reported that As exposure induced injury in the hippocampus and cerebellum of mice (Li et al. 2017; Zhang et al. 2014a). Cerebrum is another major target of As which is the vital organ in body. Impaired nerve system was observed in various As-exposed models at environmental relevant levels (Sinczuk-Walczak 2009; Zhang et al. 2013).

Taurine, a sulfur-containing-β-amino acid, presents in many mammalian tissues being a major free intracellular amino acid (Batista et al. 2013). Several studies indicated that taurine treatment reduced inflammation, fibrosis, apoptosis and hyperplasia in lungs and alveolar epithelial cells (Men et al. 2010; Schuller-Levis et al. 2003; Bhavsar, Patel and Lau-Cam 2010; Schuller-Levis et al. 1995). It protects many tissues and organs of body against apoptosis and toxicity induced by various poisonous substances(Men et al. 2010; Zhang et al. 2014b; Higuchi et al. 2012). Taurine is considered an attractive candidate to relieve arsenic-induced injury in neurons.

In the present study, the effect of As and taurine on the viability of primary cortical neurons was assessed by MTT and capase-3 assay. Bax and Bcl-2 levels were examined in taurine/arsenic-treated cells by western blot. The aim of the study was to investigate the beneficial role of taurine on arsenic-induced neurotoxicity in primary cortical neurons.

2 Methods

2.1 Primary Cortical Neuronal Culture and Groups Tested

Embryonic rats were used to isolate brains and obtain primary cortical neurons according to the methods previously reported (Teng et al. 2013) with some modifications. In brief, day 16–18 embryos were collected from pregnant rats and washed with PBS. Brains were isolated and kept in basal media eagle containing 26.8 mM glucose, 2 mM glutamine, 20% fetal bovine serum at 37 °C for 10 min with a gentle shaking. Then, a 14-G cannula was used to pass the cortices and make cell suspension. Then the suspension was centrifuged at 200 × g for 5 min and seeded on poly-D-lysine (5 μg/ml) precoated dish in incubator. Sodium arsenite in various concentrations was exposed on the second day with or without taurine for 24 h.

2.2 MTT Assay

Briefly, 10 μl of 5 mg/ml MTT solution (in PBS) was added to each well of 96-well plate, and the cells were incubated at 37 °C for 4 h. Then, 100 μl of dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan crystals. Followed by incubation for 30 min, the absorbance was read at 570 nm. Cellular viability was expressed as a percentage relative to the control (control %).

2.3 Western Blot

Cells were homogenized in lysis buffer with 1% proteinase inhibitors. The total cell lysis were loading for SDS-PAGE to separate various proteins, then transferred to PVDF membrane. The membrane was incubated with Bad, Bcl-2 or p-Akt primary antibodies (1:1000, Cell Signaling Technology, USA) overnight at 4 °C. Second horseradish peroxidase-conjugated antibody (1:5000, Sigma, USA) was used for visualizing.

2.4 Statistic Analysis

Data were analyzed with SPSS 11.0. Difference between various groups was analyzed with one-way ANOVA and LSD test. P value <0.05 was considered significant.

3 Results

3.1 As Exposure Deduced the Viability of Primary Cortical Neurons in a Concentration-Dependent Manner

The toxic effect of As on primary cortical neurons was assess by MTT assay. As shown in Fig. 1, the viability of primary cortical neurons was decreased markedly in a dose-dependent manner after 24 h As exposure. The cell viability was reduced nearly 55% with 5 μM arsenite. So, this concentration, 5 μM arsenite, was used in the following experiments.

Fig. 1
figure 1

Effect of As on the viability of primary cortical neurons. Data were presented as mean ± SD. ap < 0.05, compared with control group

Full size image

3.2 Taurine Treatment Protect Primary Cortical Neurons Against as Neurotoxicity

To confirm the protection of taurine against As neurotoxicity in primary cortical neurons, the cells were treated with 5 μM arsenite with or without taurine for 24 h. The results showed that the activity of primary cortical neurons was decreased after As exposure compared with control group, indicating arsenic has neurotoxicity in primary cortical neurons. Interestingly, the decreased viability was significantly reversed with taurine treatment. The reversion level is related with the concentration of taurine. With 4 mM taurine treatment, the viability of cells is about 90% compared with the control group. It is suggest that taurine have neuroprotection against As-exposed primary cortical neurons (Fig. 2).

Fig. 2
figure 2

Effect of taurine on the viability of As-exposed primary cortical neurons. Data were presented as mean ± SD. ap < 0.05, compared with As group

Full size image

3.3 Taurine Treatment Prohibited the Disturbance of the Level of Bax and Bcl-2 in As-Exposed Primary Cortical Neurons

The protein level of Bax and Bcl-2 in primary cortical neurons was examined with western blot. As shown in Fig. 3, Bax level was markedly increased in As exposure cells than that in control group. Interestingly, the enhancement of Bax expression was reduced apparently after taurine treatmeng. With the treatment of taurine, Bax level was significantly reduced. At the same time, compared with control group, As exposure significantly reduced the level of Bcl-2 in primary cortical neurons, which was markedly reversed after taurine administration.

Fig. 3
figure 3

Effect of taurine on the expression of Bax and Bcl-2 in As-exposed primary cortical neurons

Full size image

4 Discussion

The present study explored the protective effect of taurine against As neurotoxicity in primary cortical neurons. Our group previously reported that As exposure induced injury in the hippocampus and cerebellum of mice (Li et al. 2017, Zhang et al. 2014a). To confirm the neurotoxicity of arsenite on primary cortical neurons, various concentration of arsenite were exposed to primary cortical neurons for 24 h and the cell viability was examined by MTT assay. The results showed that the viability of primary cortical neurons was decreased in a dose-dependent manner with arsenite exposure. To identify the neuroprotective capacity of taurine against arsenic-induced injury, cells were treated with a range of doses of taurine and arsenite. We found that taurine treatment markedly enhanced cell viability in a dose-dependent way. It is suggested that arsenite exerts harmful effects on primary cortical neurons and taurine has protective effects against arsenite neurotoxicity in primary cortical neurons.

Bax and Bcl-2 are the key regulators of cell viability which take part the activation of apoptosis (Wang et al. 2013). Disturbance of Bax or Bcl-2 expression would lead the release of mitochondrial Cyt C into the cytosol, which eventually activates the caspase cascade and apoptosis. Bax is pro-apoptotic regulator and Bcl-2 is an anti-apoptotic one (Braun 2012). In the present study, the results showed that taurine significantly prohibited the disruption of Bax and Bcl-2 in As-exposed primary cortical neurons. The results demonstrate that taurine elicited protective effects on arsenic-induced injury and markedly inhibited the disturbance of Bax and Bcl-2 expression. It is suggest that taurine treatment has a protective effect on arsenic-induced primary cortical neuron injury, which is related to altered Bax/Bcl-2 ratio.

5 Conclusion

In summary, the present study shows that taurine treatment significantly inhibited the decrease of cell viability in arsenic-exposed primary cortical neurons. Taurine reversed arsenite-reduced viability of primary cortical neurons. Taurine possesses neuroprotective capacity in arsenic-exposed primary cortical neurons involving the regulation of oxidative stress.