Reversal of homocysteine-induced neurotoxicity in rat hippocampal neurons by astaxanthin: evidences for mitochondrial dysfunction and signaling crosstalk

Abstract

Elevated plasma level of homocysteine (Hcy) represents an independent risk for neurological diseases, and induction of oxidative damage is considered as one of the most important pathomechanisms. Astaxanthin (ATX) exhibits strong antioxidant activity in kinds of experimental models. However, the potential of ATX against Hcy-induced neurotoxicity has not been well explored yet. Herein, the neuroprotective effect of ATX against Hcy-induced neurotoxicity in rat hippocampal neurons was examined, and the underlying mechanism was evaluated. The results showed that ATX pre-treatment completely reversed Hcy-induced neurotoxicity through inhibiting cell apoptosis in rat primary hippocampal neurons. The mechanical investigation revealed that ATX effectively blocked Hcy-induced mitochondrial dysfunction by regulating Bcl-2 family and opening of mitochondrial permeability transition pore (MPTP). ATX pre-treatment also attenuated Hcy-induced oxidative damage via inhibiting the release of intracellular reactive oxide species (ROS) and superoxide anion through regulating MPTP opening. Moreover, normalization of MAPKs and PI3K/AKT pathways also contributed to ATX-mediated protective effects. Taken together, these results above suggested that ATX has the potential to reverse Hcy-induced neurotoxicity and apoptosis by inhibiting mitochondrial dysfunction, ROS-mediated oxidative damage and regulation of MAKPs and AKT pathways, which validated the strategy of using ATX could be a highly effective way in combating Hcy-mediated neurological disorders.

Fig. 1. ATX reverses Hcy-induced neuronal toxicity.

a Neural toxicity of Hcy towards primary neurons. Neurons were treated with 1–10 mM Hcy for 24 h, and neural viability was detected by CCK-8 assay. b ATX reversed Hcy-induced neural toxicity. Neurons were pre-treated with 0.5–5 μM ATX before Hcy co-treatment. c Morphological changes. Neural morphological changes with or without tubulin staining were observed by phase contrast and fluorescence microscope (magnification, ×200). All data and images were obtained from three random experiments. Scale bar in figures indicates 50 μm. Bars with “*”, “**” and “***” represent the P < 0.05, P < 0.01 and P < 0.001, respectively. Bars with different letters indicate the statistic difference at P < 0.05

Fig. 2. ATX inhibits Hcy-induced neurons apoptosis.

a Nuclear condensation and DNA fragmentation by TUNEL-DAPI co-staining (magnification, ×200). b Caspase-3 activity. c Time-course of Hcy on activecaspase-3 expression. d ATX attenuated Hcy-induced PARP cleavage and caspase-3 activation. Details of the experiments were conducted according to the section of methods. Scale bar in figures indicates 30 μm. All data and images were obtained from three random experiments. Bars different letters indicate the statistic difference at P < 0.05

Fig. 3. ATX Blocks Hcy-induced Mitochondrial dysfunction by regulating Bcl-2 family and opening of MPTP.

a ATX blocked Hcy-induced mitochondrial morphological changes and loss of mitochondrial membrane potential (Δψm) (magnification, ×200). b Time-course effect of Hcy on Bcl-2 and Bad expression. c ATX blocked Bcl-2 family imbalance. d Effect of CsA on Δψm in neurons. e Effect of CsA on neural viability. Neurons were pre-treated with 5 μM CsA for 2 h before combined treatment. Cell viability and Δψm were detected by CCK-8 assay and JC-1 staining, respectively. Details of experiments were conducted according to the section of methods. Scale bar in figures indicates 50 μm. All data and images were obtained from three random experiments. All images were obtained from three random experiments. Bars different letters indicate the statistic difference at P < 0.05

Fig. 4. ATX Attenuates Hcy-induced accumulation of ROS and superoxide anion.

a Detection of ROS. b Quantitative analysis of ROS generation. c Detection of superoxide anion. d Quantitative analysis of superoxide anion generation. ROS and superoxide anion were detected by HCFH-DA and DHE probes (magnification, ×200), respectively as described in the section of methods. e Effect of CsA on ROS generation in neurons. f Effect of CsA on superoxide anion in neurons. Neurons were pre-treated with 5 μM CsA for 2 h before combined treatment. Generation of ROS and superoxide anion was detected by DCFH-DA and DHE staining, respectively. Scale bar in figures indicates 50 μm. All data and images were obtained from three random experiments. Bars with different letters indicate the statistic difference at P < 0.05

Fig. 5. ATX attenuates Hcy-induced oxidative damage.

a Time-course of Hcy on p53 and histone phosphorylation. b Quantitative analysis of Ser15-p53 and Ser139-histone expression. c ATX attenuated Hcy-induced p53 and histone phosphorylation. d Effect of ATX on Hcy-induced neural toxicity. Neurons were pre-treated with 5 mM GSH for 2 h and co-treated with GSH for 24 h. Neural viability was detected by CCK-8 assay. All data and images were obtained from three random experiments. Bar with “**” or different letters indicate the statistic difference at P < 0.05

Fig. 6. Contribution of MAPKs and AKT pathways.

a Time-course of Hcy on MAPK (ERK1/2) and AKT phosphorylation. b Quantitative analysis of Thr202/Tyr204-MAPK (ERK1/2) and Ser473-AKT expression. c Effects of LY294002 (AKT inhibitor) and U0126 (ERK inhibitor) on Hcy-induced neural toxicity. Neurons were pre-treated with 10 μM LY294002 or U0126 for 2 h and co-treated with 8 mM Hcy for 24 h. d ATX attenuated Hcy-induced MAPK (ERK1/2) and AKT phosphorylation. All data and images were obtained from three random experiments. Bar with “**” or different letters indicate the statistic difference at P < 0.05