Humic Acid Increases Amyloid β-Induced Cytotoxicity by Induction of ER Stress in Human SK-N-MC Neuronal Cells

Abstract

Humic acid (HA) is a possible etiological factor associated with for several vascular diseases. It is known that vascular risk factors can directly increase the susceptibility to Alzheimer's disease (AD), which is a neurodegenerative disorder due to accumulation of amyloid β (Aβ) peptide in the brain. However, the role that HA contributes to Aβ-induced cytotoxicity has not been demonstrated. In the present study, we demonstrate that HA exhibits a synergistic effect enhancing Aβ-induced cytotoxicity in cultured human SK-N-MC neuronal cells. Furthermore, this deterioration was mediated through the activation of endoplasmic reticulum (ER) stress by stimulating PERK and eIF2α phosphorylation. We also observed HA and Aβ-induced cytotoxicity is associated with mitochondrial dysfunction caused by down-regulation of the Sirt1/PGC1α pathway, while in contrast, treating the cells with the ER stress inhibitor Salubrinal, or over-expression of Sirt1 significantly reduced loss of cell viability by HA and Aβ. Our findings suggest a new mechanism by which HA can deteriorate Aβ-induced cytotoxicity through modulation of ER stress, which may provide significant insights into the pathogenesis of AD co-occurring with vascular injury.

Figure 1

Humic acid (HA) enhances exogenous Aβ-induced apoptosis in human SK-N-MC cells. (A) Cells were treated with either 0, 10, 50, 100 or 200 μg/mL HA, and co-treated with or without 2.5 μM Aβ for 24 h followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays; (B) Cells were treated with either 0, 1.25, 2.5 or 5 μM Aβ with or without 100 μg/mL HA for 24 h followed by MTT assays. Co-treatment of the cells with HA further increased Aβ-induced cell death compared to the Aβ alone groups; (C) HA markedly increased Aβ-induced nucleus fragmentation. Apoptosis was determined by fragmented morphology in nuclei for DAPI fluorescence; (D) Aβ-induced cell death was determined by western blotting of cleaved caspase 3 and PARP levels, showing a synergistically enhanced apoptosis after HA concomitant treatments. HA, humic acid. FA, fulvic acid. All results are shown from three independent experiments, and values are presented as mean ± SEM. Significant differences was determined by using the multiple comparisons of Dunnett’s post-hoc test for ** p < 0.01. Scale bar represents 50 μm.

Figure 2

HA increases Aβ-induced ER stress. SK-N-MC cells were treated with 100 μg/mL HA with or without 2.5 μM Aβ for 0 to 24 h. (A) Immunoblotting of caspase 4 showed HA causes cleavage of caspase 4, and that co-treatment with Aβ increases this cleavage in a time-dependent manner. The lower panel displays quantitative analyses showing cleaved caspase 4/β-actin ratios; (B) Western blots showed the activation of the ER stress by monitoring the phosphorylation of PERK and eIF2α in cells treated with HA alone, or in combination with Aβ for 0 to 24 h; (C) RT-PCR analysis showed the splicing of XBP-1 mRNA for the indicated time in cells treated with HA alone, or in combination with Aβ for 0, 12 or 24 h; (D) Salubrinal, an ER stress inhibitor blocked HA and Aβ-mediated ER stress-induced PERK and eIF2α phosphorylation at 24 h; (E) Cell viabilities were significantly increased in Salubrinal-treated cells compared to HA and Aβ co-treated cells for 24 h. All results are shown from three independent experiments, and values are presented as mean ± SEM. Significant differences was determined by using the multiple comparisons of Dunnett’s post-hoc test for ** p < 0.01.

Figure 2

HA increases Aβ-induced ER stress. SK-N-MC cells were treated with 100 μg/mL HA with or without 2.5 μM Aβ for 0 to 24 h. (A) Immunoblotting of caspase 4 showed HA causes cleavage of caspase 4, and that co-treatment with Aβ increases this cleavage in a time-dependent manner. The lower panel displays quantitative analyses showing cleaved caspase 4/β-actin ratios; (B) Western blots showed the activation of the ER stress by monitoring the phosphorylation of PERK and eIF2α in cells treated with HA alone, or in combination with Aβ for 0 to 24 h; (C) RT-PCR analysis showed the splicing of XBP-1 mRNA for the indicated time in cells treated with HA alone, or in combination with Aβ for 0, 12 or 24 h; (D) Salubrinal, an ER stress inhibitor blocked HA and Aβ-mediated ER stress-induced PERK and eIF2α phosphorylation at 24 h; (E) Cell viabilities were significantly increased in Salubrinal-treated cells compared to HA and Aβ co-treated cells for 24 h. All results are shown from three independent experiments, and values are presented as mean ± SEM. Significant differences was determined by using the multiple comparisons of Dunnett’s post-hoc test for ** p < 0.01.

Figure 3

HA increases Aβ-induced mitochondria dysfunction and H₂O₂ production. (A) The mitochondrial membrane potential stained with JC-1 dye was observed by fluorescent microscopy. After co-treatment of HA and Aβ, SK-N-MC cells showed more intense green fluorescence than non-treated and Salubrinal-treated cells; (B) Intracellular oxidative bursts were determined by dichlorofluorescin diacetate (DCFH-DA) staining. Cells were treated with the indicated compounds for 24 h. After treatment, cells were stained by DCFH-DA and visualized by using fluorescence microscopy; (C) Measurements of H₂O₂ production from SK-N-MC cells by Amplex red fluorometric method. Co-treatment of HA and Aβ significantly induced H₂O₂ production parallel to decreased mitochondrial membrane potential; (D) Western blotting assays showed that co-treatment of HA and Aβ inhibits PGC1α expression, whereas the addition of Salubrinal significantly returns this inhibition; (E) The mRNA levels of SOD1, SOD2 and catalase were detected by real time PCR, showing that HA and Aβ-treated SK-N-MC cells express significantly less mRNA than non-treated and Salubrinal-treated cells. All results are shown from three independent experiments, and values are presented as mean ± SEM. Significant differences were determined by using the multiple comparisons of Dunnett’s post-hoc test for * p < 0.05 and ** p < 0.01. Scale bar represents 50 μm.

Figure 3

HA increases Aβ-induced mitochondria dysfunction and H₂O₂ production. (A) The mitochondrial membrane potential stained with JC-1 dye was observed by fluorescent microscopy. After co-treatment of HA and Aβ, SK-N-MC cells showed more intense green fluorescence than non-treated and Salubrinal-treated cells; (B) Intracellular oxidative bursts were determined by dichlorofluorescin diacetate (DCFH-DA) staining. Cells were treated with the indicated compounds for 24 h. After treatment, cells were stained by DCFH-DA and visualized by using fluorescence microscopy; (C) Measurements of H₂O₂ production from SK-N-MC cells by Amplex red fluorometric method. Co-treatment of HA and Aβ significantly induced H₂O₂ production parallel to decreased mitochondrial membrane potential; (D) Western blotting assays showed that co-treatment of HA and Aβ inhibits PGC1α expression, whereas the addition of Salubrinal significantly returns this inhibition; (E) The mRNA levels of SOD1, SOD2 and catalase were detected by real time PCR, showing that HA and Aβ-treated SK-N-MC cells express significantly less mRNA than non-treated and Salubrinal-treated cells. All results are shown from three independent experiments, and values are presented as mean ± SEM. Significant differences were determined by using the multiple comparisons of Dunnett’s post-hoc test for * p < 0.05 and ** p < 0.01. Scale bar represents 50 μm.

Figure 4

Sirt1 protects neuronal cells from HA and Aβ-induced PGC1α suppression and cytotoxicity. (A) Western blotting assays showed the co-treatment of HA and Aβ inhibits expressions of Sirt1 and PGC1α. However, the addition of Salubrinal (50 μM) significantly blocked these inhibitions; (B) Sirt1 over-expression significantly restores HA and Aβ-inhibited PGC1α levels, (C) and contributes to a better survival rate in HA and Aβ co-treated cells; (D) Sirt1 over-expression failed to inhibit the levels of p-Thr980 PERK and p-Ser51 eIF2α in HA and Aβ co-treated cells, indicating that ER stress occurs upstream of Sirt1 down-regulation. All results are shown from three independent experiments, and values are presented as mean ± SEM. Significant differences were determined by using multiple comparisons of Dunnett’s post-hoc test for ** p < 0.01.

Figure 4

Sirt1 protects neuronal cells from HA and Aβ-induced PGC1α suppression and cytotoxicity. (A) Western blotting assays showed the co-treatment of HA and Aβ inhibits expressions of Sirt1 and PGC1α. However, the addition of Salubrinal (50 μM) significantly blocked these inhibitions; (B) Sirt1 over-expression significantly restores HA and Aβ-inhibited PGC1α levels, (C) and contributes to a better survival rate in HA and Aβ co-treated cells; (D) Sirt1 over-expression failed to inhibit the levels of p-Thr980 PERK and p-Ser51 eIF2α in HA and Aβ co-treated cells, indicating that ER stress occurs upstream of Sirt1 down-regulation. All results are shown from three independent experiments, and values are presented as mean ± SEM. Significant differences were determined by using multiple comparisons of Dunnett’s post-hoc test for ** p < 0.01.