Serotonin Type 3 Receptor Is Potentially Involved in Cellular Stress Induced by Hydrogen Peroxide

Abstract

Depression is a disease with several molecular mechanisms involved, such as problems in the serotonergic pathway. This disease is very complex and prevalent, and thus important to deeply study and aim to overcome high rates of relapse and therapeutic failure. In this study, two cellular lines were used (HT-22 and SH-SY5Y cells) to gain insight about the role of the serotonin type 3 (5-HT3) receptor in cellular stress induced by hydrogen peroxide and/or corticosterone. In research, these compounds are known to mimic the high levels of oxidative stress and dysfunction of the hypothalamus-hypophysis-adrenal axis by the action of glucocorticoids, usually present in depressed individuals. The receptor 5-HT3 is also known to be involved in depression, previously demonstrated in studies that highlight the role of these receptors as promising targets for antidepressant therapy. Indeed, the drugs used in this work (mirtazapine, scopolamine, and lamotrigine) interact with this serotonergic receptor. Thus, by using cell morphology, cell viability (neutral red and MTT), and HPLC assays, this work aimed to understand the role of these drugs in the stress induced by H₂O₂/corticosterone to HT-22 and SH-SY5Y cell lines. We concluded that the antagonism of the 5-HT3 receptor by these drugs may be important in the attenuation of H₂O₂-induced oxidative stress to the cells, but not in the corticosterone-induced stress.

Keywords: 5-HT3 receptor; corticosterone; hydrogen peroxide; lamotrigine; mirtazapine; scopolamine; serotonin.

Figure 1

Representative images (400× total magnification) of (A) HT-22 and (B) SH-SY5Y cells after immunostaining with DAPI (blue) and primary antibody rabbit polyclonal anti-5-HT3AR/donkey anti-rabbit 488 secondary antibody (green). Scale bar: 100 µm.

Figure 2

HT-22 and SH-SY5Y viability after incubation of mirtazapine (0.01 µM–20 µM) for 48 h, obtained by MTT (left) and NR (right) assays. The results represent the mean ± SEM of 3 independent assays. For SH-SY5Y, these results were previously developed by our Research Group [21].

Figure 3

HT-22 and SH-SY5Y viability after incubation of scopolamine (0.01 µM–20 µM) for 48 h, obtained by MTT (left) and NR (right) assays. The results represent the mean ± SEM of 3 independent assays.

Figure 4

HT-22 and SH-SY5Y viability after incubation of lamotrigine (0.01 µM–20 µM) for 48 h, obtained by MTT (left) and NR (right) assays. The results represent the mean ± SEM of 3 independent assays.

Figure 5

HT-22 and SH-SY5Y morphology after incubation of (A,E) vehicle, (B,F) mirtazapine 20 µM, (C,G) scopolamine 20 µM, and (D,H) lamotrigine 20 µM, for 48 h (100× total magnification). Scale bar: 50 µm.

Figure 6

HT-22 and SH-SY5Y viability after incubation of H₂O₂ (50 µM–300 µM) for 48 h, obtained by MTT (left) and NR (right) assays. The results represent the mean ± SEM of 3–6 independent assays (%). Statistical significance at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. vehicle.

Figure 7

HT-22 and SH-SY5Y viability after incubation of corticosterone (100 µM–500 µM) for 48 h, obtained by MTT (left) and NR (right) assays. The results represent the mean ± SEM of 3–6 independent assays. Statistical significance at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. vehicle.

Figure 8

HT-22 and SH-SY5Y morphology after 48 h-incubation of (A,D) vehicle, (B,E) H₂O₂ 10⁵ µM and 132 µM, respectively, and (C,F) corticosterone 35 µM and 322 µM, respectively (100× total magnification). Scale bar: 50 µm (A,B,D,E) and 179.3 µm (C,F).

Figure 9

HT-22 and SH-SY5Y viability after incubation of H₂O₂ (10⁵ µM and 132 µM) combined with mirtazapine, scopolamine, and lamotrigine (20 µM) for 48 h, obtained by MTT assay. The results represent the mean ± SEM of 3–6 independent assays. Statistical significance at * p < 0.05, and **** p < 0.0001 vs. vehicle.

Figure 10

HT-22 and SH-SY5Y morphology after 48 h-incubation of (A,E) vehicle, (B,F) H₂O₂ 105 µM (for HT-22)/132 µM (for SH-SH5Y) combined with mirtazapine 20 µM, (C,G) H₂O₂ 105 µM (for HT-22)/132 µM (for SH-SH5Y) combined with scopolamine 20 µM, and (D,H) H₂O₂ 105 µM (for HT-22)/132 µM (for SH-SH5Y) combined with lamotrigine 20 µM (100× total magnification). Scale bar: 50 µm.

Figure 11

HT-22 and SH-SY5Y viability after incubation of corticosterone (35 µM and 322 µM) combined with mirtazapine, scopolamine, and lamotrigine (20 µM) for 48 h, obtained by MTT assay. The results represent the mean ± SEM of 3–6 independent assays. Statistical significance at ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. vehicle.

Figure 12

HT-22 and SH-SY5Y morphology after 48 h-incubation of (A,E) vehicle (B,F) corticosterone 35 µM (for HT-22)/322 µM (for SH-SH5Y) combined with mirtazapine 20 µM, (C,G) corticosterone 35 µM (for HT-22)/322 µM combined with scopolamine 20 µM, and (D,H) corticosterone 35 µM (for HT-22)/322 µM combined with lamotrigine 20 µM (100× total magnification). Scale bar: 179.3 µm.

Figure 13

Comparison between the attenuation of H₂O₂ and corticosterone-induced stress by mirtazapine, scopolamine, and lamotrigine, in HT-22 and SH-SY5Y cells. The results represent the % of stress attenuation of H₂O₂/corticosterone (IC50 values for each cell line) combined with mirtazapine, scopolamine, and lamotrigine (20 µM) in comparison with H₂O₂/corticosterone alone (IC50 values for each cell line).

Figure 14

Concentrations of 5-HT (nM) in the extracellular medium of mirtazapine, scopolamine, and lamotrigine-treated HT-22 cells (above) and SH-SY5Y cells (below), determined by HPLC.

Figure 15

Concentrations of 5-HT (nM) in the extracellular medium of HT-22 cells (above) and SH-SY5Y cells (below) treated with corticosterone, H₂O₂, and their combinations with mirtazapine, scopolamine, and lamotrigine, determined by HPLC.