2-Iodo-4'-Methoxychalcone Attenuates Methylglyoxal-Induced Neurotoxicity by Activation of GLP-1 Receptor and Enhancement of Neurotrophic Signal, Antioxidant Defense and Glyoxalase Pathway

Abstract

Methylglyoxal (MG) acts as a reactive precursor of advanced glycation end products (AGEs). This compound is often connected with pathologies such as diabetes, neurodegenerative processes and diseases of aging. 2-iodo-4'-methoxychalcone (CHA79), a synthetic halogen-containing chalcone derivative, has been reported its anti-diabetic activity. This study aims to investigate the potential protective capability of CHA79 against MG-mediated neurotoxicity in SH-SY5Y cells. Results indicated CHA79 increased viability of cells and attenuated the rate of apoptosis in MG-exposed SH-SY5Y. CHA79 up-regulated expression of anti-apoptotic protein (Bcl-2) and down-regulated apoptotic proteins (Bax, cytochrome c, caspase-3, caspase-9). Moreover, CHA79 significantly up-regulated expression of neurotrophic factors, including glucagon-like peptide-1 receptor (GLP-1R), brain derived neurotrophic factor (BDNF), p75NTR, p-TrkB, p-Akt, p-GK-3β and p-CREB. CHA79 attenuated MG-induced ROS production and enhanced the antioxidant defense including nuclear factor erythroid 2-related factor 2 (Nrf2), HO-1, SOD and GSH. Furthermore, CHA79 attenuated MG-induced reduction of glyoxalase-1 (GLO-1), a vital enzyme on removing AGE precursors. In conclusion, CHA79 is the first novel synthetic chalcone possessing the GLP-1R and GLO-1 activating properties. CHA 79 also exhibits neuroprotective effects against MG toxicity by enhancing neurotrophic signal, antioxidant defense and anti-apoptosis pathway.

Keywords: antioxidant defense; glyoxalase pathway; halogen-containing chalcones; methylglyoxal; neuroprotection; neurotrophic effect.

Figure 1

Chemical structure of 2-iodo-4′-methoxychalcone (CHA79).

Figure 2

Effects of CHA79 on cell viability (A), nuclear condensation (B), and Annexin V-positive cell numbers (C) in MG-treated SH-SY5Y dopaminergic neurons. Cells were pre-treated with CHA79 (0.1–1 μM) for 1 h, and MG (500 μM) was then treated for 24 h. Cell viability was determined by MTT assay. Nuclear condensation (white arrow) was determined by Hoechst 33342 and observed by a fluorescent microscope. Scale bar = 50 μM. Annexin V-positive cell numbers were counted by flow cytometer and were represented as the percentage of total cell numbers. # p < 0.05 versus the control group (vehicle control: 0.1% DMSO). * p < 0.05 versus the MG group.

Figure 3

Effects of CHA79 on expressions of Bcl-2 (A), Bax (B), cytosolic cytochrome c (c-cyt c) (C), cleaved caspase-9 (D), and cleaved caspase-3 (E) in MG-treated SH-SY5Y dopaminergic neurons. Cells were pre-treated with CHA79 (0.1–1 μM) for 1 h, and MG (500 μM) was then treated for 24 h. Densitometry analyses are presented as the relative ratio of protein/β-actin protein, and are represented as percentages of control group (vehicle control: 0.1% DMSO). Bars represent the mean ± SEM (n = 6). # p < 0.05 versus the control group (vehicle control: 0.1% DMSO). * p < 0.05 versus the MG group.

Figure 4

CHA79 up-regulated the expression of GLP-1R in MG-treated SH-SY5Y dopaminergic neurons. One-hour pre-treatment with CHA79 (0.1–1 μM) followed by 24 h MG (500 μM) exposure was carried out. WB data are presented as % of control follow by normalization with internal standard (β-actin). Data are shown as mean ± SEM (n = 6). # p < 0.05 versus the control group (vehicle control: 0.1% DMSO). * p < 0.05 versus the MG group.

Figure 5

CHA79 up-regulated BDNF (A), p75NTR (B), p-TrkB (C), p-Akt (D), p-GSK-3β (E), and p-CREM (F) expression in MG-treated SH-SY5Y dopaminergic neurons. One-hour pre-treatment with CHA79 (0.1–1 μM) followed by 24 h MG (500 μM) exposure was carried out. WB data are presented as % of control follow by normalization with internal standard (β-actin or Lamin B). Data are shown as mean ± SEM (n = 6). # p < 0.05 versus the control group (vehicle control: 0.1% DMSO). * p < 0.05 versus the MG group.

Figure 6

CHA79 down-regulated ROS production (A), up-regulated nuclear-Nrf2 (n-Nrf2) expression (B), HO-1 expression (C), SOD activity (D), total GSH level (E) in MG-treated SH-SY5Y dopaminergic neurons. One-hour pre-treatment with CHA79 (0.1–1 μM) followed by 24 h MG (500 μM) exposure was carried out. Fluorescent DCF was measured a by flow cytometer as the indicator of ROS production. WB data are presented as % of control follow by normalization with internal standard (β-actin or Lamin B). Commercial kits were used for detecting GSH and SOD. Data are shown as mean ± SEM (n = 6). # p < 0.05 versus the control group (vehicle control: 0.1% DMSO). * p < 0.05 versus the MG group.

Figure 7

CHA79 up-regulated expression of GLO-1 in MG-treated SH-SY5Y dopaminergic neurons. One-hour pre-treatment with CHA79 (0.1–1 μM) followed by 24 h MG (500 μM) exposure was carried out. WB data are presented as % of control follow by normalization with internal standard (β-actin). Data are shown as mean ± SEM (n = 6). # p < 0.05 versus the control group (vehicle control: 0.1% DMSO). * p < 0.05 versus the MG group.