SOD3 Ameliorates Aβ25-35-Induced Oxidative Damage in SH-SY5Y Cells by Inhibiting the Mitochondrial Pathway

Abstract

This study was designed to investigate the protective effects of extracellular superoxide dismutase (SOD3) against amyloid beta (Aβ_25-35)-induced damage in human neuroblastoma SH-SY5Y cells and to elucidate the mechanisms responsible for this beneficial effect. SH-SY5Y cells overexpressing SOD3 were generated by adenoviral vector-mediated infection and Aβ_25-35 was then added to the cell culture system to establish an in vitro model of oxidative stress. Cell viability, the generation of intracellular reactive oxygen species (ROS), the expression and activity of antioxidant enzymes, the levels of lipid peroxidation malondialdehyde (MDA), the expression of mitochondrial apoptosis-related genes and calcium images were examined. Following Aβ_25-35 exposure, SOD3 overexpression promoted the survival of SH-SY5Y cells, decreased the production of ROS, decreased MDA and calcium levels, and decreased cytochrome c, caspase-3, caspase-9 and Bax gene expression. Furthermore, SOD3 overexpression increased the expression and activity of antioxidant enzyme genes and Bcl-2 expression. Together, our data demonstrate that SOD3 ameliorates Aβ_25-35-induced oxidative damage in neuroblastoma SH-SY5Y cells by inhibiting the mitochondrial pathway. These data provide new insights into the functional actions of SOD3 on oxidative stress-induced cell damage.

Keywords: Antioxidant enzymes; Extracellular superoxide dismutase; Mitochondrial pathway; Oxidative stress; Reactive oxygen species.

Fig. 1

SOD3 was highly expressed in infected SH-SY5Y cells, and SOD3 overexpression prevented Aβ_25–35-induced cell death. a SOD3 was highly expressed in neuroblastoma SH-SY5Y cells following infection with the Ad-SOD3-EGFP adenoviral vector. b SOD3 expression level in SH-SY5Y cells was normalized to the β-actin expression level, as determined by densitometric analysis (t _0.05,6 = 5.112, p < 0.001). c SH-SY5Y cells were exposed to increasing concentrations of Aβ_25–35 (20, 40, 60, 80, 100 μM) for 6 h, and cell death was detected by CCK8. d SH-SY5Y cells were exposed to Aβ_25–35 (40 μM) for 6 h in the presence or absence of SOD3 overexpression. Data are expressed as the mean ± SD (n = 6). **p < 0.001

Fig. 2

SOD3 overexpression prevents the Aβ_25–35-induced activation of the mitochondria-mediated apoptosis pathway in SH-SY5Y cells. a The expression of mitochondrial apoptotic-related genes was examined by RT-PCR and cytochrome c (b), caspase-3 (c), caspase-9 (d), Bax (e) and Bcl-2 (f) were normalized to the expression level of β-actin (F _0.05,6 = 3.223, p = 0.027). Data are expressed as the mean ± SD (n = 6). *p < 0.05, **p < 0.001

Fig. 3

SOD3 overexpression modulates the redox state of SH-SY5Y cells exposure to Aβ_25–35. a Malondialdehyde (MDA) level in the cell supernatant was measured by a UV–visible spectrophotometer. b The enzymatic activities of total SOD (T-SOD), CAT and GPx in the cell supernatant were determined. c The expression of intracellular antioxidant enzyme genes was detected by RT-PCR, and the expression levels of SOD1 (d), SOD2 (e), SOD3 (f), GPx (g) and CAT (h) were normalized to the expression level of β-actin (F _0.05,6 = 12.877, p < 0.001). Data are expressed as the mean ± SD (n = 6). *p < 0.05; **p < 0.001

Fig. 4

SOD3 overexpression decreases the generation of intracellular ROS in SH-SY5Y cells exposure to Aβ_25–35. The generation of reactive oxygen species was analysed by flow cytometry in normal control cells (a), Aβ_25–35 single-treated SH-SY5Y cells (b) and SOD3-infected SH-SY5Y cells. c The results were compared across the different treatments (d). Data are expressed as the mean ± SD (n = 3). *p < 0.05; **p < 0.001

Fig. 5

SOD3 overexpression prevents intracellular Ca²⁺ overloading in SH-SY5Y cells exposure to Aβ_25–35. [Ca²⁺]_i was analysed using Ca²⁺ imaging in control cells (a), Aβ_25–35 single-treated SH-SY5Y cells (b) and SOD3 infected SH-SY5Y cells (c). The results were compared between different treatments (d). Data are expressed as the mean ± SD (n = 3). *p < 0.05; **p < 0.001