Sophora flavescens Aiton Decreases MPP+-Induced Mitochondrial Dysfunction in SH-SY5Y Cells

Abstract

Sophora flavescens Aiton (SF) has been used to treat various diseases including fever and inflammation in China, South Korea and Japan. Several recent reports have shown that SF has anti-inflammatory and anti-apoptotic effects, indicating that it is a promising candidate for treatment of Parkinson's disease (PD). We evaluated the protective effect of SF against neurotoxin 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced mitochondrial dysfunction in SH-SY5Y human neuroblastoma cells, an in vitro PD model. SH-SY5Y cells were incubated with SF for 24 h, after which they were treated with MPP⁺. MPP⁺-induced cytotoxicity and apoptosis were confirmed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assay. MitoSOX red mitochondrial superoxide indicator, tetramethylrhodamine methyl ester perchlorate and Parkin, PTEN-induced putative kinase 1 (PINK1), and DJ-1 immunofluorescent staining were conducted to confirm the mitochondrial function. In addition, western blot was performed to evaluate apoptosis factors (Bcl-2, Bax, caspase-3 and cytochrome c) and mitochondrial function-related factors (Parkin, PINK1 and DJ-1). SF suppressed MPP⁺-induced cytotoxicity, apoptosis and collapse of mitochondrial membrane potential by inhibiting the increase of reactive oxidative species (ROS) and DNA fragmentation, and controlling Bcl-2, Bax, caspase-3 and cytochrome c expression. Moreover, it attenuated Parkin, PINK1 and DJ-1 expression from MPP⁺-induced decrease. SF effectively suppressed MPP⁺-induced cytotoxicity, apoptosis and mitochondrial dysfunction by regulating generation of ROS, disruption of mitochondrial membrane potential, mitochondria-dependent apoptosis and loss or mutation of mitochondria-related PD markers including Parkin, PINK1 and DJ-1.

Keywords: MPP+; Parkinson’s disease; SH-SY5Y; Sophora flavescens Aiton; mitochondrial dysfunction.

Figure 1

Cell viability after 1-methyl-4-phenylpyridinium ion (MPP⁺) or Sophora flavescens Aiton (SF) treatment of SH-SY5Y cells. Dose-dependent effects were observed in SH-SY5Y cells treated with MPP⁺ (A), SF (B), or both SH-SY5Y and MPP⁺ (C). Values are the means ± SD (n = 6). In (A,B), ***p < 0.001, compared with control (untreated group). In (C), ^##p < 0.01 and ^###p < 0.001, compared with the group treated by MPP⁺ alone.

Figure 2

SF attenuated MPP⁺-induced apoptosis in SH-SY5Y cells. Cells were treated with 0.01 mg/mL SF for 24 h, then exposed to 1 mM MPP⁺ for 24 h and stained with DAPI and TUNEL. TUNEL staining showed that MPP⁺ increased apoptotic DNA fragmentation, but SF suppressed it. Scale bar = 50 μm. Values are the means ± SD (three slides per group). ^a–cMeans with different letters are significantly different (p < 0.05) by Duncan’s multiple-range test.

Figure 3

SF attenuated MPP⁺-mediated reactive oxidative species (ROS) generation in SH-SY5Y cells. Cells were exposed to 1 mM MPP⁺ for 24 h in the absence or presence of 0.01 mg/mL SF for 24 h, then stained with DAPI, MitoSOX and Mitotracker. MPP⁺ increased mitochondria-derived ROS generation, which was suppressed by SF. Scale bar = 50 μm. Values are the means ± SD (three slides per group). ^a–cMeans with different letters are significantly different (p < 0.05) by Duncan’s multiple-range test.

Figure 4

SF attenuated MPP⁺-mediated collapse of mitochondrial membrane permeability in SH-SY5Y cells. Cells were exposed to 1 mM MPP⁺ for 24 h in the absence or presence of 0.01 mg/mL SF for 24 h, stained with tetramethylrhodamine methyl ester perchlorate (TMRM), then visualized by fluorescence microscopy and the intensity of fluorescence was calculated. MPP⁺ collapsed mitochondrial membrane permeability, which was prevented by SF. Scale bar = 50 μm. Values are the means ± SD (three slides per group). ^a,bMeans with different letters are significantly different (p < 0.05) by Duncan’s multiple-range test.

Figure 5

SF regulated MPP⁺-induced changes in Bcl-2 and Bax expression in SH-SY5Y cells. Cells were exposed to 1 mM MPP⁺ for 24 h in the absence or presence of 0.01 mg/mL SF for 24 h, then harvested and subjected to Western blotting using the antibodies against Bcl-2 and Bax. SF alleviated MPP⁺-induced down-regulation of Bcl-2 and up-regulation of Bax. Values are the means ± SD (n = 3). ^a–cMeans with different letters are significantly different (p < 0.05) by Duncan’s multiple-range test.

Figure 6

SF inhibited MPP⁺-induced release of cytochrome c and activation of caspase-3 in SH-SY5Y cells. Cells were exposed to 1 mM MPP⁺ for 24 h in the absence or presence of 0.01 mg/mL SF for 24 h. Cells were harvested and subjected to Western blotting using antibodies against cytochrome c and cleaved caspase-3. SF suppressed MPP⁺-induced release of cytochrome c and activation of caspase-3. Values are the means ± SD (n = 3). ^a–cMeans with different letters are significantly different (p < 0.05) by Duncan’s multiple-range test.

Figure 7

SF suppressed MPP⁺-mediated loss of Parkin and PTEN-induced putative kinase 1 (PINK1) in SH-SY5Y cells. Cells were exposed to 1 mM MPP⁺ for 24 h in the absence or presence of 0.01 mg/mL SF for 24 h, after which they were subjected to immunofluorescent staining using antibodies against Parkin and PINK1. MPP⁺ suppressed Parkin and PINK1 expression, while SF enhanced them. Scale bar = 50 μm. Values are the means ± SD (three slides per group). ^a–cMeans with different letters are significantly different (p < 0.05) by Duncan’s multiple-range test.

Figure 8

SF attenuated MPP⁺-mediated loss of DJ-1 in SH-SY5Y cells. Cells were exposed to 1 mM MPP⁺ for 24 h in the absence or presence of 0.01 mg/mL SF for 24 h, then subjected to immunofluorescent staining using antibody against DJ-1. SF enhanced the MPP⁺-induced decrease of DJ-1. Scale bar = 50 μm. Values are the means ± SD (three slides per group). ^a–cMeans with different letters are significantly different (p < 0.05) by Duncan’s multiple-range test.

Figure 9

SF inhibited loss of Parkinson’s disease (PD) related markers Parkin, PINK1 and DJ-1 in MPP⁺-induced SH-SY5Y cells. Cells were exposed to 1 mM MPP⁺ for 24 h in the absence or presence of 0.01 mg/mL SF for 24 h, then harvested and subjected to Western blotting using antibodies against Parkin, PINK1 and DJ-1 with whole cell lysate. MPP⁺ suppressed Parkin, PINK1 and DJ-1 expression, while SF enhanced them. Values are the means ± SD (n = 3). ^a–dMeans with different letters are significantly different (p < 0.05) by Duncan’s multiple-range test.