Junqiang Yan1,2, Liang Qiao3, Jiannan Wu4, Hua Fan5, Jiachun Sun6, Yude Zhang4. 1. Neurological Diseases Institute, The First Affiliated Hospital, College of Clinical Medicine of Henan University of Science and Technology, Luoyang, Chinayanjq20062007@126.com. 2. Department of Neurology, The First Affiliated Hospital, College of Clinical Medicine of Henan University of Science and Technology, Henan, Chinayanjq20062007@126.com. 3. Department of Neurology, The First Affiliated Hospital, College of Clinical Medicine of Henan University of Science and Technology, Henan, China. 4. Neurological Diseases Institute, The First Affiliated Hospital, College of Clinical Medicine of Henan University of Science and Technology, Luoyang, China. 5. Department of Pharmacy, The First Affiliated Hospital, College of Clinical Medicine of Henan University of Science and Technology, Luoyang, China. 6. Department of Oncology, The First Affiliated Hospital, College of Clinical Medicine of Henan University of Science and Technology, Luoyang, China.
Abstract
BACKGROUND/AIMS: Many clinical studies have demonstrated that statins, especially simvastatin, can decrease the incidence of Parkinson's disease (PD). However, the specific underlying mechanism remains unclear. This study aimed to investigate how simvastatin affects experimental parkinsonian models via the regulation of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated activation of the anti-oxidant system. METHODS: l-Methyl-4-phenylpyridine ion (MPP+)-treated SH-SY5Y cells and substantia nigra neurons were used to investigate the neuroprotective effect of simvastatin. After incubation with MPP+ and/or simvastatin for 24 h, the MTT assay was used to assess cell viability. Reactive oxygen species (ROS) levels were measured using 2',7'-dichlorofluorescin diacetate, while cellular superoxide dismutase (SOD) levels were determined based on the blue formazan produced by the reduction of nitroblue tetrazolium. The level of cellular grade micro-reduced glutathione (GSH) was measured with 5,5'-dithiobis-(2-nitrobenzoic acid). Meanwhile, the malondialdehyde content released from SH-SY5Y cells and substantia nigra neuronal cells exposed to different culture media was calculated based on the condensation reaction involving thiobarbituric acid. The mRNA levels of genes encoding nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase 1 (HO-1), and NAD(P)H dehydrogenase (quinone) 1 (NQO-1) were determined by a quantitative polymerase chain reaction assay, while the ERK, Nrf2, HO-1, NOX2, and NQO-1 protein levels were analyzed by western blot. Additionally, ERK small interfering RNA (siRNA) was used to investigate the mechanisms underlying MPP+-induced oxidative stress and the regulation of the endogenous anti-oxidant system. RESULTS: Simvastatin (1.5 μM) enhanced the viability of SH-SY5Y cells and primary neurons treated with MPP+, and significantly alleviated the oxidative stress induced by MPP+ in SH-SY5Y cells by regulating the production of SOD, analytical grade micro-reduced GSH, and ROS, which may be associated with the activation of the Nrf2 anti-oxidant system. An analysis involving ERK1/2 siRNA revealed that simvastatin can inhibit NOX2 expression via the activation of ERK1/2 in the MPP+-treated PD cell model. CONCLUSION: Our results provide strong evidence that ERK1/2-mediated modulation of the anti-oxidant system after simvastatin treatment may partially explain the anti-oxidant activity in experimental parkinsonian models. These findings contribute to a better understanding of the critical roles of simvastatin via the ERK1/2-mediated modulation of the anti-oxidant system, which may be relevant for treating PD.
BACKGROUND/AIMS: Many clinical studies have demonstrated that statins, especially simvastatin, can decrease the incidence of Parkinson's disease (PD). However, the specific underlying mechanism remains unclear. This study aimed to investigate how simvastatin affects experimental parkinsonian models via the regulation of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated activation of the anti-oxidant system. METHODS:l-Methyl-4-phenylpyridine ion (MPP+)-treated SH-SY5Y cells and substantia nigra neurons were used to investigate the neuroprotective effect of simvastatin. After incubation with MPP+ and/or simvastatin for 24 h, the MTT assay was used to assess cell viability. Reactive oxygen species (ROS) levels were measured using 2',7'-dichlorofluorescin diacetate, while cellular superoxide dismutase (SOD) levels were determined based on the blue formazan produced by the reduction of nitroblue tetrazolium. The level of cellular grade micro-reduced glutathione (GSH) was measured with 5,5'-dithiobis-(2-nitrobenzoic acid). Meanwhile, the malondialdehyde content released from SH-SY5Y cells and substantia nigra neuronal cells exposed to different culture media was calculated based on the condensation reaction involving thiobarbituric acid. The mRNA levels of genes encoding nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase 1 (HO-1), and NAD(P)H dehydrogenase (quinone) 1 (NQO-1) were determined by a quantitative polymerase chain reaction assay, while the ERK, Nrf2, HO-1, NOX2, and NQO-1 protein levels were analyzed by western blot. Additionally, ERK small interfering RNA (siRNA) was used to investigate the mechanisms underlying MPP+-induced oxidative stress and the regulation of the endogenous anti-oxidant system. RESULTS:Simvastatin (1.5 μM) enhanced the viability of SH-SY5Y cells and primary neurons treated with MPP+, and significantly alleviated the oxidative stress induced by MPP+ in SH-SY5Y cells by regulating the production of SOD, analytical grade micro-reduced GSH, and ROS, which may be associated with the activation of the Nrf2 anti-oxidant system. An analysis involving ERK1/2 siRNA revealed that simvastatin can inhibit NOX2 expression via the activation of ERK1/2 in the MPP+-treated PD cell model. CONCLUSION: Our results provide strong evidence that ERK1/2-mediated modulation of the anti-oxidant system after simvastatin treatment may partially explain the anti-oxidant activity in experimental parkinsonian models. These findings contribute to a better understanding of the critical roles of simvastatin via the ERK1/2-mediated modulation of the anti-oxidant system, which may be relevant for treating PD.