p-Hydroxybenzyl Alcohol Antagonized the ROS-Dependent JNK/Jun/Caspase-3 Pathway to Produce Neuroprotection in a Cellular Model of Parkinson's Disease

Abstract

Parkinson's disease (PD) is a progressive disorder that affects brain nerve cells responsible for body motion and remains incurable. p-Hydroxybenzyl alcohol (HBA) is the primary phenolic compound in Gastrodiae Rhizoma, known for its therapeutic benefits against neurodegeneration. However, the protective effect of HBA against Parkinson's disease (PD) remains unclear. The objective of this study was to evaluate the neuroprotective effects of HBA in vitro 6-hydroxydopamine (6-OHDA)-induced PD model in SH-SY5Y cells. SH-SY5Y cells were pretreated with various concentrations of HBA for 1 h and incubated with 100 μmol/L 6-OHDA for 24 h to induce cellular lesions. 2,5-Diphenyl-2H-tetrazolium bromide was used to detect cellular viability. 2',7'-dichlorofluorescin oxidation detects reactive oxygen species (ROS). The enzyme-linked immunosorbent assay was used to determine the activities of superoxide dismutase, catalase, and glutathione peroxidase. The cellular mitochondrial function was identified through the collapse of the mitochondrial membrane potential, the release of cytochrome c, and the synthesis of mitochondrial ATP. Expression of pro-and anti-apoptotic factors was measured by Western blot. HBA enhanced cell viability, blocked ROS overproduction, and reduced antioxidant activities induced by 6-OHDA. HBA also reduced mitochondrial dysfunction and cell death caused by 6-OHDA. Moreover, HBA reversed the 6-OHDA-mediated activation of c-Jun N-terminal kinase, the downregulation of the Bcl-2/Bax ratio, the Apaf-1 upregulation and the induction of caspase-9, caspase-3, and PARP cleavage. This study shows that the protective effects of HBA against 6-OHDA-induced cell injury provide the potential preventive effects of HBA, making it a promising preventive agent for PD.

Keywords: 6-OHDA; Parkinson’s disease; SH-SY5Y cells; neurodegeneration; p-hydroxybenzyl alcohol.

Figure 1

HBA rescues SH-SY5Y cells from the neurotoxicity induced by 6-OHDA. (A) Cell viability was assessed for various concentrations of 6-OHDA that were incubated with SH-SY5Y cells over a 24 h period. (B) Cell viability was evaluated for SH-SY5Y cells incubated with 100 μmol/L 6-OHDA for a period. (C) Cell viability was evaluated when SH-SY5Y cells were incubated with various concentrations of HBA, 1 mmol/L NALC, or 10 µmol/L SP600125 for 24 h without exposure to 100 μmol/L 6-OHDA. (D) Cell viability was evaluated when SH-SY5Y cells were pretreated with different concentrations of HBA, 1 mmol/L NALC, or 10 µmol/L SP600125 for 1 h and then exposed to 100 μmol/L of 6-OHDA for another 24 h. Cell viability was determined with an MTT assay and expressed as a percentage of untreated cells, considering the control group. The results are shown as the mean ± SD of five independent experiments (n = 5) performed in triplicate. ^a p < 0.05 and ^b p < 0.01 compared to the data from untreated control group (control); ^c p < 0.05 and ^d p < 0.01 compared to the data from cells exposed to 6-OHDA with no treatment.

Figure 2

HBA reduces SH-SY5Y from the oxidative stress induced by 6-OHDA. SH-SY5Y cells were pretreated with HBA (120 µmol/L), NALC (1 mmol/L), or SP600125 (10 µmol/L) for 1 h and then exposed to 6-OHDA (100 μmol/L) for another 24 h. (A) Fluorescence images representative of intracellular ROS production were detected using DCFH-DA fluorescent probes (200× g magnification). The ROS fluorescence intensity is represented as a percentage in comparison with untreated control cells. (B) The activities of SOD, GSH-Px, and CAT were identified using commercial analysis packages. The results are shown as the mean ± SD of five independent experiments (n = 5) performed in triplicate. ^a p < 0.05 and ^b p < 0.01 compared to the data from untreated control group (control); ^c p < 0.05 and ^d p < 0.01 compared to the data from cells exposed to 6-OHDA with no treatment.

Figure 3

HBA reduces SH-SY5Y cells of 6-OHDA-induced mitochondrial dysfunction. SH-SY5Y cells were pretreated with HBA (120 µmol/L), NALC (1 mmol/L), or SP600125 (10 µmol/L) for 1 h and then exposed to 6-OHDA (100 μmol/L) for another 24 h. (A) The potential of the mitochondrial membrane was measured using the JC-1 fluorescence probe. (B) The ADP/ATP ratio in cells was measured using a commercial assay kit based on the bioluminescent detection of ADP and ATP levels. (C) Cytochrome c concentrations were determined by immunoassay for mitochondrial and cytosolic fractions. (D) The extent of apoptotic DNA fragmentation was quantified by using the cell death ELISA kit. The results are shown as the mean ± SD of five independent experiments (n = 5) performed in triplicate. ^a p < 0.05 and ^b p < 0.01 compared to the data from untreated control group (control); ^c p < 0.05 and ^d p < 0.01 compared to the data from cells exposed to 6-OHDA with no treatment.

Figure 4

HBA reduces mitochondrial Apaf-1/caspase-9 pathway-mediated caspase-3 activation induced by 6-OHDA. SH-SY5Y cells were pretreated with HBA (120 µmol/L), NALC (1 mmol/L), or SP600125 (10 µmol/L) for 1 h and then exposed to 6-OHDA (100 μmol/L) for another 24 h. (A) Western blot was carried out to detect the expression of Apaf-1, cleaved caspase-9 (Casp 9), cleaved caspase-3 (Casp 3), and cleaved PARP-1. Band densities of proteins have been normalized to β-actin. (B) Caspase-9, caspase-3, and PARP activities were evaluated using the commercial colorimetric assay kits. The results are shown as the mean ± SD of five independent experiments (n = 5) performed in triplicate. ^a p < 0.05 and ^b p < 0.01 compared to the data from untreated control group (control); ^c p < 0.05 and ^d p < 0.01 compared to the data from cells exposed to 6-OHDA with no treatment.

Figure 5

HBA reduces activation of the JNK/c-Jun signal induced by 6-OHDA. SH-SY5Y cells were pretreated with HBA (120 µmol/L), NALC (1 mmol/L), or SP600125 (10 µmol/L) for 1 h and then exposed to 6-OHDA (100 μmol/L) for another 24 h. Photographs representing Western transfer analysis for p-JNK, JNK, p-c-Jun, c-Jun, Bax, and Bcl-2. The ratio between phosphoprotein and total protein was calculated for JNK (p-JNK/JNK) and c-Jun (p-c-Jun/c-Jun). The ratio between the relative intensities of Bcl-2 and Bax (Bcl-2/Bax) has been indicated. The results are shown as the mean ± SD of five independent experiments (n = 5) performed in triplicate. ^a p < 0.05 and ^b p < 0.01 compared to the data from untreated control group (control); ^c p < 0.05 and ^d p < 0.01 compared to the data from cells exposed to 6-OHDA with no treatment.