Neurosupportive Role of Vanillin, a Natural Phenolic Compound, on Rotenone Induced Neurotoxicity in SH-SY5Y Neuroblastoma Cells

Abstract

Vanillin, a phenolic compound, has been reported to offer neuroprotection against experimental Huntington's disease and global ischemia by virtue of its antioxidant, anti-inflammatory, and antiapoptotic properties. The present study aims to elucidate the underlying neuroprotective mechanism of vanillin in rotenone induced neurotoxicity. Cell viability was assessed by exposing SH-SY5Y cells to various concentrations of rotenone (5-200 nM) for 24 h. The therapeutic effectiveness of vanillin against rotenone was measured by pretreatment of vanillin at various concentrations (5-200 nM) and then incubation with rotenone (100 nM). Using effective dose of vanillin (100 nM), mitochondrial membrane potential, levels of reactive oxygen species (ROS), and expression patterns of apoptotic markers were assessed. Toxicity of rotenone was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, release of cyt-c, and enhanced expressions of proapoptotic and downregulation of antiapoptotic indices via the upregulation of p38 and JNK-MAPK pathway proteins. Our results indicated that the pretreatment of vanillin attenuated rotenone induced mitochondrial dysfunction, oxidative stress, and apoptosis. Thus, vanillin may serve as a potent therapeutic agent in the future by virtue of its multiple pharmacological properties in the treatment of neurodegenerative diseases including PD.

Figure 1

Structure of vanillin.

Figure 2

Effect of vanillin on rotenone induced cytotoxicity (MTT assay) in SH-SY5Y neuroblastoma cells. Cell viability was determined by measuring MTT method. (a) shows the dose-dependent effect of rotenone (5, 10, 50, 100, and 200 nM) induced cell toxicity after 24 h. An approximately half-maximal inhibition of cell viability was obtained at 100 nM rotenone concentration. (b) shows the dose-dependent effect of vanillin at various concentrations. Low concentrations (5, 10, 20, 50, 100, and 200 nM) did not induce any toxicity after 24 h treatment, whereas slight toxicity was induced at 500 μM concentration. Values are expressed as the percentage of the untreated control and represented as mean ± SD of four independent experiments in each group.

Figure 3

The protective effect of vanillin (5, 10, 20, 50, and 100 nM) against rotenone induced cell death was determined by MTT assay. Values are expressed as the percentage of the untreated control and represented as mean ± SD of four independent experiments in each group.

Figure 4

(a) Vanillin reduced ROS formation as stained by 1 μM CM-H2 DCFDA. (a) Photomicrograph showing the preventive effect of vanillin (100 nM) against rotenone induced ROS generation. (A) Control, (B) rotenone, (C) vanillin + rotenone, and (D) vanillin. (b) Rotenone (100 nM) treatment significantly increased the levels of ROS as compared to control cells, while vanillin (100 nM) pretreatment significantly decreased the levels of ROS as compared to rotenone alone treated cells. Values are given as mean ± SD of four independent experiments in each group. ^∗ p < 0.05 compared to control and ^# p < 0.05 compared to rotenone group (DMRT).

Figure 5

Vanillin stabilizes MMP as stained by Rh-123. Rotenone (100 nM) significantly decreased mitochondria membrane potential, while cells that were pretreated with vanillin (100 nM) significantly increased MMP. Values are given as mean ± SD of four independent experiments in each group. ^∗ p < 0.05 compared to control; ^# p < 0.05 compared to rotenone groups (DMRT).

Figure 6

Vanillin protects SH-SY5Y cells against rotenone induced apoptosis. (a) Photomicrograph showing the antiapoptotic effect of vanillin (100 nM) against rotenone at a concentration of 100 nM effective dose. (A) Control, (B) rotenone, (C) vanillin + rotenone, and (D) vanillin. (b) Rotenone (100 nM) treatment induced cell apoptosis compared to control cells; pretreatment with vanillin (100 nM) suppresses these apoptotic features. Values are given as mean ± SD of four independent experiments in each group. ^∗ p < 0.05 compared to control and ^# p < 0.05 compared to rotenone group (DMRT).

Figure 7

The effect of vanillin on the expressions of apoptotic and signaling markers. Lane 1: control; 2: rotenone; 3: vanillin + rotenone; and 4: vanillin. (a), (b), and (c) show the expressions of Bax; caspase-3, caspase-8, and caspase-9 cyt-c in cytosol were increased while the expressions of Bcl-2 and cyt-c in mitochondria were significantly decreased by the rotenone treated group as compared with control. Pretreatment with vanillin gradually restored the imbalanced expression profile of these proteins. (d) Rotenone treatment stimulates the expressions of p-JNK, p-P38, and p-ERK as compared with control. Pretreatment with vanillin decreases the expressions of p-JNK, p-P38, and p-ERK significantly. Immunoblots are representative of at least four independent experiments. Values are given as mean ± SD in each group. ^∗ p < 0.05 compared to control and ^# p < 0.05 compared to rotenone group (DMRT).