Teaghrelin Protects SH-SY5Y Cells against MPP+-Induced Neurotoxicity through Activation of AMPK/SIRT1/PGC-1α and ERK1/2 Pathways

Abstract

The prevalence and incidence of Parkinson's disease (PD), an age-related neurodegenerative disease, are higher among elderly people. Independent of etiology, dysfunction and loss of dopaminergic neurons are common pathophysiological changes in PD patients with impaired motor and non-motor function. Currently, preventive or therapeutic treatment for combating PD is limited. The ghrelin axis and ghrelin receptor have been implicated in the preservation of dopaminergic neurons and have potential implications in PD treatment. Teaghrelin, a compound originating from Chin-Shin Oolong tea, exhibits ghrelin agonist activity. In this study, the neuroprotective potential of teaghrelin against PD was explored in a cell model in which human neuroblastoma SH-SY5Y cells were treated with the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP⁺). Upon MPP⁺ exposure, SH-SY5Y cells exhibited decreased mitochondrial complex I activity and apoptotic cell death. Teaghrelin activated AMP-activated protein kinase (AMPK)/sirtuin 1(SIRT1)/peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α (PGC-1α) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways to antagonize MPP⁺-induced cell death. Herein, we propose that teaghrelin is a potential candidate for the therapeutic treatment of PD.

Keywords: Parkinson’s disease; neurodegeneration; teaghrelin.

Figure 1

Teaghrelin (TG) protected cells against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity. Cells were treated with different concentrations of teaghrelin or MPP⁺ for 24 h ((A,B) respectively). Cells were treated with or without TG, ghrelin, and MPP⁺ for 24 h (C). Cell viability was measured using 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. The results were analyzed using one-way analysis of variance. Values are presented as the mean ± standard deviation (n = 4). * p < 0.05, compared with the control group, ^# p < 0.05 compared with MPP⁺ alone. -: untreated with drugs or compounds, +: treated with drugs or compounds.

Figure 2

Teaghrelin (TG) attenuated 1-methyl-4-phenylpyridinium (MPP⁺)-induced mitochondrial dysfunction and apoptosis in SH-SY5Y cells. SH-SY5Y cells were treated with TG or ghrelin in the presence or absence of MPP⁺ for 24 h. After various treatments, mitochondrial complex I activity was measured at 450 nm using a microplate reader (A). Cells were harvested and analyzed through Western blotting using antibodies against phosphatase and tensin homologue-induced kinase 1 (PINK1) (B), cytochrome c (C), and caspase-3 (D). The expression of β-actin was used as the internal control. Results were analyzed with one-way analysis of variance. Values are presented as the mean ± standard deviation (n = 3). * p < 0.05 compared with the control group, ^# p < 0.05 compared with MPP ⁺ alone. -: untreated with drugs or compounds, +: treated with drugs or compounds.

Figure 3

Teaghrelin (TG) attenuated 1-methyl-4-phenylpyridinium (MPP⁺)-induced loss of tyrosine hydroxylase (TH) expression in SH-SY5Y cells. (A) Immunofluorescence staining of tyrosine hydroxylase (green) and DAPI (blue) in each group of SH-SY5Y cells with various treatments (scale bar, 50 µm). (B) SH-SY5Y cells treated with or without TG, ghrelin, and MPP⁺ for 24 h. The expression of TH was analyzed through Western blotting. The expression of β-actin was used as the internal control. The results were analyzed through one-way analysis of variance. Values are presented as the mean ± standard deviation (n = 3). * p < 0.05 compared with the control group, ^# p < 0.05 compared with MPP ⁺ alone. -: untreated with drugs or compounds, +: treated with drugs or compounds.

Figure 4

Substance P (SP-analog) attenuated the protective effects of teaghrelin (TG) on 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity. SH-SY5Y cells were treated with TG or SP-analog in the presence or absence of MPP⁺ for 24 h. Cell viability was measured using2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. The results were analyzed using one-way analysis of variance. Values are presented as the mean ± standard deviation (n = 4). * p < 0.05 compared with the control group, ^# p < 0.05 compared with MPP⁺ alone. -: untreated with drugs or compounds, +: treated with drugs or compounds.

Figure 5

The protective effects of teaghrelin (TG) on 1-methyl-4-phenylpyridinium (MPP⁺)-induced mitochondrial dysfunction was not blocked by substance P (SP-analog). SH-SY5Y cells were treated with TG or SP analog in the presence or absence of MPP⁺ for 24 h. After various treatments, mitochondrial complex I activity was measured at 450 nm using a microplate reader (A). Cells were harvested and analyzed through Western blotting using antibodies against phosphatase and tensin homologue-induced kinase 1 (PINK1) (B). The expression of β-actin was used as the internal control. The results were analyzed using one-way analysis of variance. Values are presented as the mean ± standard deviation (n = 3). * p < 0.05 compared with the control group, ^# p < 0.05 compared with MPP⁺ alone. -: untreated with drugs or compounds, +: treated with drugs or compounds.

Figure 6

Teaghrelin (TG) attenuated 1-methyl-4-phenylpyridinium (MPP⁺)-induced neurotoxicity through activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway. SH-SY5Y cells were treated with TG or substance P (SP-analog) in the presence or absence of MPP⁺ for 24 h. Cells were harvested and analyzed through Western blotting using antibodies against ERK1/2, p-ERK1/2, cytochrome c, caspase-3, and cleaved caspase-3. The expression of β-actin and was used as the internal control. Values are presented as the mean ± standard deviation (n = 4). * p < 0.05 compared with the control group, ^# p < 0.05 compared with MPP⁺ alone. ^$ p < 0.05 compared with the MPP⁺ + TG group. -: untreated with drugs or compounds, +: treated with drugs or compounds.

Figure 7

Teaghrelin (TG) attenuated 1-methyl-4-phenylpyridinium (MPP⁺)-induced neurotoxicity through activated AMP-activated protein kinase (AMPK)/sirtuin 1(SIRT1)/peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α (PGC-1α). SH-SY5Y cells were treated with TG, SP-analog, respectively, in the presence or absence MPP⁺ for 24 h. Cells were harvested and determined by Western blotting using antibodies against AMPK, p-AMPK, SIRT, PGC-1α. The expression of β-actin was used as internal control. Values are presented as mean ± SD (n = 4). * p < 0.05 compared with control group, # p < 0.05 compared with MPP + alone. ^$ p < 0.05 compared with MPP⁺+TG group. -: untreated with drugs or compounds, +: treated with drugs or compounds.