Neuroprotective Effect of Chebulagic Acid via Autophagy Induction in SH-SY5Y Cells

Abstract

Autophagy is a series of catabolic process mediating the bulk degradation of intracellular proteins and organelles through formation of a double-membrane vesicle, known as an autophagosome, and fusing with lysosome. Autophagy plays an important role of death-survival decisions in neuronal cells, which may influence to several neurodegenerative disorders including Parkinson's disease. Chebulagic acid, the major constituent of Terminalia chebula and Phyllanthus emblica, is a benzopyran tannin compound with various kinds of beneficial effects. This study was performed to investigate the autophagy enhancing effect of chebulagic acid on human neuroblastoma SH-SY5Y cell lines. We determined the effect of chebulagic acid on expression levels of autophago-some marker proteins such as, DOR/TP53INP2, Golgi-associated ATPase Enhancer of 16 kDa (GATE 16) and Light chain 3 II (LC3 II), as well as those of its upstream pathway proteins, AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and Beclin-1. All of those proteins were modulated by chebulagic acid treatment in a way of enhancing the autophagy. Additionally in our study, chebulagic acid also showed a protective effect against 1-methyl-4-phenylpyridinium (MPP(+)) - induced cytotoxicity which mimics the pathological symptom of Parkinson's disease. This effect seems partially mediated by enhanced autophagy which increased the degradation of aggregated or misfolded proteins from cells. This study suggests that chebulagic acid is an attractive candidate as an autophagy-enhancing agent and therefore, it may provide a promising strategy to prevent or cure the diseases caused by accumulation of abnormal proteins including Parkinson's disease.

Keywords: 1-Methyl-4-phenylpyridinium (MPP+); Autophagy; Chebulagic acid; Parkinson’s disease.

Fig. 1.

Induction of autophagosomes by CA in SH-SY5Y cells. SH-SY5Y cells were treated with CA at the concentrations of 1.56, 3.13, 6.25, 12.5 and 25 μM for 24 hr. (A) The cell viability was measured by MTT assay and the results were expressed as the percentile of absorbance of treated samples compared to that of the control. (B, C) SH-SY5Y cells were determined by measuring expression levels, DOR early autophagosome marker, LC3 protein the autophagosome marker, and GATE16 autophagosome membrane protein using an immunoblotting assay. The expression levels of three kinds of proteins were evaluated by densitometric analysis and data was expressed as folds of the control. GAPDH was used as an equal loading control. Each data represents the mean ± SD (n=3). Significantly different (^**p<0.01) from control.

Fig. 2.

Induction of autophagosome upstream pathway by CA in SH-SY5Y cells. The induction of autophagy by CA in SH-SY5Y cells was determined by measuring the p-AMPK, mTOR and Beclin-1 protein levels using an immunoblotting assay with antibody against p-AMPK, mTOR and Beclin-1. The expression levels of three kinds of proteins were evaluated by densitometric analysis and data was expressed as folds of the control. GAPDH was used as an equal loading control. Each data represents the mean ± SD (n=3). Significantly different (^*p<0.05, ^**p<0.01) from control.

Fig. 3.

Inhibition effect of 3MA on CA-induced autophagy in SHSY5Y cells. Inhibition effect of 3MA on CA-induced autophagy was determined by measuring the LC3 protein levels using an immunoblotting assay with antibody against LC3 in SH-SY5Y cells. Rapamycin (10 μM) was used as a positive control. The expression levels of three kinds of proteins were evaluated by densitometric analysis and data were expressed as folds of the control. GAPDH used as an equal loading control. Each data represents the mean ± SD (n=3). Significantly different (^**p<0.01, ^***p<0.001) from control. Significantly different (^#p<0.05, ^##p<0.01) from CA treated group.

Fig. 4.

CA-induced LC3 expression in autophagosomes. SH-SY5Y cells were treated with 25 μM of CA, 10 μM of rapmycin and 5 mM of 3MA for 24 h and then, were labeled with 40, 6-diamidino-2-phenylindole (DAPI, blue), and Alexa 488 secondary antibody against LC3 (green). LC3 fluorescent puncta were observed in cells by using the confocal microscope.

Fig. 5.

Neuroprotective effect of CA on MPP⁺-induced cytotoxicity in SH-SY5Y cells. Neuroprotective effect of CA on MPP⁺-induced cytotoxicity was determined by MTT assay. Cells were treated with MPP⁺ 600 μM alone, or MPP⁺ and CA for 48 h. SH-SY5Y cells were pre-treated with CA at the concentrations of 3.13, 6.25, 12.5 and 25 μM for 2 h followed by addition of MPP⁺ 600 μM for 48 hr. The cells viability were measured by MTT assay. Each data represents the mean ± SD (n=3). Significantly different (^**p<0.01) from control. Significantly different (^#p<0.05, ^##p<0.01) from MPP⁺ treated group.

Fig. 6.

Regulation of autophagy key regulators, AMPK, mTOR, Beclin-1 and LC3, by CA treatment. CA causes activation of AMPK, Beclin-1 and LC3, and inhibition of mTOR followed by the induction of autophagy.