Protective effects of baicalin in a Caenorhabditis elegans model of Parkinson's disease

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder of the central nervous system. However, the pathogenetic mechanisms of PD are far from understood. The aim of this study was to determine the protective effect of baicalin in a Caenorhabditis elegans model of PD. C. elegans worms were stimulated for 24 h with 6-hydroxydopamine (6-OHDA, 50 mM) and treated with or without baicalin (1, 10, or 100 μM). At all tested concentrations, baicalin improved the reversal and omega turn behavioral phenotypes, as well as the survival, of 6-OHDA-stimulated worms. It also inhibited 6-OHDA-induced oxidative stress by decreasing malondialdehyde levels, increasing superoxide dismutase, glutathione reductase, catalase, and glutathione levels and up-regulating mRNA expression of the antioxidant-related genes sod-1, sod-2, sod-3, daf-2, and daf-16. Additionally, it significantly decreased the expression of the apoptosis-related gene ced-3 and increased that of the anti-apoptosis-related gene ced-9. The expression levels of cleaved caspase-3 and B-cell lymphoma 2 in 6-OHDA-treated worms were reversed by baicalin. Apoptosis was suppressed by 6-OHDA in loss-of-function strains via the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, the apoptotic effects of 6-OHDA were blocked in sek-1 and pmk-1 mutants. Finally, the mRNA expression of sek-1 and pmk-1 and the protein expression of p38 MAPK and stress-activated protein kinase/extracellular signal-regulated kinase 1 were up-regulated by 6-OHDA and reversed by baicalin. Baicalin may protect against 6-OHDA injury by inhibiting apoptosis and decreasing oxidative stress through the p38 MAPK signaling pathway.

Keywords: 6-hydroxydopamine; Caenorhabditis elegans; Parkinson’s disease; baicalin; oxidative stress; p38 mitogen-activated protein kinase.

Figure 1

Effects of baicalin on the (a) survival, (b) omega turns, and (c) reversals of C. elegans. The worms were used as a model of PD by treating them with 10 mM 6-OHDA. (a) Survival rate and the frequencies of (b) omega turns and (c) reversals were significantly decreased by 6-OHDA but significantly increased by baicalin (1, 10, or 100 μM). Data are presented as mean ± SEM. ### indicates P < 0.001 when data are compared to those for the vehicle-treated group. ^***, ^**, and ^* indicate P < 0.001, 0.01, and 0.05, respectively, when data are compared to those for the worms treated with both 6-OHDA and the vehicle.

Figure 2

Baicalin ameliorates apoptosis in 6-OHDA-treated C. elegans. The levels of (a) c-caspase-3 and (b) Bcl-2 proteins in C. elegans were evaluated by western blotting after treating the worms with 6-OHDA. Baicalin significantly reversed 6-OHDA-induced increase in c-caspase-3 and decrease in Bcl-2 levels. Results of the RT-PCR analysis of (c) ced-3 and (d) ced-9 gene expression in C. elegans after treatment with 6-OHDA. Baicalin effectively reversed 6-OHDA-induced changes in the gene expression of ced-3 and ced-9. Data are presented as mean ± SEM. ###, ##, and # indicate P < 0.001, 0.01, and 0.05, respectively, when data are compared to those for the vehicle-treated group. ^***, ^**, and ^* indicate P < 0.001, 0.01, and 0.05, respectively, when data are compared to those for the worms treated with both 6-OHDA and the vehicle.

Figure 3

Baicalin ameliorates oxidative stress in 6-OHDA-treated C. elegans. The levels of (a) SOD, (b) MDA, (c) CAT, (d) GSH, and (e) GR were measured after the worms were treated with 6-OHDA. Baicalin significantly reversed 6-OHDA-induced changes in the levels of the enzymes. Data are presented as mean ± SEM. ### and ## indicate P < 0.001 and 0.01, respectively, when data are compared to those for the vehicle-treated group. ^***, ^**, and ^* indicate P < 0.001, 0.01, and 0.05, respectively, when data are compared to those for the worms treated with both 6-OHDA and the vehicle.

Figure 4

Effects of baicalin on the mRNA expression levels of (a) sod-1, (b) sod-2, (c) sod-3, and (d) daf-16 in 6-OHDA-treated C. elegans. The mRNA levels of these genes were decreased by 6-OHDA; however, the decreases were significantly reversed by baicalin. Data are presented as mean ± SEM. ## indicates P < 0.01 when data are compared to those for the vehicle-treated group. ^** and ^* indicate P < 0.01 and 0.05, respectively, when data are compared to those for the worms treated with both 6-OHDA and the vehicle.

Figure 5

Effects of baicalin on the expression of proteins and genes involved in the p38 MAPK signaling pathway in C. elegans. (a) The survival rates of pmk-1(km25) and sek-1(km4) mutants were not changed after treatment with 6-OHDA. The mRNA levels of (b) pmk-1 and (c) sek-1 were determined by RT-PCR. The protein levels of (d) Sek-1 and (e) p-p38 were determined by western blotting after treating the worms with 6-OHDA. Data are presented as mean ± SEM. ### and ## indicate P < 0.001 and 0.01, respectively, when data are compared to those for the vehicle-treated group. ^***, ^**, and ^* indicate P < 0.001, 0.01, and 0.05, respectively, when data are compared to those for the worms treated with both 6-OHDA and the vehicle.