Dried Rehmannia root protects against glutamate-induced cytotoxity to PC12 cells through energy metabolism-related pathways

Abstract

Rehmannia has been shown to be clinically effective in treating neurodegenerative diseases; however, the neuroprotective mechanisms remain unclear. In this study, we established a model of neurodegenerative disease using PC12 cytotoxic injury induced by glutamate. The cells were treated with 20 mM glutamate in the absence or presence of water extracts of dried Rehmannia root of varying concentrations (70%, 50% and 30%). The different concentrations of Rehmannia water extract significantly increased the activity of glutamate-injured cells, reduced the release of lactate dehydrogenase, inhibited apoptosis, increased the concentrations of NADH, NAD and ATP in cells, ameliorated mitochondrial membrane potential, and reduced the levels of light chain 3. Taken together, our findings demonstrate that Rehmannia water extracts exert a cytoprotective effect against glutamate-induced PC12 cell injury via energy metabolism-related pathways.

Keywords: PC12 cells; Rehmannia water extracts; autophagy; energy metabolism; glutamate; nerve regeneration; neural regeneration.

Figure 1

The multiple parallel perfused microbioreactor (TissueFlex^®) used for the experiments.

Figure 2

Morphology, viability and lactate dehydrogenase (LDH) release rate of PC12 cells before and after treatment with glutamate. (A, B) Morphology of PC12 cells in the control group (A) and treated with 20 mM glutamate for 24 hours (B). The cells changed from a spindle-shaped to a round morphology. Scale bars: 200 μm. (C, D) Cell viability (C) and LDH release rate (D) of PC12 cells treated with different concentrations of glutamate for 24 hours. Data are expressed as the mean ± SD of at least three independent experiments. *P < 0.05, ***P < 0.001, vs. control group.

Figure 3

Different concentrations of water extract of dried Rehmannia root reduced the apoptosis rate. PC12 cells were exposed to 20 mM glutamate in the three-dimensional perfusion microbioreactor. (A) Water extract processed with AB-8 resin, consisting of 70% ethanol elution; (B) water extract processed with AB-8 resin, consisting of 50% ethanol elution; (C) water extract processed with AB-8 resin, consisting of 30% ethanol elution; (D) water extract.

Figure 4

Effects of dried Rehmannia root on PC12 cell injury induced by 20 mM glutamate. (A) PC12 cell injury was induced by glutamate for 24 hours. Concentrations of 70%, 50% and 30% of dried Rehmannia root were tested for their protective effect against glutamate injury. (B) LDH release rate in the glutamate group was 46.51%, which was significantly different compared with the control group. The 70% and 50% concentrations of dried Rehmannia root had significant protective effects against glutamate cytotoxicity. The 30% concentration of dried Rehmannia root had no significant protective effect. Data are expressed as the mean ± SD of at least three independent experiments. ***P < 0.001, vs. control group; ##P < 0.01, ###P < 0.001, vs. 20 mM glutamate group; †P < 0.05, vs. 30% concentration group. LDH: Lactate dehydrogenase; MTT: 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Figure 5

Effects of dried Rehmannia root on energy metabolism in glutamate-treated PC12 cells. (A) NADH, (B) NAD⁺, (C) ratio of NAD⁺/NADH. The three different concentrations of dried Rehmannia root alleviated glutamate-induced cytotoxicity to varying degrees. The 70% and 50% concentrations of dried Rehmannia root had significant effects compared with the glutamate group. The 30% concentration of dried Rehmannia root had no significant effect. The assay was done on three separate mitochondrial isolations. Data are expressed as the mean ± SD of at least three independent experiments. ***P < 0.001, vs. control group; ###P < 0.001, vs. 20 mM glutamate group.

Figure 6

Effects of dried Rehmannia root on adenosine triphosphate (ATP) concentration after glutamate-induced PC12 cell injury (fluorescence assay). After PC12 cells were treated with glutamate, the cellular ATP concentration significantly decreased. All three concentrations of dried Rehmannia root improved ATP levels to varying degrees. The 70% and 50% concentrations had significant effects compared with the glutamate group. The assay was done on three separate mitochondrial isolations. Data are expressed as the mean ± SD of at least three independent experiments. ***P < 0.001, vs. control group; ###P < 0.001, vs. 20 mM glutamate group.

Figure 7

Mitochondrial membrane potential detected using the TMRE fluorescent probe under the fluorescence microscope. (A–D) Glutamate group: PC12 cells were treated with 20 mM glutamate. The 70% and 50% concentrations of dried Rehmannia root increased TMRE density. MitoTracker derivatives of the orange-fluorescent tetramethylrosamine dye were used. Scale bar: 100 μm. (E) Quantitative analysis of TMRE density values. Data are expressed as the mean ± SD of at least three independent experiments. ***P < 0.001, vs. control group, ###P < 0.001, vs. 20 mM glutamate group. (F–I) The various concentrations of the water extract of dried Rehmannia root ameliorated mitochondrial membrane potential. Flow cytometry was used to assess mitochondrial cardiolipid oxidation. Representative results are from three independent experiments. (A, F) Control group; (B, G) glutamate-treated group; (C, H) 70% concentration of dried Rehmannia root and 20 mM glutamate group; (D, I) 50% Concentration of dried Rehmannia root group and 20 mM glutamate group.

Figure 8

Autophagosomal LC3 protein and nuclei observed with laser confocal microscopy. (A–F) Changes in autophagosomal LC3 protein and nuclei. (A, C, E) LC3 protein (green fluorescence); (B, D, F) nuclei (blue fluorescence). (A, B) Control group. (C, D) PC12 cells treated with glutamate. (E, F) 70% concentration of dried Rehmannia root with 20 mM glutamate. Scale bar: 100 μm. (G) LC3 protein levels were assessed by western blot assay. LC3 II and LC3 I are different forms of LC3 protein, and are indicators of the degree of activation of LC3 protein. GAPDH was used as a loading control. The effect of dried Rehmannia root on autophagy in PC12 cells treated with 20 mM glutamate was observed. The expression levels of LC3 II and GAPDH were assessed by western blot assay at 24 hours. 3-MA was used to examine whether autophagy pathways play a role in the neuroprotection. Data are presented as the mean ± SD (n = 3). **P < 0.01, vs. 0 h group. h: Hour(s); LC3: light chain 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 9

Effects of the autophagy inhibitor 3-MA on the cytoprotective action of dried Rehmannia root in PC12 cells. The effect of 3-MA on apoptotic changes in PC12 cells after exposure to 20 mM glutamate for 24 hours. The cells were treated with the dried Rehmannia root for 0, 6, 12, 18 or 24 hours. Data are expressed as the mean ± SD of at least three independent experiments. **P < 0.01, vs. control (20 mM glutamate group); #P < 0.05, ##P < 0.01. h: Hour(s).