Neuroprotective Effect of Ginseng Fibrous Root Enzymatic Hydrolysate against Oxidative Stress

Abstract

Oxidative stress is one of the potential causes of nervous system disease. Ginseng extract possesses excellent antioxidant activity; however, little research on the function of the ginseng fibrous root. This study aimed to investigate the neuroprotective effects of ginseng fibrous root to alleviate the pathogenesis of Alzheimer's disease (AD) against oxidative stress. Ginseng fibrous root enzymatic hydrolysate (GFREH) was first prepared by digesting ginseng fibrous roots with alkaline protease. In vitro, the GFREH showed antioxidant activities in free radical scavenging mechanisms. With a cellular model of AD, GFREH inhibited the increase in Ca²⁺ levels and intracellular ROS content, maintained the balance of mitochondrial membrane potential, and relieved L-glutamic acid-induced neurotoxicity. In vivo, GFREH improved the survival rate of Caenorhabditis elegans (C. elegans) under oxidative stress, upregulated SOD-3 expression, and reduced reactive oxygen species (ROS) content. Therefore, our findings provide evidence for the alleviation effect of GFREH against oxidative stress in neuroprotection, which may accelerate the development of anti-Alzheimer's drugs and treatments in the future.

Keywords: Alzheimer’s disease; antioxidants; ginseng; neuroprotection; oxidative stress.

Figure 1

Schematic experiments of GFREH’s effect on oxidative stress and neurotoxicity. In cell assays, AD model was established by treating L-Glu. GFREH administration increased cell viability, which may be achieved by reversing intracellular Ca²⁺ influx, maintaining MMP stability, and upregulating the expression of antioxidant enzymes to eliminate intracellular ROS. In vivo, GFREH treatment increased nematodes’ survival rate under OS conditions, upregulated the expression of SOD-3, and decreased intracellular ROS in nematodes exposed to juglone. GFREH treatment also reduced lipid and lipofuscin accumulation. (GFREH: ginseng fibrous root enzymatic hydrolysate; OS: oxidative stress; L-Glu: L-glutamic acid; ROS: reactive oxygen species; MMP: mitochondrial membrane potential; SOD: superoxide dismutase; CAT: catalase; LDH: lactate dehydrogenase; MDA: Malondialdehyde).

Figure 2

The role of GFREH in the scavenging of free radicals. (A) GFREH effectively scavenged ABTS free radicals in vitro (n = 3). (B) GFREH greatly scavenged DPPH free radicals in vitro (n = 3). (C) GFREH effectively scavenged OH∙ free radicals in vitro (n = 3). (D) GFREH effectively scavenged superoxide anion in vitro (n = 3).

Figure 3

(A) The effect of L-Glu on apoptosis of SH-SY5Y cells, (n = 6). (B) GFREH treatment alone for 24 h caused no decrease in the viability of SH-SY5Y cells, (n = 6). (C) GFREH pretreatment for 4 h and incubation with L-Glu for an additional 24 h improved cell viability, (n = 6). The data are presented as the mean value ± SD. ### p < 0.001 vs. CTRL, *** p < 0.001 vs. L-Glu-treated cells. CTRL: untreated cells, L-Glu, cells treated with L-Glu.

Figure 4

Fluorescence microscopy images of intracellular calcium levels. (A) Intracellular Ca²⁺ overload caused by L-Glu was reversed by GFREH pretreatment, as detected by Fluo-4-AM staining. The scale bar is 100 μm. (B) Fluorescence quantification by ImageJ software (version: 1.51), (n = 3). The data are presented as the mean value ± SD. ### p < 0.001 vs. CTRL, ** p < 0.01, *** p < 0.001 vs. L-Glu-treated cells.

Figure 5

(A) The effect of MMP caused by 24 h L-Glu exposure was strongly restored by 4 h GFREH pretreatment, as analyzed by JC-1. Scale bar is 100 μm. (B) Fluorescence quantification by ImageJ software (version: 1.51), (n = 3). The data are presented as the mean value ± SD. ^# p < 0.01 and ^### p < 0.001 vs. CTRL, * p < 0.01 and *** p < 0.001 vs. L-Glu-treated cells.

Figure 6

ROS are detected by DCFH-DA, which produces green fluorescence in the presence of ROS. (A) ROS contents in SH-SY5Y cells detected by a flow cytometer. (B) Cartogram of ROS in SH-SY5Y cells under different treatments, (n = 3). The data are expressed as the mean value ± SD. ^### p < 0.001 vs. CTRL, *** p < 0.001 vs. L-Glu-treated cells. (C) Pictures of SH-SY5Y cells are imaged by fluorescence microscopy. Scale bar is 100 μm.

Figure 7

Effect of GFREH antioxidative enzymes in SH-SY5Y cells. The (A) SOD activity and (B) CAT activity induced by L-Glu were significantly increased by pretreatment with GFREH. The (C) MDA levels and (D) LDH release levels induced by L-Glu were significantly decreased by pretreatment with GFREH (n = 5). The data are presented as the mean value ± SD. ^### p < 0.001 vs. CTRL, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. L-Glu-treated cells.

Figure 8

The protective effect of GFREH on C. elegans under stress conditions. (A) Different concentrations of GFREH reduced the cumulative mortality of C. elegans under oxidative stress, and the optimal concentration was 1 mg/mL, (n = 30). (B) GFREH reduced the ROS level in C. elegans, (n = 30). (C) The picture of C. elegans imaged by fluorescence microscopy. (D) GFREH upregulated the expression of SOD-3 in nematodes, (n = 30). Scale bar is 100 μm. The data in A, B, and D are presented as the mean value ± SD. *** p < 0.001 vs. juglone group. CTRL: untreated worms, Juglone: juglone-treated worms, Juglone + GFREH: Juglone, and 1 mg/mL GFREH-treated group.

Figure 9

C. elegans N2 pictures imaged by fluorescence microscope. (A) Oil red O staining image of lipid accumulation in C. elegans N2 and (B) quantitation of the fluorescence by ImageJ software (version: 1.51) (n = 6). (C) Autofluorescence image of lipofuscin accumulation in C. elegans N2 and (D) quantitation of the fluorescence by ImageJ software (version: 1.51) (n = 6). Scale bar is 100 μm. The data in B and D are presented as the mean value ± SD. ^### p < 0.001 vs. CTRL, ** p < 0.01 and *** p < 0.001 vs. the Juglone group.