Lab Anim Res.  2016 Jun;32(2):105-115. 10.5625/lar.2016.32.2.105.

Beneficial effect of diosgenin as a stimulator of NGF on the brain with neuronal damage induced by Aβ-42 accumulation and neurotoxicant injection

• Koh EK
• Yun WB
• Kim JE
• Song SH
• Sung JE
• Lee HA
• Seo EJ
• Jee SW
• Bae CJ
• Hwang DY

• KMID: 2309084
• DOI: http://doi.org/10.5625/lar.2016.32.2.105

Abstract

To investigate the beneficial effects of diosgenin (DG) on the multiple types of brain damage induced by Aβ-42 peptides and neurotoxicants, alterations in the specific aspects of brain functions were measured in trimethyltin (TMT)-injected transgenic 2576 (TG) mice that had been pretreated with DG for 21 days. Multiple types of damage were successfully induced by Aβ-42 accumulation and TMT injection into the brains of TG mice. However, DG treatment significantly reduced the number of Aβ-stained plaques and dead cells in the granule cells layer of the dentate gyrus. Significant suppression of acetylcholinesterase (AChE) activity and Bax/Bcl-2 expression was also observed in the DG treated TG mice (TG+DG group) when compared with those of the vehicle (VC) treated TG mice (TG+VC group). Additionally, the concentration of nerve growth factor (NGF) was dramatically enhanced in TG+DG group, although it was lower in the TG+VC group than the non-transgenic (nTG) group. Furthermore, the decreased phosphorylation of downstream members in the TrkA high affinity receptor signaling pathway in the TG+VC group was significantly recovered in the TG+DG group. A similar pattern was observed in p75NTR expression and JNK phosphorylation in the NGF low affinity receptor signaling pathway. Moreover, superoxide dismutase (SOD) activity was enhanced in the TG+DG group, while the level of malondialdehyde (MDA), a marker of lipid peroxidation, was lower in the TG+DG group than the TG+VC group. These results suggest that DG could exert a wide range of beneficial activities for multiple types of brain damage through stimulation of NGF biosynthesis.

Keyword

Diosgenin; neurodegenerative disorder; Aβ-42; trimethyltin; acetylcholinesterase; nerve growth factor

MeSH Terms

Acetylcholinesterase
Animals
Brain*
Dentate Gyrus
Diosgenin*
Lipid Peroxidation
Malondialdehyde
Mice
Nerve Growth Factor*
Neurodegenerative Diseases
Neurons*
Peptides
Phosphorylation
Superoxide Dismutase
Acetylcholinesterase
Diosgenin
Malondialdehyde
Nerve Growth Factor
Peptides
Superoxide Dismutase

Figure

• 

  Figure 1 Deposition of Aβ peptides. Accumulation of Aα peptides in the brains of trimethyltin (TMT)-treated transgenic 2576 (TG) mice was detected by immunohistochemical staining (A) and dot blot assay (B) using specific antibody for total Aâ peptide. The data shown represent the means ± SD of three replicates. *P<0.05 relative to he non-trangenic (nTG) group. #P<0.05 relative to the vehicle (VC) treated TG group (TG+VC group). §P<0.05 relative to the diosgenin (DG) treated TG group (TG+DG group).

• 

  Figure 2 Alteration of the numbers of dead cells. (A) Apoptotic cells in the hippocampus of the brain were detected by Nissl staining. Low intensity was observed in the hippocampus (CA2 and CA3) of the diosgenin (DG) treated TG group (TG+DG group) relative to the vehicle (VC) treated TG group (TG+VC group) (100× magnification, scale bar=200 µm). Detailed histological features of several regions of the hippocampus are shown at 400× magnification (scale bar=50 µm). (B) Total numbers of dead cells were counted in specific areas. The data shown represent the means ± SD of three replicates. *P<0.05 relative to the non-treansgenic (nTG) group. #P<0.05 relative to the VC treated TG group (TG+VC group).

• 

  Figure 3 Measurement of acetylcholine esterase (AChE) activity and Bax/Bcl-2 protein expression. (A) AChE activity was measured using the homogenate of hippocampus tissue collected from transgenic 2576 (TG) mice. (B) Bax and Bcl-2 protein in the lysate mixture prepared from the brain tissue was detected using the specific primary antibody. The expression level of β-actin was used as an endogenous control. The data shown represent the means ± SD of three replicates. *P<0.05 relative to the non-treansgenic (nTG) group. #P<0.05 relative to the vehicle (VC) treated TG group (TG+VC group). §P<0.05 relative to the diosgenin (DG) treated TG group (TG+DG group).

• 

  Figure 4 Measurement of nerve growth factor (NGF) concentration. After final treatment, brain tissues were collected from subset groups. NGF concentration in the brain homogenate was measured using an anti-NGF ELISA kit. The data shown represent the means ± SD of three replicates. *P<0.05 relative to the non-treansgenic (nTG) group. #P<0.05 relative to the vehicle (VC) treated TG group (TG+VC group). §P<0.05 relative to the diosgenin (DG) treated TG group (TG+DG group).

• 

  Figure 5 Alteration of the downstream signaling pathway of high affinity nerve growth factor (NGF) receptor. The phosphorylation level of three components including p-TrkA, p-ERK and p-Akt in the lysate mixture prepared from the cortex and hippocampus tissue was detected using the specific primary antibody. The expression level of β-actin was used as an endogenous control. The data shown represent the means ± SD of three replicates. *P<0.05 relative to the non-treansgenic (nTG) group. #P<0.05 relative to the vehicle (VC) treated TG group (TG+VC group). §P<0.05 relative to the diosgenin (DG) treated TG group (TG+DG group).

• 

  Figure 6 Alteration of the downstream signaling pathway of low affinity nerve growth factor (NGF) receptor. The level of p-JNK and JNK in the lysate mixture prepared from the cortex and hippocampus tissue was detected using the specific primary antibody. The expression level of β-actin was used as an endogenous control. The data shown represent the means ± SD of three replicates. *P<0.05 relative to the non-treansgenic (nTG) group. #P<0.05 relative to the vehicle (VC) treated TG group (TG+VC group). §P<0.05 relative to the diosgenin (DG) treated TG group (TG+DG group).

• 

  Figure 7 Status of oxidant stress in the brain. (A) The level of malondialdehyde (MDA) was determined in the tissue collected from the brains of mice using a lipid peroxidation assay kit that could detect MDA at 0.1 nmole/mg to 20 nmole nmole/mg. (B) superoxide dismutase (SOD) activity was measured in the homogenate of the cortex and hippocampus tissue collected from the subset groups. Three samples were assayed in triplicate by MDA and SOD assay. The data shown represent the means ± SD of three replicates. *P<0.05 relative to the non-treansgenic (nTG) group. #P<0.05 relative to the vehicle (VC) treated TG group (TG+VC group).

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