Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

Abstract

Neurite outgrowth and neuronal differentiation play a crucial role in the development of the nervous system. Understanding of neurotrophins induced neurite outgrowth was important to develop therapeutic strategy for axon regeneration in neurodegenerative diseases as well as after various nerve injuries. It has been reported that extension of neurite and differentiation of sympathetic neuron-like phenotype was modulated by nerve growth factor (NGF) in PC12 cells. In this study, NGF mediated neurite outgrowth was investigated in PC12 cells after liquiritin exposure. Liquiritin is a kind of flavonoids that is extracted from Glycyrrhizae radix, which is frequently used to treat injury or swelling for its life-enhancing properties as well as detoxification in traditional Oriental medicine. The result showed that liquiritin significantly promotes the neurite outgrowth stimulated by NGF in PC12 cells in dose dependant manners whereas the liquiritin alone did not induce neurite outgrowth. Oligo microarray and RT-PCR analysis further clarified that the neurotrophic effect of liquiritin was related to the overexpression of neural related genes such as neurogenin 3, neurofibromatosis 1, notch gene homolog 2, neuromedin U receptor 2 and neurotrophin 5. Thus, liquiritin may be a good candidate for treatment of various neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease.

Fig. 1

Effect of liquiritin on the cell survival in PC12 cells. PC12 cells were treated with various concentrations of liquiritin in the presence of NGF (2 ng/mL) for 3 or 5 days. The cell viability was determined by MTT (a) or LDH release assay (b). The results were expressed as percentage of surviving cells over control cells (a) or total LDH release (b). The data are the mean ± SD (n = 3), and are representative of three or more independent experiments. * p < 0.05; significantly different compared to the control treated

Fig. 2

Liquiritin promotes neurite outgrowth induced through NGF (2 ng/mL) in PC12 cells. a, b PC12 cells were treated with various concentrations of liquiritin in the presence or absence of NGF (2 ng/mL) for 3 days. NGF 50 ng/mL was used as a positive control. a Phase contrast microphotographs (100×). b Total cell neurite length or c cells with neurite processes longer than the one or three cell body diameter (CBD) were counted, and the percentage of neurite-containing cells was determined. The data are the mean ± SD (n = 3), and are representative of three or more independent experiments. More than 100 cells/condition were analyzed in each experiment.* p < 0.05; significantly different compared to the control treated. LQ Liquiritin

Fig. 3

Differential expression of selected genes on OHS-057 analyzed by oligo SuperArrays specific for human Alzheimer’s disease. Labeled RNA extracted from PC12 cells was used to hybridize membrane-based DNA microarrays. The house-keeping genes in four corners were used as quality control in each array. Arrowed spots showed mRNA overexpression on OHS-057. A representative experiment out of two is shown in this figure. a liquiritin (20 μg/mL), b control

Fig. 4

Confirmation of oligo microarray results of up-regulated genes by RT-PCR. Five genes, Neurog 3, Nf 1, Notch 2, Nmur 2, and Ntf 5 were analyzed by RT-PCR with total RNA from control and liquiritin (20 μg/mL) treated PC12 cells. As an internal control, β-actin was amplified. The data are representative of results obtained from three independent experiments