The Protective Effects of Icariin against the Homocysteine-Induced Neurotoxicity in the Primary Embryonic Cultures of Rat Cortical Neurons

Abstract

Icariin, an ingredient in the medicinal herb Epimedium brevicornum Maxim (EbM), has been considered as a potential therapeutic agent for neurodegenerative diseases such as Alzheimer's disease (AD). Hyperhomocysteinaemia is a risk factor for AD and other associated neurological diseases. In this study we aim to investigate whether icariin can reverse homocysteine (Hcy)-induced neurotoxicity in primary embryonic cultures of rat cortical neurons. Our findings demonstrated that icariin might be able restore the cytoskeleton network damaged by Hcy through the modulation of acetyl-α-tubulin, tyrosinated-α-tubulin, and phosphorylation of the tubulin-binding protein Tau. In addition, icariin downregulated p-extracellular signal-regulated kinase (ERK) which is a kinase targeting tau protein. Furthermore, icariin effectively restored the neuroprotective protein p-Akt that was downregulated by Hcy. We also applied RT² Profiler PCR Arrays focused on genes related to AD and neurotoxicity to examine genes differentially altered by Hcy or icariin. Among the altered genes from the arrays, ADAM9 was downregulated 15 folds in cells treated with Hcy, but markedly restored by icariin. ADAM family, encoded α-secreatase, plays a protective role in AD. Overall, our findings demonstrated that icariin exhibits a strong neuroprotective function and have potential for future development for drug treating neurological disorders, such as AD.

Keywords: RT2 Profiler PCR array; homocysteine; icariin; neuroprotection.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1

Photos of Epimedium brevicornum Maxim (A) The plant (B) The dry leaves (C) The chemical structure of icariin.

Figure 2

Cytotoxicity of Hcy and icariin measured by LDH assay. Cortical neurons were treated with Hcy (A), Icariin (B) and Hcy plus icariin (C) and performed LDH assay. The treatment duration for Hcy or Icariin was 12 h. In the co-treatment, cells were pretreated 1 h before the addition of Hcy. Results were quantified and expressed as fold of control. Data represents mean ± SE from at least three independent experiments. The significance of differences among treatment groups was determined by one-way ANOVA. *: p < 0.05 compared to control. “#”: p < 0.05. “+” and “-”: with and without the compound.

Figure 3

Icariin restored the Hcy-reduced acetylation and phosphorylation of tubulin in cultured neuronal cells. (A–F): Primary cortical neuronal cultures were treated with 200 µM Hcy for 48 h. For the co-treatment, cells were pretreated with icariin for 1 h, before incubating with Hcy for 48 h. The treated cells were subjected for western blot analyses and quantified for the detection of Ac- and Tyr-tubulin with anti-Ac-α-tubulin and anti-tyr-antibodies. (G–H) Western blot analysis for the detection of p-tau and total tau with anti-p-tau and anti-tau antibodies. Cells were treated 200αM Hcy alone for 2 h. For the co-treatment, cells were pretreated with icariin for 1 h, followed by co-incubation with Hcy for another 2 h. α-tubulin was used as internal control. Data represent mean ± SE from at least three independent experiments and was quantified using densitometry. *: p < 0.05 compared to control. ** and #: p < 0.05 compared to cultures treated with 200 μM Hcy. “+” and “-”: with and without the compound.

Figure 4

Microscopic images of IHC stained cells hybridized with anti-Ac-α-tubulin, anti-tyr-α-tubulin, and anti-p-tau (Ser400/Thr403/Ser404) antibodies. Method for IHC staining was described under Materials and Methods.

Figure 5

Icariin counteracts the effects of Hcy on ERK (A,B), JNK (C,D) and AKT (E,F) signaling molecules assessed using the western blot analysis. Cells were treated with either Hcy, Icariin, or both according to the treatment illustrated in Figure 3 legend. The α-tubulin was used as internal control. Statistical analysis was performed with one-way ANOVA. *: p < 0.05 compared to control. # and **: p < 0.05 compared to cultures treated with Hcy (200 μM). Data represent mean ± SE from at least 3 independent experiments. “+” and “-”: with and without the compound.

Figure 6

The scatter plots of AD array and the neurotoxicity array. cDNAs were prepared from Hcy, icariin, Icariin + Hcy and the control groups and each cDNA sample was applied for AD and neurotoxicity arrays. The scatter plots of each treatment groups were generated by normalized against the control according the Qiagen online data analysis for RT2 Profile PCR Arrays.

Figure 7

Verification of the mRNA expressions of four differentially altered genes identified from the AD and neurotoxicity arrays by using qPCR reactions with each specific primers. The mRNA levels of Adam9 (A), Apbb2 (B), Bdnf (C) and Cd8b (D) were verified using real-time RT-PCR with SYBR Green Mater and specific sets of primers for each gene (Table 1). All data was expressed in fold changes compared to the control. The house-keeping genes were used as internal control. Statistical analysis was performed with one-way ANOVA. *: p < 0.05 compared to control. ** and #: p < 0.05 compared to cultures treated with 200 μM Hcy.

Figure 8

Signaling molecules and factors contributed to the neuroprotective effects of icariin in the primary embryonic cultures of rat cortical neurons. The red arrows indicate the Hcy-induced changes of the protein molecules, while the green arrows indicate the changes of protein molecules altered by the co-treatment of Hcy and icariin. ↑: upregulation; ↓: downregulation.