Icariside II promotes the differentiation of human amniotic mesenchymal stem cells into dopaminergic neuron-like cells

Abstract

The purpose of this study is to observe the effect of icariside II (ICS II) on the differentiation of human amniotic mesenchymal stem cells (hAMSCs) into dopaminergic neuron-like cells, the involvement of PI3K signaling pathway inhibitors. After identifying hAMSCs by flow cytometry, hAMSCs were induced and treated with ICS II at 10 μmol/L, 3 μmol/L, 1 μmol/L, and 0 μmol/L. hAMSCs in the LY294002+3μM ICS II group were pretreated with 20 μmol/L LY294002, a PI3K-specific inhibitor, for 1 h, and then hAMSCs were induced with 3 μmol/L ICS II. On the 21st day of induction, immunofluorescence was used to detect expression of the neuronal nuclei (NeuN), neuron-specific enolase (NSE), microtubule-associated protein-2 (MAP-2), glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) antigens in each induced cell group. Western blotting was used to detect the relative protein expression of NSE, MAP-2, GFAP, and TH. ELISA was used to detect the dopamine concentration in the induction medium supernatant of each group. After 21 d of ICS II induction, immunofluorescence showed that GFAP expression was not obvious in any hAMSC group. The NeuN, NSE, MAP-2, and TH fluorescent proteins were expressed in each group. NeuN was expressed in the nucleus and cytoplasm, while NSE, MAP-2, and TH were mainly expressed in the cytoplasm. The positive cell rates of NeuN, NSE, MAP-2, and TH in the 10 μmol/L, 3 μmol/L, and 1 μmol/L ICS II groups were higher than those in the LY294002+3μM ICS II and control groups. After 21 d of induction, the Western blot results showed that the protein expression levels of NSE, MAP-2, and TH in the 10 μmol/L, 3 μmol/L, and 1 μmol/L ICS II groups were significantly higher than those in the LY294002+3μM ICS II and control groups. The MAP-2 protein expression levels in the 10 μmol/L and 3 μmol/L groups were higher than that in the 1 μmol/L group. After 21 d of induction, the dopamine concentrations in the culture supernatants of the 10 μmol/L, 3 μmol/L, and 1 μmol/L ICS II groups were higher than those in the LY294002+3μM ICS II and control groups. In our experiment, ICS II induced hAMSCs to differentiate into dopaminergic neuron-like cells, and the optimal concentration range of ICS II was 3–10 μmol/L. Moreover, the PI3K signaling pathway is involved in the above differentiation process.