NBP Cytoprotective Effects Promoting Neuronal Differentiation in BMSCs by Inhibiting the p65/Hes1 Pathway

Bone marrow-derived mesenchymal stem cell (BMSC) transplantation has become an effective method for treating neurodegenerative diseases. Objectives: This study investigated the effect of 3-N-butylphthalide (NBP) on the neuronal differentiation of BMSCs and its potential mechanism.

We confirmed that NBP exhibited potential therapeutic properties in the stem cell transplantation treatment of neurodegenerative diseases by protecting cells and promoting BMSC neuronal differentiation by inhibiting the p65/HES 1 pathway.

In this study, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to detect cell proliferation and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was conducted to detect the apoptosis of BMSCs. Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to detect the messenger RNA (mRNA) and protein expression levels, respectively. An enzyme-linked immunosorbent serologic assay assessed the levels of interleukin-1β, tumor necrosis factor-α, and cyclic adenosine monophosphate (cAMP). Moreover, a flow cytometry assay was used to detect the proportion of active β-tubulin III (TUJ-1) cells, and TUJ-1 expression was observed by immunofluorescence assay.

The results showed that a low concentration of NBP promoted the proliferation and induction of BMSC neuronal differentiation while inhibiting apoptosis, the production of inflammatory factors, and p65 expression. Compared with differentiation induction alone, combined NBP treatment increased the levels of nestin, neuron-specific enolase (NSE), TUJ-1, and microtubule-associated protein 2 (MAP2) protein, as well as the ratio of TUJ-1-positive cells and cAMP expression. Furthermore, p65 overexpression weakened the effect of NBP, and the overexpression of hairy and enhancer of split homolog-1 (HES1) reversed the effect of NBP in the induction of BMSC neuronal differentiation in vitro. Conclusions: We confirmed that NBP exhibited potential therapeutic properties in the stem cell transplantation treatment of neurodegenerative diseases by protecting cells and promoting BMSC neuronal differentiation by inhibiting the p65/HES 1 pathway.