Objectives: To explore the effect of exogenous recombinant high mobility group protein box1 (rHMGB1) on proliferation and differentiation of neural stem cells (NSCs) and the related mechanism.
Methods: SD rat cerebral cortex cells were cultured in serum-free medium, extending the culture and purification of neural stem cells. NSCs were identified by detecting nestin-label with immunofluorescence method.The NSCs proliferation activity after adding different concentrations of rHMGB1 was determined by CCK-8 assay and the optimal concentration of rHMGB1 for the follow-up experiments was selected.The effect of rHMGB1 on NSCs differentiation was detected by immunofluorescence assay. The mRNA and protein expression of involved factors were studied by real-time PCR and Western blot separately.
Results: The neural cells isolated from the cortex of rat embryos showed the expression of nestin antigen and the neural stem cells purity could reach more than 99% when cultured to the third generation. Under the stimulation of 10 ng/mL rHMGB1, neural stem cells proliferation activity were the highest, therefore, 10 ng/mL rHMGB1 was selected to treat the experimental group. The expression of TUJ1 in the experimental group was higher than that in the control group (P<0.05). Real-time PCR and Western blot confirmed rHMGB1 could improve the expression of receptor for advanced glycation end products (RAGE), Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), matrix metalloproteinase 9 (MMP-9) and nerve growth factor(NGF) respectively at the level of mRNA and protein expression.
Conclusions: Exogenous rHMGB1 promoted rat NSCs proliferation and differentiation into neuronsin vitro by activating RAGE, TLRs, MMP-9 signaling.
Keywords: Matrix metalloproteinase 9; Nerve growth factor; Neural stem cells; Receptor for advanced glycation end products; Recombinant high mobility group protein box 1; Toll-like receptors.