Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as ‘find/eat-me’ signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW) communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme), suramin (broad-spectrum P2 receptor antagonist), 2-APB (IP3 receptor blocker) and thapsigargin (endoplasmic reticulum calcium pump inhibitor) potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 μM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.

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To clarify nanoparticle-protein interaction and their action characteristics, the interactions between MTO-CHP NPs and human serum albumin (HSA) were studied by isothermal titration calorimetry (ITC), fluorescence spectroscopy, dynamic light scattering (DLS), and circular dichroism spectroscopy (CD). Hydrophobically modified pullulan (CHP) nanoparticles (NPs) loaded with mitoxantrone (MTO) were prepared (MTO-CHP NPs) with size 166.9 nm. The spherical shape was verified by transmission electron microscopy (TEM). The ITC results demonstrated an interaction between MTO-CHP NPs mainly by hydrophobic interaction force, electrostatic force, and hydrogen bonding. The mean binding constant KA was 0.832×104 M−1 and mean HSA coverage 0.939±0.302. MTO-CHP NPs could quench the fluorescence intensity of HSA, which gradually decreased to be balanced in 9 h and indicated the completion of the complexation. The size and zeta potential changes of the combined particle were dynamically detected with DLS at 0, 3, 6, 9, 12, 15, and 18 h. When the reaction was completed at 9 h, the particle size and potential remained stable, accompanied by a size change from 89.91 to about 145 nm and potential change from -15 to -3 mV, respectively. The results of CD measurement showed that the change in ellipticity of HSA at 208 nm was similar to the fluorescence spectra and DLS measurements with MTO-CHP NPs combined with HSA. At the beginning of the reaction, the proportion of α-helix was 52.3% to 43.7%, which decreased by 39.1% at compound stabilization. The release of MTO from MTO-CHP NPs at pH=5.6 was significantly accelerated, whereas that of MTO from HSA-MTO-CHP NPs was significantly reduced, and the drug release was significantly slowed down even under acidic conditions, which indicates the beneficial effect of HSA on the persistence and stability of the HSA-MTO-CHP NP compound.

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