P2Y12-mediated HIV gp120 and ddC-induced neuropathic pain improved by esculin.

Abstract

We studied whether esculin (ES) has the effect of alleviating peripheral neuropathic pain (NP) in rat models of HIV glycoprotein 120 (gp120) together with zalcitabine (2',3'-dideoxycytidine; ddC) treatment and explored the possible mechanism of it. The rats pain behaviors were evaluated by observing the paw withdrawal threshold (PWT) and the paw withdrawal latency (PWL). The rats were divided into a control group, sham group, gp120 combined with a ddC treatment group (gp120& ddC group), gp120&ddC combined with ES treatment group (gp120&ddC+ES group), which ES was administered intragastrically, and gp120&ddC combined with short hair RNA of P2Y12 receptor (rP2Y12) treatment group (gp120&ddC+shP2Y12 group), which shRNA of rP2Y12 was injected intrathecally with a dose of 25 µg/20 µl for every rat, and a negative control plasmid was administered to the gp120&ddC+nc group. Western blotting was used to measure the protein expression levels of the rP2Y12, the nuclear factor of activated T-cells type c1 (NFATc1), phospho-NFATc1 and the C-C motif chemokine ligand 3 (CCL3) in the L4-L6 dorsal root ganglia (DRG). Real-time quantitative PCR (RT-qPCR) was used to test the mRNA expression level of the CCL3. Double-labeling immunofluorescence was used to identify the co-localization of the rP2Y12 with glial fibrillary acidic protein (GFAP) in DRG. Fluorescence imaging with calcium indicator fluo-3 AM (7.5 μM) was performed to observe the change of intracellular calcium concentration ([Ca2+]i). Molecular docking was performed to identify the interaction between rP2Y12 and the ligand ES. We found that accompanied by the attenuation of mechanical allodynia and thermal hyperalgesia, rP2Y12 expression in the gp120+ddC+ES group of rats was downregulated compared with the gp120+ddC ones, as was the coexpression of the rP2Y12 and GFAP of satellite glial cells (SGCs) in DRG, and the CCL3 mRNA levels and protein expression were both decreased. In addition, mechanistic studies have found that there is a docking pocket between ES and the rP2Y12 protein, which causes ES to decrease the [Ca2+]i, thus increasing the phosphorylation level of NFATc1. Taken together, the results suggest that ES can combine with the rP2Y12, inhibit DRG SGCs activation caused by gp120&ddC, reduce [Ca2+]i, and prevent the NFATc1-mediated gene transcription of CCL3, finally relieving NP in rats treated with gp120&ddC.