Fig 2: Sciatic nerve explants cultured for 6 DIV after P2RX4 antagonist treatment exhibited myelination upregulation. (A) Representative images of semithin sections showing that P2RX4 antagonist treatment (PSB12062) blocked the degradation of myelin proteins in nerve segments maintained for 6 DIV. Scale bar = 20 μm. The lower panel shows the number of intact myelin layers. n = 6. (B) Immunofluorescence analysis showing that the P2RX4 antagonist PSB12062 (10 μM) prevented the degradation of MPZ (green). Myelin was stained with MPZ (green, left panel) and nuclei with DAPI (blue, right panel). Scale bar = 50 μm. (C) Western blot showing that PSB12062 blocked degradation of the myelin protein MPZ in nerve segments maintained for 6 DIV. Uncut indicates freshly isolated nerves. Western blotting was carried out for LAMP1, p75, MPZ, and GAPDH. The lower panel shows the Western blot results. n = 3. (D) RT-qPCR analysis of MPZ and MBP mRNA levels in nerve segments maintained for 6 DIV treated with PSB12062, compared with the untreated control. Data are mean ± SD (error bars). n = 3. (E) Overexpression of P2RX4 by pcDNA-P2RX4-DYKDDDDK tag plasmid increased the MPZ, KROX20, and MBP levels. Left, level of P2RX4 after transfection with pcDNA or pcDNA-P2RX4-DYKDDDDK-tagged plasmid. n = 5. Right, levels of MPZ, KROX20, and MBP. n = 4. (F) RT-qPCR analysis of miRNA 363−5p expression in nerve segments maintained for 6 DIV and treated with PSB12062, compared with the untreated control. Uncut indicates freshly isolated nerves. n = 3. Data are the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: P2X4-BDNF axis in the corneal epithelium after DLGR. (A) The expression pattern of P2X4 in corneas of rat (A1), rabbit (A2), and human (A3). Protein expression was imaged by immunofluorescent staining with P2X4 antibody. In each species, P2X4 was detected at the corneal epithelium (yellow arrows) but not in corneal stroma or endothelium (green arrows). (B) Tear production was measured before (D0) and 2 weeks after (2wks) DLGR. (C–E) Expression of P2X4 (C) and BDNF (D) was detected by immunofluorescence and quantified by fluorescence intensity (E). ****P < 0.001; n = 12/group. Scale bar: 100 µm or 25 µm (inset).

Fig 4: P2X4 and BDNF expression in the treated cornea. (A, B) Corneal P2X4 (A) and BDNF (B) expression was detected by immunofluorescence in the vehicle-, cyclosporine-, and BAY-776–treated groups. Panels A1 to A3 show corneas immunohistochemically stained with DAPI and P2X4, with enlarged images in A4 to A6. Panels B1 to B3 show corneas immunohistochemically stained with DAPI and BDNF, with enlarged images in B4 to B6. (C) Quantification of P2X4 and BDNF immunofluorescence density. *P < 0.05, **P < 0.01; n = 3 animals/group (10 images/animal). Scale bar: 100 µm or 25 µm.

Fig 5: P2X4R antagonist alleviated CAP-induced upregulation of P2X4R-p38-TRPV1 pathway, Iba-1 and IL-1β