Fig. 5

RhoA/ROCK2 signaling, which acts downstream of P2Y12R in the TNC, was increased following NTG administration and contributed to NTG-induced CGRP expression. a, b Representative immunoblots of GTP-RhoA and ROCK2 in the TNC at various time points after NTG administration. The band intensities of GTP-RhoA and ROCK2 are presented relative to those of total RhoA and β-actin, respectively. Quantitative analysis indicated a significant increase on day 9 after NTG injection. Data are presented as mean ± SEM; n = 6 per group; *p < 0.05 compared with the sham group. c, d Western blot showing that the P2Y12R inhibitors MRS2395 and clopidogrel attenuated the upregulation of GTP-RhoA and ROCK2 expression. Values are presented as mean ± SEM; n = 6 per group; *p < 0.05 and **p < 0.01 compared with the sham group, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared with the NTG group. The ROCK inhibitor fasudil reversed the upregulation of ROCK2 (e) in the TNC induced by NTG administration but had no effect on P2Y12R (f) and GTP-RhoA (g) expression in the TNC. Data are presented as mean ± SEM; n = 6 per group; *p < 0.05 and **p < 0.01 compared with the sham group, ^#p < 0.05 compared with the NTG group. h Immunofluorescence staining images of CGRP in the TNC. Scale bar: 100 μm. i Representative immunoblots of CGRP in the TNC after NTG and fasudil administration. The band intensity is presented relative to that of β-actin. Data are presented as mean ± SEM; n = 6 per group; **p < 0.01 compared with the sham group; ^#p < 0.05 compared with the NTG group. j Quantitative analysis of CGRP immunoreactivity in the TNC. Data are presented as mean ± SEM; n = 4 per group; six sections per mouse; ***p < 0.001 compared with the sham group; ^###p < 0.001 compared with the NTG group