Fig. 8: The antinociceptive effects of JH-ENT-01 depend on A1R.

a–c Patch clamp recordings showing the effects of JH-ENT-01 on synaptic transmission (sEPSCs) in spinal cord slices of STZ-treated in mice at 1 week. a Schematic of patch clamp recordings in spinal cord slice. Created in BioRender. He, W. (2024) BioRender.com/d34r338. b sEPSC traces before, during, and after the inhibitor perfusion (3 μM, 3 min). The lower traces are shorter segments of the upper trace, marked as 1 (before the perfusion) and 2 (after perfusion). c Quantification of sEPSC frequency (top) and amplitude (bottom) before and after JH-ENT-01 treatment (n = 13 neurons from 3 male mice, paired Student’s t test, two-sided). d, e Reversal of JH-ENT-01’s analgesia by A1R antagonist DPCPX in STZ-treated mice (d) and CFA-treated mice (e) via intrathecal route. DPCPX (10 nmol) was given 30 min prior to the injection of ENT1 inhibitor (10 nmol). f The reversal of JH-ENT-01’s analgesia on the CFA-induced inflammatory pain is observed only with the A1R-specific antagonist DPCPX (10 nmol), not the A2AR-specific inhibitor ZM241385 (10 nmol), the A2BR-specific inhibitor PSB603 (10 nmol), and the A3R-specific inhibitor MRS3777 (10 nmol). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-Way ANOVA followed by Bonferroni posthoc comparison. n = 6 males. g sEPSC traces before, during, and after the A1R antagonist DPCPX perfusion (1 μM, green) and DPCPX together with JH-ENT-01 (3 μM, blue). h Quantification of sEPSC frequency (left) and amplitude (right) of g (n = 16 neurons from 3 mice–2 males and 1 female; paired Student’s t test, two-sided). All data were expressed as mean ± s.e.m.