RCAN1.4 In Spinal Dorsal Horn Contributes To Pain Hypersensitivity Induced By Peripheral Inflammation

Objective:Regulator of calcineurin 1 is an endogenous regulator of calcineurin.This study aimed to investigate the possible role of RCAN1 in the spinal dorsal horn of mice in modification of pain.Method:We established the inflammatory pain model by subcutaneous injection of complete Freund’s adjuvant into the hind plantar of mice.The immunohisto-chemistry was conducted to observe the location of RCAN1 in the spinal dorsal horn.The separate subcellular were separated by gradient centrifugation to examine the contents RCAN1 in Homogenates and PSD-enriched fractions.The expression of AMPA receptor and GluA1 phosphorylation at Ser845 was investigated by western blot.The co-immunoprecipitation was conducted to examine interaction of RCAN1.4and CaN.Paw withdrawal thresholds,dynamic scoring,acetone stimulation and paw withdrawal latencies were used to detect the effects of overexpression and knockdown of RCAN1.4 in the spinal dorsal horn of mice on pain threshold.Results:（1）RCAN1 is co-localized with NeuN that is the marker of neurons in the spinal dorsal horn,but not co-labeled with the microglial marker OX42 and the astrocyte marker GFAP;most of RCAN1 positive cells were expressed on the dorsolateral side of lamina Ⅱ labeled by CGRP positive（CGRP⁺）fibers.（2）The spinal dorsal horn mainly expressed RCAN1.1and RCAN1.4;CFA induced peripheral inflammation can significantly increase the total protein and synaptic expression of RCAN1.4 without affecting the expression of RCAN1.1.（3）Peripheral inflammation induced by CFA can increase the interaction of RCAN1.4 and CaN in neuron.（4）Overexpression of RCAN1.4 in intact mice can evoke the mechanical allodynia,cold allodynia and thermal hyperalgesia.（5）Knockdown of RCAN1.4 in inflamed mice can effectively alleviate the mechanical allodynia,cold allodynia and thermal hyperalgesia.（6）Overexpression of RCAN1.4 in intact mice also increased the synaptic expression of the GluA1 subunit and phosphorylation at the Ser845.（7）Knockdown of RCAN1.4 in inflamed mice can obviously reduce the synaptic expression of the GluA1 subunit and the phosphorylation at the Ser845.Conclusion:RCAN1 is mainly distributed in the superficial neurons of spinal dorsal horn.Peripheral inflammation induced upregulation of RCAN1.4 may be involved in the formation of pain sensitization by increasing the phosphorylation and synaptic expression of AMPA receptor GluA1 subunit.Intervention of the expression of RCAN1.4 can effectively inhibit symptoms of inflammatory pain.