Angiotensin II type 2 receptor pharmacological agonist, C21, reduces the inflammation and pain hypersensitivity in mice with joint inflammatory pain

Primary and secondary hyperalgesia develop in response to chronic joint inflammation due to peripheral and central mechanisms. Synovial macrophage and spinal microglia are involved in pain sensitization in arthritis. The level of angiotensin II type 2 receptor (AT₂R) is related to the severity of arthritis. This study aimed to determine the role of AT₂R in primary and secondary hyperalgesia in joint inflammatory pain in mice. After intra-articular CFA injection, primary hyperalgesia in the ipsilateral knee joint was measured by pressure application meter and gait analysis, secondary hypersensitivity in ipsilateral hind-paw was measured by von-Frey and Hargreaves tests following a combination of global AT₂R-deficient (Agtr2^-/-) mice and AT₂R pharmacological agonist C21. Synovial macrophage and spinal microglia were collected for flow cytometry. Morphological reconstruction of microglia was detected by immunostaining. AT₂R expression was investigated by quantitative polymerase chain reaction and western blot. Neuronal hyperactivity was evaluated by c-Fos and CGRP immunostaining. We found that pain hypersensitivity and synovial inflammation in Agtr2^-/- mice were significantly exacerbated compared with wild-type mice; conversely, systemically administrated C21 attenuated both of the symptoms. Additionally, spinal microglia were activated, and an abundant increase of spinal AT₂R was expressed on activated microglia in response to peripheral joint inflammation. Intrathecally-administrated C21 reversed the secondary hypersensitivity, accompanied by alleviation of spinal microglial activation, spinal neuronal hyperactivity, and Calcitonin gene-related peptide content. These findings revealed a beneficial role of AT₂R activating stimulation against pain hypersensitivity in joint inflammatory pain via direct modulation of synovial macrophage and spinal microglial activity.