The Role Of Angiotensin Ⅱ Type 2 Receptor In Mice With Knee Joint Inflammatory Pain

Part Ⅰ Angiotensin Ⅱ type 2 receptors are involved in the process of chronic knee joint inflammatory painObjective:To find an ideal animal model of mono-arthritic chronic pain for the further study of the interaction between synovial inflammation and the nervous system.To investigate whether AT2R is involved in joint inflammatory pain in mice using Agtr2-/-mice and to verify its role with pharmacological tools.According to the results,the appropriate therapeutic dose of C21 was selected for subsequent studies.Methods:Male wild-type C57BL/6J mice were injected intra-articularly into the right knee joint with 10μ1 of saline or different doses of CFA(5 μg,10μg,30μg,50μg).On days 1,3,7,14,21 and 28 after CFA injection,the changes in pain behavior were detected by the electronic Von Frey and Hargreaves test.Additionally,the diameter of the ipsilateral knee joint was measured at the corresponding time points to evaluate joint edema.On day 28,the knee joint was harvested for hematoxylin eosin(H&E)staining to observe the changes in inflammation in the knee joint.Saline(0.2 ml),the AT2R agonist C21(0.1,0.3,0.9 mg/kg/day),or the AT2R agonist PD123319(3 mg/kg/day)was injected intraperitoneally on day 8 after CFA intra-articular injection and continuously administered for 7 days.After the treatment,the changes in pain behavior and knee joint diameter were measured to assess the pharmacological effects.Before and 15 days after CFA injection,electronic Von Frey,Hargreaves and P.A.M.tests were used to detect changes in pain behavior,and Vernier calipers were used to measure changes in knee diameter in male C57BL/6J mice and Agtr2-/-mice with Agtr2 gene knockout on a C57/6J background.After finishing the behavioral test,the knee joints were harvested for H&E staining,which was used to evaluate joint inflammation.Results:1.Unilateral knee arthritis was generated via a single intra-articular injection of CFA(10 μg/10 μl)into the right knee joint cavity.Behavioral measurements of hyperalgesia showed that the PWT and PWL of the ipsilateral hind paw of mice in the CFA(10 μg,30 μg,50 μg)groups decreased from the first day after CFA injection,reached the lowest level on the third day,and tended to be stable after 1 week,forming a persistent state of chronic pain,which could last for approximately four weeks.The PWT and PWL of the CFA(5 μg)group began to decrease on the first day,lasted for approximately 1 week,and then began to increase on the 14th day.Although the value was still low,there was no significant difference compared with the saline control.Additionally,the joint diameter of each group increased significantly on the first day after CFA injection,especially in the CFA(10μg,30 μg,50 μg)groups,and then gradually decreased,which was stable on the 14th day.H&E staining showed a massive infiltration of mononuclear cells,synovial hyperplasia and meniscus displacement in the joint cavity of CFA groups.Compared with the saline control,the synovitis score of the mice in CFA(10 μg,30 μg,50 μg)groups was higher.2.Genetical absence of Agtr2 exacerbated pain hypersensitivity and knee joint inflammationTo detect the role of AT2R in inflammatory pain,we compared pain-related behavior between WT and Agtr2-/-mice.There was no difference between WT and Agtr2-/-mice at baseline.A single intra-articular injection of CFA caused primary and secondary hypersensitivity in the ipsilateral hind limbs of both WT and Agtr2-/-mice over a period of 15 days post-injection.We found that knee mechanical hypersensitivity(LWT),ipsilateral hind paw mechanical(PWT)and thermal(PWL)hypersensitivity were lower in Agtr2-/-mice.Additionally,after intra-articular CFA injection,Agtr2-/-mice displayed exacerbated histological responses in the knee joint over 15 days compared with WT mice.The correlation analysis showed that there were positive correlations between pain hypersensitivity and knee inflammation.3.The systemic AT2R pharmacological agonist,C21,alleviates pain hypersensitivity and knee joint inflammationThe LWT,PWT and PWL in each CFA subgroup decreased significantly on the 8th day after CFA intra-articular injection.After treatment,the AT2R pharmacological agonist C21(0.3 and 0.9 mg/kg)significantly alleviated pain hypersensitivity allodynia.Consistent with the attenuation of pain sensitivity,C21 reduced joint diameter.However,C21 0.1 mg/kg,PD123319 3 mg/kg and C21 0.3 mg/kg+PD123319 3 mg/kg did not change the pain sensitivity in mice with unilateral knee arthritis,and the same phenomenon was observed in the joint diameter.Conclusion:1.Unilateral knee arthritis was generated via a single intra-articular injection of CFA(10μg/10 μl)into the right knee joint.Chronic pain lasted for at least 28 days.2.The absence of the Agtr2 gene facilitated both pain hypersensitivity and knee inflammation in CFA-induced unilateral knee arthritis.3.Systemic AT2R pharmacological agonist C21 attenuates pain hypersensitivity and knee inflammation.A dose of 0.3 mg/kg C21 was chosen for further study.Part Ⅱ Angiotensin Ⅱ type 2 receptor is involved in peripheral sensitization in mice with chronic joint inflammatory painObjective:To investigate the effect of an AT2R pharmacological agonist,C21,on the changes in the infiltration of mononuclear cells and the expression of calcitonin gene-related peptide(CGRP)in the synovium,and on the changes in macrophages and neuronal excitability in the ipsilateral dorsal root ganglion(DRG)in mice with chronic joint inflammatory pain.Methods:Male wild-type C57BL/6J mice were randomly divided into the saline+vehicle group,CFA+vehicle group,and CFA+C21 group.A total of 0.2 ml of C21(0.3 mg/kg/day)was given intraperitoneally to the mice in the CFA+C21 group,and 0.2 ml of saline was given intraperitoneally to the mice in the saline+vehicle and CFA+vehicle groups on day 8 post CFA injection and lasted for 7 days.After treatment,the primary and secondary hyperalgesia in mice were detected by electronic Von Frey,Hargreaves,P.A.M.and DigiGait system.Synovial macrophages and T cells,and DRG macrophages were collected for flow cytometry.Immunostaining and qPCR were used to detect the expression of CGRP in the synovium.Whole-cell patch-clamp recordings were used to measure the excitability of DRG neurons.Results:1.The AT2R pharmacological agonist,C21,promotes the recovery of motor function in miceIn the first part of the study,C21(0.3 mg/kg)alleviated primary and secondary hyperalgesia in mice with joint inflammatory pain.Meanwhile,the locomotor deficits of the affected knee joint were tested by gait analysis.The swing duration,stride duration and swing/stride were prolonged,and the brake duration and brake/stride were decreased on day 15 post-CFA injection.These changes were reversed by C21 treatment.2.The AT2R pharmacological agonist,C21,can alleviate synovial inflammation and neurogenesisIncreased synovial hyperplasia,mononuclear cell infiltration,and synovitis scores were observed after CFA injection.C21 treatment reduced the CFA-induced histological changes in the affected joint.The level of synovial macrophages,which were identified as CD45+CD11b+F4/80+cells,was rapidly increased after CFA intra-articular injection,which was reversed by C21 treatment.Correlation analysis showed that the percentage of macrophages in synovial cells was positively correlated with the mechanical pain response of the affected knee joint.In addition,the expression of MHC-Ⅱ,a marker of activated macrophages,was significantly increased accordingly.The level of MHC-Ⅱ in CD45+CD11 b+F4/80+macrophages was significantly increased after CFA intra-articular injection,and this increase could be inhibited by C21.At the same time,the percentages of total CD3+CD4+T cells and activated CD4 T cells(CD4+CD62L-)in synovial tissue were significantly increased,which was reversed by C21 treatment.Immunostaining and qPCR further showed that CGRP(+)nerve fibers in the synovium were significantly increased post-CFA injection,and these were significantly decreased after C21 treatment.3.Effect of C21 on macrophage infiltration and neuronal excitability in the DRGFlow cytometry showed that CD45+CD11b+ macrophages in the DRG were significantly increased after CFA intra-articular injection and were inhibited by C21.Meanwhile,the absolute value of the threshold of the action potential in small neurons of the DRG decreased in mice with CFA-induced knee joint inflammatory pain and increased after C21 treatment.Conclusion:1.The AT2R pharmacological agonist,C21,can alleviate synovial inflammation,reduce the infiltration of inflammatory cells,regulate the function of inflammatory cells and reduce their pro-inflammatory response.2.Activation of AT2R can alleviate primary hyperalgesia and motor dysfunction in mice with chronic joint inflammatory pain,which may be related to the reduction in synovial inflammation and nerve fiber proliferation.3.The AT2R agonist C21 can significantly alleviate inflammation in the ipsilateral DRG,including reduced macrophage infiltration,and can also reduce neural excitability in the ipsilateral DRG.Part Ⅲ The mechanism of angiotensin Ⅱ type 2 receptor in pain regulation and central sensitization in chronic joint inflammatory painObjective:To investigate whether spinal microglia are involved in central sensitization in chronic joint inflammatory pain and the potential mechanism of the analgesic effect of AT2R at the spinal cord level.Methods:On day 7 after CFA intraarticular injection,L3-5 spinal dorsal horn tissues were harvested from saline and CFA mice.Ang Ⅱ levels were detected by EIA;the expression of AT2R was detected by WB;spinal microglia were sorted by flow cytometry,and Agtr2 gene levels in spinal cord tissues or microglia were detected by qPCR.The colocalization of AT2R with CD206 or MHC Ⅱ was detected by immunofluorescence staining in microglia cells ex vivo.Then,minocycline(10 mg/ml,10 μl)was injected intrathecally on day 5 and day 6 after CFA intra-articular injection.The pain behavior test was performed on day 7.Then,L3-5 spinal dorsal horn tissues were harvested for the analysis of the changes in microglia in the spinal dorsal horn,which were detected by immunostaining.Male wild-type C57BL/6J mice were randomly divided into the saline+vehicle group,CFA+vehicle group and CFA+C21 group.On days 5 and 6 after CFA intra-articular injection,C21(0.12 mg/ml,10 μl)was given intrathecally in the CFA+C21 group,and the same volume of artificial cerebrospinal fluid(ACSF)was given intrathecally in the saline+vehicle group and CFA+vehicle group.Pain-related behavior was tested by the electronic Von Frey test and Hargreaves test on day 7.The L3-5 spinal cord was harvested for further analysis.cFos and NeuN colocalization and the expression of CGRP and Iba-1 in the dorsal horn of the spinal cord were detected by immunofluorescence staining.Flow cytometry was used to detect changes in microglia.The expression of NF-κB p65,NF-κB p-p65 and CGRP was detected by WB.The levels of TNF-α and IL-1β were detected by qPCR.Results:1.AT2R increased significantly in microglia from the spinal cord under conditions of chronic joint inflammatory painOn day 7 after CFA injection,the staining of L3-5 spinal cord sections with Iba-1 showed that microglia were activated,including the number and morphological alterations,which was consistent with previous studies.Intrathecal injection of minocycline inhibited the number and morphological changes of activated microglia and subsequently alleviated the hind paw mechanical and thermal hypersensitivity of the affected hind limbs.The expression of the Agtr2 gene and AT2R protein in the L3-5 spinal dorsal horn increased significantly on day 7 after CFA intra-articular injection.Microglia from the spinal cord were isolated and sorted for further analysis.The results showed that the expression of the Agtr2 gene in microglia increased significantly.We found that AT2R was expressed in microglia cells ex vivo,with different phenotypes.However,enzyme immune assay results showed that the level of angiotensin Ⅱ in the spinal dorsal horn was not altered following joint inflammation.This discordance suggests that the increase in spinal AT2R might not be fully activated because of insufficient endogenous ligand angiotensin Ⅱ.2.Intrathecal AT2R pharmacological agonist,C21,inhibits microglial activation and alleviates secondary hyperalgesiaThe pain-related behavior results demonstrated that intrathecal injection of C21 significantly alleviated the mechanical and thermal hypersensitivity of the hind paw in CFA-induced knee joint inflammatory pain mice.Immunofluorescence staining showed that intrathecal injection of C21 could change the morphology of activated microglia.Flow cytometry was used to assess the numerical changes in microglia in the spinal dorsal horn.Intrathecal injection of C21 significantly inhibited the number of activated microglia CD45hiCD11b+ cells.Correlation analysis showed that activated microglia in the spinal cord were positively correlated with secondary hypersensitivity in the ipsilateral hind paw.Intrathecal injection of C21 reversed the increased levels of TNF-α and IL-1β induced by CFA,and AT2R activation significantly inhibited the phosphorylation of NF-κB p65.3.Intrathecal injection of the AT2R pharmacological agonist,C21,reduces neuronal excitability in the spinal dorsal hornTo explore whether the excitability changes of spinal neurons were in line with microglial activation,the release of CGRP protein and the colabeling of neurons with cFos in layers Ⅰ and Ⅱ of the spinal dorsal horn were detected.The enhancement of CGRP and c-Fos immunoactivity was well displayed in lamella Ⅰ and Ⅱ in the SDH at day 7 post CFA injection,which could be suppressed by intrathecal C21 treatment.Conclusion:1.Spinal microglia are involved in central sensitization in chronic joint inflammatory pain.2.The expression of AT2R in the spinal dorsal horn was upregulated in mice with unilateral knee joint inflammatory pain,and the upregulated AT2R was mainly derived from activated microglia of the spinal cord.3.Intrathecal injection of the AT2R pharmacological agonist,C21,inhibited microglial activation while alleviating secondary hypersensitivity.4.Intrathecal injection of the AT2R pharmacological agonist,C21,inhibited neuronal excitability in layers Ⅰ and Ⅱ of the spinal dorsal horn.