| Background:Chronic inflammatory pain is a common chronic clinical condition that has become a public health concern.Currently,patients suffering from pain are still unable to achieve the desired effect of treatment and often suffer from mood disorders such as anxiety,which seriously affects the quality of life of patients.The complexity and variability of the pathogenesis of chronic inflammatory pain make clinical treatment more difficult.Numerous publications have reported that microglia activation is an important driver of spinal nerve inflammation leading to central sensitization.Activation of cannabinoid receptor 2 inhibits microglia activation and induces the conversion of activated microglia from a pro-inflammatory to an anti-inflammatory phenotype,alleviating the neuroinflammatory response.Trifluoro-icaritin(ICTF),a structural derivative of Icaritin(ICT),has anti-inflammatory,analgesic and neuroprotective effects.In addition,molecular docking data showed that ICTF has a more stable docking conformation with cannabinoid receptor 2(CB2).However,the anti-inflammatory and analgesic effects of ICTF on chronic inflammatory pain and the improvement of motor dysfunction and anxiety-like mood induced by ICTF remain to be further validated.Therefore,the present experiments were proposed to investigate the spinal cord level of CFA-induced chronic inflammatory pain rats,the cascade relationship of ICTF on CB2 receptors with P2Y12 receptors and NLRP3 inflammatory vesicles in microglia,the polarizing effect of microglia and the linkage of microglia IL-10/β-endorphin action.To provide new analgesic targets and pathways for the antiinjurious effects of ICTF on chronic inflammatory pain.Purposes:1.To observe the effects of ICTF on mechanical pain sensitivity,thermal pain sensitivity,toe swelling,gait and open field experiments in CFA-induced chronic inflammatory pain rats.2.To observe the effects of ICTF on CFA-induced chronic inflammatory pain rats spinal CB2 receptors on microglia and their activation phenotype and the expression of CB2,P2Y12,NLRP3,ASC,Caspase-1,IL-1β,IL-10,β-endorphin,CD11 b,i NOS and CD206 proteins.3.To observe the effects of ICTF on mechanical pain sensitivity,thermal pain sensitivity and gait parameters in CFA-induced chronic inflammatory pain rats after intrathecal injection of CB2 receptor antagonist AM630.4.To observe the effects of ICTF on the expression of CB2,P2Y12,NLRP3,ASC,Caspase-1,IL-1β,IL-10,β-endorphin,CD11 b,i NOS and CD206 proteins in the spinal cord of CFA-induced chronic inflammatory pain rats after intrathecal injection of CB2 receptor antagonist AM630.Methods:1.ICTF alleviates CFA-induced mechanical and thermal pain sensitization in rats with chronic inflammatory pain.2.To apply dynamic paw tactometry and plantar tester to detect mechanical and thermal nociception in CFA-induced chronic inflammatory pain rats,and to observe the effect of ICTF on the nociceptive behavior of CFA-induced chronic inflammatory pain rats.3.Application of limb swelling measurement to detect the effect of ICTF on CFA-induced toe swelling in rats with chronic inflammatory pain.4.Application of Cat Walk gait analysis system to detect the effect of ICTF on the gait behavior of CFA-induced chronic inflammatory pain rats.5.The effect of ICTF on CFA-induced anxiety-like behavior in rats with chronic inflammatory pain was analyzed by applying the open-field experiment.6.apply correlation analysis of the relationship between mechanical pain threshold,thermal pain threshold and toe swelling degree.7.Western-blot was applied to detect the expression levels of ICTF on CB2,P2Y12,NLRP3,ASC,Caspase-1,IL-1β,IL-10,β-endorphin,CD11 b,i NOS and CD206 protein molecules in the spinal cord of CFA rats.8.Immunofluorescence was applied to observe the co-localization of ICTF on CB2 receptors,P2Y12 receptors,IL-10 and β-endorphin with microglia in the spinal cord of CFA rats.9.Application of dynamic paw tactile and plantar tester to detect the effect of intrathecal injection of AM630 on anti-mechanical and thermal nociceptive sensitization in ICTF treated rats.10.Application of Cat Walk gait analysis system to detect the effect of intrathecal injection of CB2 receptor antagonist AM630 on the abnormal gait of ICTF in CFA rats.11.Western-blot was applied to detect the effect of intrathecal injection of CB2 receptor antagonist AM630 on the expression of CB2,P2Y12,NLRP3,ASC,Caspase-1,IL-1β,IL-10,β-endorphin,CD11 b,i NOS and CD206 protein molecules in the spinal cord of CFA rats by ICTF.12.Application of immunofluorescence to detect the effect of intrathecal injection of CB2 receptor antagonist AM630 on the co-localization of ICTF on the expression of CB2 receptor,P2Y12 receptor,IL-10 and β-endorphin with microglia in the spinal cord of CFA rats.Results:1.ICTF alleviates CFA-induced mechanical and thermal pain sensitization in rats with chronic inflammatory pain.Twenty-one days after successful CFA modeling,the CFA rats showed decreased mechanical and thermal pain thresholds compared to the Ctrl group;both the 3 mg/kg ICTF group and the 10 mg/kg ICTF group increased the mechanical and thermal pain thresholds of the rats compared to the CFA group at days 14 and 21.ICTF effectively reduces paw swelling in CFA-induced chronic inflammatory pain rats.2.ICTF effectively reduces toe swelling in CFA-induced chronic inflammatory pain ratsAfter the CFA model was successfully established,the left hind paw volume was significantly higher in CFA rats compared with Ctrl;the swelling of the left hind paw was significantly lower in the 3 mg/kg ICTF group on days 10,14 and 21 and in the 10mg/kg ICTF group on days 14 and 21 compared with the CFA group.3.ICTF effectively improves abnormal gait in CFA-induced chronic inflammatory pain ratsWe performed the assay analysis by Cat Walk gait analysis system on day 21.Compared with the Ctrl group,the left hind maximal strength,mean strength,standing time,compared to support and swing speed of rats in the CFA group decreased significantly,while swing time increased significantly;compared with the CFA group,the left hind paw maximal strength,mean strength,standing time,compared to support and swing speed of rats in the 3 mg/kg ICTF and 10 mg/kg ICTF groups increased significantly,while swing time was shortened.4.Correlation analysis between mechanical and thermal pain sensitivity and toe swellingWe explored the correlation between mechanical pain thresholds,thermal pain thresholds and toe swelling by correlation analysis.The analyzed data showed that there was a negative correlation between mechanical pain threshold and thermal pain threshold and toe swelling.5.ICTF alleviates anxiety-like behaviors induced by chronic inflammatory pain in CFA-induced ratsWe examined anxiety-like behavior by the open field experiment,and there was no significant difference in the total distance rats moved between the groups on day 21.Compared with the Ctrl group,the rats in the CFA group showed reduced dwell time in the central region and prolonged latency into the central region and dwell time in the peripheral region;compared with the CFA group,the rats in the 3 mg/kg ICTF group showed prolonged dwell time in the central region and reduced latency into the central region and dwell time in the peripheral region.6.Strong interaction of ICTF with CB2 receptorThe molecular docking data showed that ICTF has a more stable binding conformation with CB2 with binding free energy(ΔG)and inhibition constant(Ki)of-8.73 kcal-mol-1 and 401.14 nmol-L-1,respectively.7.Intrathecal injection of AM630 reverses the antinociceptive effect of ICTF on chronic inflammatory pain ratsCompared with the CFA+ICTF+DMSO group,intrathecal administration of AM630 followed by 3 mg/kg ICTF significantly decreased the mechanical and thermal pain thresholds in CFA rats on day 21.8.Intrathecal injection of AM630 effectively reversed the effect of ICTF on the improvement of abnormal gait in CFA ratsWe applied the Cat Walk gait analysis system to see whether AM630 affected the effect of ICTF on abnormal gait in CFA rats.On day 21,rats with intrathecal injection of AM630 showed a significant decrease in stance time,support time compared with the CFA+ICTF+DMSO group,and swing speed,while swing time was increased.9.ICTF may act on CB2 receptors to inhibit the activation of NLRP3 inflammatory vesicles by P2Y12 receptor-activated microglia mediated spinal neuroinflammationWestern-blot and immunofluorescence results showed that at day 21,the spinal cord CB2 receptor protein expression and the percentage of positive cells co-localized with Iba-1 were reduced in CFA rats compared to the Ctrl group,whereas the percentage of positive cells co-localized with Iba-1 increased after ICTF treatment;compared to the Ctrl group,the spinal cord of CFA rats Compared with the Ctrl group,protein expression of P2Y12 receptor,CD11 b,i NOS and NLRP3 inflammatory vesicles complex and the percentage of positive cells co-localized with Iba-1 increased in the spinal cord of CFA rats,while protein expression of P2Y12 receptor,CD11 b,i NOS and NLRP3 inflammatory vesicles complex and the percentage of positive cells colocalized with Iba-1 in the spinal cord of CFA rats treated with ICTF The percentage of positive cells was significantly reduced.To further verify whether ICTF mediates the anti-spinal neuroinflammatory effect of CB2 receptors,Western-blot and immunofluorescence results showed that on day 21 after intrathecal injection of AM630,the protein expression of spinal CB2 receptors and the percentage of positive cells co-localized with Iba-1 were reduced in the inhibitor group compared with the CFA+ICTF+DMSO group;the protein expression of spinal P2Y12 receptors,CD11 b,i NOS and NLRP3 inflammatory vesicle complexes and the percentage of positive cells co-localized with Iba-1 were reduced compared with the CFA +ICTF+DMSO group,the protein expression of spinal P2Y12 receptor,CD11 b,i NOS and NLRP3 inflammatory vesicle complex and the percentage of positive cells co-localized by P2Y12 with Iba-1 were significantly up-regulated in the inhibitor group compared with the CFA+ICTF+DMSO group.10.ICTF may exert analgesic effects by upregulating CB2 receptor-mediated spinal microglial IL-10/β-endorphin signaling pathway.We found by Western-blot and immunofluorescence assay that at day 21,compared with the Ctrl group,the spinal cord IL-10,POMC and CD206 protein expression was down-regulated in the CFA rats,and the expression ratio of both IL-10 and β-endorphin co-localized with Iba-1 was reduced;compared with the CFA group,the spinal cord IL-10,β endorphin and CD206 were upregulated and the percentage of IL-10 and β-endorphin co-localized with Iba-1 was significantly higher in the ICTF group compared with the CFA group.To again corroborate that ICTF mediates IL-10/β-endorphin signaling in spinal microglia via CB2 receptors.On day 21 of intrathecal administration of AM630 compared with the CFA+ICTF+DMSO group,protein expression of IL-10,POMC and CD206 in the spinal cord of the inhibitor group rats was decreased,and the percentage of IL-10 and β-endorphin co-localized with Iba-1 was reduced.Conclusions:1.ICTF has analgesic and anti-swelling effects on CFA-induced chronic inflammatory pain in rats.2.ICTF effectively improved motor dysfunction and anxiety-like behavior induced by CFA rats.3.ICTF alleviates central sensitization by upregulating microglia CB2 receptors to inhibit P2Y12 receptor activation of microglia to NLRP3 inflammatory vesicle activation-mediated spinal neuroinflammation;secondly,it exerts anti-injurious effects by increasing microglia IL-10/β-endorphin molecular signaling and promoting microglia polarization to M2 type. |
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