小胶质细胞P2Y6受体通过NLRP3-caspase-1-IL-1β通路介导福尔马林诱导的小鼠急性炎性疼痛OA
Microglial P2Y6 receptor mediates formalin-induced acute inflammato-ry pain in mice via NLRP3-caspase-1-IL-1β signaling pathway
目的:探讨小胶质细胞P2Y6受体在福尔马林诱导的小鼠急性炎性疼痛中的作用及其分子机制.方法:使用C57BL/6小鼠足底注射福尔马林诱导急性炎性疼痛模型,随机分为假手术(sham)组、疼痛模型组、P2Y6受体激动剂尿苷二磷酸(uridine diphosphate,UDP)组和抑制剂MRS2578组,每组15只;P2Y6敲除与P2Y6野生型的C57BL/6小鼠,每组15只;构建P2Y6-/-:CX3CR1GFP/+与P2Y6+/+:CX3CR1GFP/+基因型的C57BL/6小鼠,每组9只.小胶质细胞系BV2细胞分别使用P2Y6受体激动剂及抑制剂处理,分为对照组、UDP组、UDP+MRS2578组及MRS2578组.通过疼痛行为学分析、双光子活体成像技术动态观察脊髓背角小胶质细胞的分支运动、免疫荧光染色和Western blot技术检测小胶质细胞密度、炎症因子白细胞介素1β(interleukin-1β,IL-1β)的表达以及NLRP3-caspase-1信号通路关键分子的变化.结果:激活P2Y6受体加剧福尔马林诱导的小鼠疼痛行为,抑制或敲除该受体则显著缓解疼痛(P<0.05);双光子成像显示P2Y6敲除有效抑制福尔马林引发的脊髓小胶质细胞分支动态增强(P<0.05);在疼痛发生24 h后,P2Y6敲除显著降低脊髓背角小胶质细胞的聚集密度(P<0.01);在体及离体实验均证实,P2Y6基因缺失或药理抑制能够显著抑制核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)炎症小体活化及caspase-1的表达,并同时降低小胶质细胞中IL-1β的释放(P<0.05).结论:在小鼠急性炎性疼痛模型中,P2Y6受体能够调控小胶质细胞分支的动态运动,并增加其在脊髓背角部位的密度,同时激活NLRP3-caspase-1-IL-1β信号通路,从而参与福尔马林诱导的小鼠炎性疼痛在急性期的形成与发展.
AIM:To investigate the role of the microglial P2Y6 receptor in formalin-induced acute inflamma-tory pain and its underlying molecular mechanisms in mice.METHODS:An acute inflammatory pain model was estab-lished by plantar injection of formalin in C57BL/6 mice.The animals were randomly divided into the following groups:the sham group,pain model group,P2Y6 receptor agonist uridine diphosphate(UDP)group,and P2Y6 receptor inhibitor(MRS2578)group,with 15 mice in each group.Additionally,P2Y6-/-and P2Y6+/+C57BL/6 mice were used,with 15 mice in each group.Transgenic C57BL/6 mice with P2Y6-/-:CX3CR1GFP/+and P2Y6+/+:CX3CR1GFP/+genotypes were con-structed,with 9 mice in each group.BV2 microglia were divided into 4 groups:control,UDP,UDP+MRS2578,and MRS2578.Pain-related behaviours were analysed,and two-photon in vivo imaging was employed to dynamically observe microglial branch motility in the spinal dorsal horn.Immunofluorescence staining and Western blot were used to assess mi-croglial density,the expression of the inflammatory cytokine interleukin-1β(IL-1β),and changes in key molecules of the nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)-caspase-1 signaling pathway.RESULTS:Pharmacological activation of the P2Y6 receptor exacerbated formalin-induced pain behaviours,whereas its inhibition or genetic deletion significantly alleviated pain in mice(P<0.05).Two-photon imaging revealed that P2Y6 knockout effec-tively suppressed the enhanced microglial process dynamics in the spinal cord induced by formalin(P<0.05).At 24 hours post-pain induction,P2Y6 knockout significantly reduced the density of accumulated microglia in the spinal dorsal horn(P<0.01).Both in vivo and in vitro experiments confirmed that P2Y6 gene deletion or pharmacological inhibition significant-ly suppressed NLRP3 inflammasome activity and caspase-1 activation,concurrently reducing the release of IL-1β from mi-croglia(P<0.05).CONCLUSION:In a mouse model of acute inflammatory pain,the P2Y6 receptor regulates the dy-namic motility of microglial branches and promotes their subsequent aggregation in the spinal dorsal horn.Moreover,it ac-tivates the NLRP3-caspase-1-IL-1β signaling pathway,thereby contributing to the development and maintenance of forma-lin-induced inflammatory pain during the acute phase.
陈洪懿;朱烨杰;余嘉慧;焦子墨;沈晓华;朱佳
嘉兴大学医学院微生物学与免疫学教研室,浙江 嘉兴 314001嘉兴大学医学院微生物学与免疫学教研室,浙江 嘉兴 314001嘉兴大学医学院微生物学与免疫学教研室,浙江 嘉兴 314001嘉兴大学医学院微生物学与免疫学教研室,浙江 嘉兴 314001嘉兴市中医医院检验与病理科,浙江 嘉兴 314001嘉兴大学医学院微生物学与免疫学教研室,浙江 嘉兴 314001
医药卫生
P2Y6受体急性炎性疼痛福尔马林小胶质细胞NLRP3-caspase-1-IL-1β信号通路
P2Y6 receptoracute inflammatory painformalinmicrogliaNLRP3/caspase-1/IL-1β signaling pathway
《中国病理生理杂志》 2026 (3)
487-496,10
嘉兴市科技计划项目(No.2025CGZ028)浙江省医药卫生科技计划项目(No.2025KY1628)嘉兴大学省级大学生创新训练计划项目(No.S202510354059)
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