基于FKN/CX3CR1信号通路的壮骨止痛解郁方抗类风湿关节炎伴抑郁症作用机制研究  

Study on Mechanism of Zhuanggu Zhitong Jieyu Prescription Against Rheumatoid Arthritis Accompanied by Depression Based on FKN/CX3CR1 Signaling Pathway

作　　者：刘检[1] 蔺晓源[1] 赵洪庆[2,3] 杨蕙[1] 周梓洋 刘平安 王宇红[2,3,4] LIU Jian;LIN Xiaoyuan;ZHAO Hongqing;YANG Hui;ZHOU Ziyang;LIU Ping'an;WANG Yuhong(The First Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Institute of Innovation and Applied Research,Hunan University of Chinese Medicine,Changsha 410208,China;Hunan University of Chinese Medicine,Changsha 410208,China;Hunan Key Laboratory of Traditional Chinese Medicine Prevention&Treatment of Depressive Diseases,Changsha 410208,China)

机构地区：[1]湖南中医药大学第一附属医院,湖南长沙410007 [2]湖南中医药大学科技创新中心,湖南长沙410208 [3]湖南中医药大学,湖南长沙410208 [4]抑郁类疾病中医药防治湖南省重点实验室,湖南长沙410208

出　　处：《中国中医药信息杂志》2023年第1期96-102,共7页Chinese Journal of Information on Traditional Chinese Medicine

基　　金：国家自然科学基金(82104793、82104836);湖南省发展和改革委创新研发项目(201930);湖南省中医药科研计划项目(202019、2021181);湖南省教育厅项目(19K066);湖南省中药粉体与创新药物省部共建国家重点实验室培育基地开放基金(21PTKF1008);湖南中医药大学中医学一流学科开放基金重点项目(2022ZYX16)。

摘　　要：目的基于FKN/CX3CR1信号通路研究壮骨止痛解郁方抗类风湿关节炎伴抑郁症(RAD)的作用机制。方法诱导滑膜细胞炎症模型并建立脑微血管内皮细胞、小胶质细胞及海马神经元共培养体系,将细胞分为正常组、模型组、CX3CR1阻断剂组和壮骨止痛解郁方组,分别予相应处理。采用细胞成像分析系统观察滑膜细胞、脑微血管内皮细胞、小胶质细胞及海马神经元形态变化;ELISA检测滑膜细胞上清液肿瘤坏死因子-α(TNF-α)、类风湿因子(RF)、趋化因子Fractalkine(FKN)含量;电阻仪测量跨内皮细胞电阻(TEER);免疫荧光染色检测脑微血管内皮细胞闭合蛋白(Occludin)、闭锁小带蛋白1(ZO-1),小胶质细胞趋化因子C-X3-C-基元受体1(CX3CR1)、腺苷A2A受体(A2AR),海马神经元谷氨酸受体2A(NR2A)、谷氨酸受体2B(NR2B)蛋白表达;尼氏染色观察海马神经元突触可塑性变化。结果与正常组比较,模型组滑膜细胞、脑微血管内皮细胞、小胶质细胞及海马神经元明显损伤;滑膜细胞上清液TNF-α、RF、FKN含量明显增加(P<0.01,P<0.05);脑微血管内皮细胞TEER明显减少(P<0.01),Occludin、ZO-1蛋白表达明显降低(P<0.01);小胶质细胞CX3CR1、A2AR蛋白表达明显升高(P<0.01);海马神经元NR2A、NR2B蛋白表达明显升高(P<0.05,P<0.01),尼氏小体、树突和树突棘分支数量减少,海马神经元网络明显断裂。与模型组比较,壮骨止痛解郁方组滑膜细胞、脑微血管内皮细胞、小胶质细胞及海马神经元损伤均有所减轻;滑膜细胞上清液TNF-α、RF、FKN含量明显减少(P<0.01,P<0.05);脑微血管内皮细胞TEER明显增加(P<0.01),Occludin、ZO-1蛋白表达明显升高(P<0.01);小胶质细胞CX3CR1、A2AR蛋白表达明显降低(P<0.01,P<0.05);海马神经元NR2A、NR2B蛋白表达明显降低(P<0.01),海马神经元尼氏小体、树突和树突棘分支数量减少且神经网络断裂情况明显改善。结论壮骨止痛解郁方对体外模拟的RAD�Objective To investigate the mechanism of Zhuanggu Zhitong Jieyu Prescription against rheumatoid arthritis accompanied by depression(RAD)based on FKN/CX3CR1 signaling pathway.Methods The inflammation model of synovial cells was induced,the co-culture system of cerebral microvascular endothelial cells,microglia and hippocampal neurons in vitro were established.The cultured cells were randomly divided into normal group,model group,CX3CR1 blocker group,and Zhuanggu Zhitong Jieyu Prescription group,and received relevant administration.The cell morphology of synovial cells,cerebral microvascular endothelial cells,microglia and hippocampal neurons were ovserved by cell imaging analysis;the levels of TNF-α,RF and FKN were detected by ELISA;the transendothelial electric resistance(TEER)was detected by resistance meter;the proteins expression of Occludin,ZO-1,CX3CR1,A2AR,NR2A and NR2B were detected by immunofluorescent staining;the synaptic plasticity of hippocampus neurons was detected by Nissl’s staining.Results Compared with the normal group,the synovial cells,cerebral microvascular endothelial cells,microglia and hippocampal neurons were significantly damaged in the model group;the contents of TNF-α,RF and FKN in the supernatant of synovial cells significantly increased(P<0.01,P<0.05);the TEER of cerebral microvascular endothelial cells significantly reduced(P<0.01),and the proteins expression of Occludin and ZO-1 significantly decreased(P<0.01);the proteins expression of CX3CR1 and A2AR in microglia significantly increased(P<0.01);the expressions of NR2A and NR2B protein in hippocampal neurons significantly increased(P<0.05,P<0.01),the number of Nissl bodies,dendrites and dendritic spine branches decreased,and the hippocampal neuronal network was significantly broken.Compared with the model group,the damages of synovial cells,brain microvascular endothelial cells,microglia and hippocampal neurons in Zhuanggu Zhitong Jieyu Prescription group were alleviated;the contents of TNF-α,RF and FKN in the supernatant of s

关 键 词：壮骨止痛解郁方 类风湿关节炎伴抑郁症 趋化因子FRACTALKINE C-X3-C-基元受体1 突触可塑性 

分 类 号：R285.5[医药卫生—中药学]