温肾通督方通过减轻铁过载促进脊髓损伤修复  

Wenshen Tongdu Recipe Promotes Spinal Cord Injury Repairment by Reducing Iron Overload

作　　者：陈思娴 马勇[1,2,3] 石磊[1,2] 吴承杰 赵瑞华 郭杨[2,3] 潘娅岚 吴毛[3] 刘孟敏 CHEN Si-xian;MA Yong;SHI Lei;WU Cheng-jie;ZHAO Rui-hua;GUO Yang;Pan Ya-lan;WU Mao;LIU Meng-min(School of Chinese Medicine,School of Integrated Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing,210023;Laboratory of New Techniques of Restoration&Reconstruction,Institute of Traumatology&Orthopedics,Nanjing University of Chinese Medicine,Nanjing,210023;Jiangsu CM Clinical Innovation Center of Degenerative Bone&Joint Disease,Wuxi TCM Hospital Affiliated to Nanjing University of Chinese Medicine,Jiangsu,214071)

机构地区：[1]南京中医药大学中医学院·中西医结合学院,南京210023 [2]南京中医药大学骨伤修复与重建新技术实验室,南京210023 [3]南京中医药大学无锡附属医院,江苏省中医退行性骨关节病临床医学创新中心,江苏214071

出　　处：《中国中西医结合杂志》2024年第11期1339-1345,共7页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金面上项目(No.81973885,No.82174400);南京中医药大学自然科学基金青年项目(No.NZY81704100);江苏省高校自然科学基金项目(No.21KJB360006);江苏省中医退行性骨关节病临床医学创新中心资助项目(No.苏中医科教[2021] 4号);2022江苏省研究生科研与实践创新计划项目(No.KYCX22_1942);2023年江苏省研究生科研创新计划项目(No.KYCX23_2205,No.KYCX23_2207)。

摘　　要：目的 探究温肾通督方通过减轻脊髓损伤后铁过载现象促进微血管内皮细胞(MECs)吞噬和清理髓鞘碎片,进而改善脊髓损伤后炎症微环境的作用机制。方法 50只C57BL/6小鼠采用随机数字表法分为5组,即:假手术组、模型组、甲磺酸去铁胺(DFO)组、温肾通督方组、温肾通督方+DFO组,每组各10只。采用改良Allen's法制备脊髓损伤小鼠模型,通过HE染色和尼氏染色观察脊髓的病理改变;采用铁离子比色法检测试剂盒测定血清中铁离子含量;利用ELISA法检测脊髓匀浆和血清中单核细胞趋化蛋白-1(MCP-1)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的表达水平;通过3D激光共聚焦观察脊髓中MECs吞噬和清理髓鞘碎片情况。结果 实验过程中模型组、DFO组及温肾通督方组各死亡1只。与模型组比较,HE染色结果表明,DFO组和温肾通督方组能够减少脊髓空洞和神经纤维排列紊乱现象;尼氏染色分析显示,DFO组和温肾通督方组可改善尼氏体形态和神经元固缩现象;铁离子比色法检测发现,DFO组和温肾通督方组小鼠血清中铁离子浓度显著减少(P<0.01);ELISA结果显示,与假手术组比较,模型组血清及脊髓组织匀浆中MCP-1、IL-6、IL-1β和TNF-α浓度含量均显著上升(P<0.01)。与模型组比较,DFO、温肾通督方组能够显著降低血清中MCP-1、IL-6、IL-1β、TNF-α浓度(P<0.05,P<0.01);温肾通督方+DFO组可显著降低血清中MCP-1和IL-1β浓度(P<0.01)。与温肾通督方组比较,温肾通督方+DFO组显著提高MCP-1、IL-6及IL-1β的浓度(P<0.01)。与模型组比较,DFO、温肾通督方、温肾通督方+DFO组能够显著降低脊髓组织中MCP-1、IL-6、IL-1β(除温肾通督方组)和TNF-α浓度(P<0.05,P<0.01);3D激光共聚焦结果表明,DFO组和温肾通督方组可促进MECs吞噬和清理髓鞘碎片。结论 温肾通督方可能通过减轻铁过载促进MECs吞噬髓鞘碎片,改善炎性微环境,促Objective To study the action mechanism of Wenshen Tongdu Recipe(WSTDR)in promoting microvascular endothelial cells(MECs)phagocytosis and clearing myelin debris by reducing iron overload phenomenon after spinal cord injury,thereby improving the infiammatory microenvironment after spinal cord injury.Methods Fifty C57BL/6 mice were divided into 5 groups by random number table method:shamoperation group,model group,deferoxamine(DFO)group,WSTDR group,WSTDR+DFO group,10 mice in each group.A mouse model of spinal cord injury was prepared by modified Allen's method.The pathological changes of the spinal cord were observed by HE staining and Nissl staining.The iron ion content in serum was determined by the iron ion colorimetric assay kit.The expression levels of monocyte chemoattractant protein-1(MCP-1),interleukin(IL)-6,IL-1β,and tumor necrosis factor-α(TNF-α)in the homogenate and serum of the spinal cord were detected by ELISA.The phagocytosis and clearance of myelin debris by MECs in the spinal cord were observed by 3D laser confocal.Results One mouse died in the model group,DFO group,and WSTDR group,respectively.The results of HE staining showed that compared with the model group,spinal cord cavities were reduced with nerve fiber disorder in DFO group and WSTDR group.Nissl staining analysis showed that compared with the model group,Nissl morphology and neuronal pyknosis were improved in DFO group and WSTDR group.Ferric ion colorimetric assay showed that compared with the model group,serum iron ion concentration were significantly reduced in DFO group and WSTDR group(P<0.01).ELISA results showed that compared with the sham-operation group,the levels of MCP-1,IL-6,IL-1β,and TNF-αin serum and spinal cord tissues significantly increased in the model group(P<0.01).Compared with the model group,serum levels of MCP-1,IL-6,IL-1β,and TNF-αwere significantly lowered in DFO group and WSTDR group(P<0.05,P<0.01).Serum concentrations of MCP-1 and IL-1βwere significantly lowered in WSTDR+DFO group(P<0.01).Compared with WST

关 键 词：脊髓损伤 温肾通督方 微血管内皮细胞 髓鞘碎片 铁过载 中药复方 

分 类 号：R269[医药卫生—中西医结合]