米诺环素对脊神经结扎大鼠脊髓内M1/M2型小胶质细胞极化的影响  

Effect of minocycline on polarization of types M1/M2 microglia in spinal cord in rats after spinal nerve ligation

作　　者：成志红 冯松 王霞 倪锐男 郭洋[1] 向宇[1] 杨正伟[1] 彭彬[1] CHENG Zhihong;FENG Song;WANG Xia;NI Ruinan;GUO Yang;XIANG Yu;YANG Zhengwei;PENG Bin(Institute of Basic Medicine and Forensic Medicine,North Sichuan Medical College,Nanchong,Sichuan Province,637000;Department of Neurosurgery,Dazhou Third People’s Hospital,Dazhou,Sichuan Province,635000,China;Faculty of Clinical Medicine,North Sichuan Medical College,Nanchong,Sichuan Province,637000)

机构地区：[1]川北医学院基础医学与法医学研究所,四川南充637000 [2]达州市第三人民医院神经外科,四川达州635000 [3]川北医学院临床医学院,四川南充637000

出　　处：《陆军军医大学学报》2024年第15期1740-1750,共11页Journal of Army Medical University

基　　金：南充市科学技术局博士、硕士学位授权点和“一流学科”建设专项(20SXQT0304);四川省大学生创新创业训练计划项目(S202310634104)。

摘　　要：目的探讨米诺环素(minocycline,Mino)对脊神经结扎(spinal nerve ligation,SNL)致神经病理性疼痛(neuropathic pain,NP)大鼠脊髓内M1型(促炎型)和M2型(抗炎型)小胶质细胞极化的影响及其机制。方法36只成年雄性SD大鼠按体质量分层随机抽样分为Sham组、SNL组和M+SNL组(鞘内注射米诺环素),于术前1 d和术后14 d内测定大鼠后足机械痛阈值和冷痛敏阈值。术后14 d于L5脊髓取材,采用免疫组化技术和体视学技术检测L5脊髓背角内小胶质细胞的总数以及M1/M2型小胶质细胞的数量;利用生物信息学技术筛选出脊髓小胶质细胞内的核心靶点Cst7,并采用Western blot检测小胶质细胞标志物Iba-1、M1型小胶质细胞标志物iNOS、M2型小胶质细胞标志物CD206、Cst7基因编码的蛋白质胱抑素F(cystatin F,CF)及CatS/CX3CL1/CX3CR1通路蛋白的表达水平;采用ELISA检测脊髓内TNF-α、IL-6和IL-10的蛋白表达量。结果M+SNL组大鼠术后7~14 d手术侧的机械痛和冷痛敏阈值均比SNL组显著升高(P<0.01)。SNL组大鼠手术侧脊髓背角内小胶质细胞的总数,M1型、M2型小胶质细胞的数量以及脊髓CatS、CX3CL1、CX3CR1、TNF-α、IL-6和IL-10蛋白表达较Sham组均显著增加,而CF含量显著降低(P<0.01);与SNL组比较,M+SNL组小胶质细胞的总数、M1型小胶质细胞的数量以及CatS、CX3CL1、CX3CR1、TNF-α、IL-6蛋白表达均显著减少,而M2型小胶质细胞的数量以及CF、IL-10的蛋白表达均显著增加(P<0.05)。结论米诺环素改善SNL致神经病理性疼痛的机制可能是通过上调小胶质细胞内CF蛋白表达,进而抑制CatS/CX3CL1/CX3CR1信号通路,促进小胶质细胞由M1型向M2型转化,以平衡M1/M2小胶质细胞极化失衡、减轻神经炎症。Objective To investigate the effect of minocycline(Mino)on the polarization of types M1/M2 microglia(pro-and anti-inflammatory type)in the spinal dorsal horn of rats with neuropathic pain(NP)induced by spinal nerve ligation(SNL)and its underlying mechanism.Methods A total of 36 adult male SD rats were randomly stratified into Sham-operation(Sham)group,SNL group and Mino+SNL group by stratified random sampling based on body weight.Mechanical pain threshold and cold nociceptive thresholds of rat hind paw were measured in 1 d before and 14 d after modelling.Spinal cord tissue at the lumbar 5(L5)segment was taken at 14 d after modelling,and the total number of microglia as well as the numbers of M1 and M2 microglia in the spinal dorsal horn were measured with immunohistochemistry and stereology.With aid of bioinformatics techniques,the core target in the spinal cord,Cst7,was selected.Then,the protein levels of microglia marker Iba-1,M1 microglia marker iNOS,M2 microglia marker CD206,Cst7 encoded protein cystatin F(CF)and pathway CatS/CX3CL1/CX3CR1 were detected with Western blotting.The expression levels of TNF-α,IL-6 and IL-10 in the spinal cord tissues were measured with ELISA.Results The mechanical pain and cold nociceptive thresholds were both significantly higher in the M+SNL group than the SNL group at 7~14 d after modelling(P<0.01).The total number of microglia and the numbers of M1/M2 microglia in the spinal dorsal horn as well as the expression levels of CatS,CX3CL1,CX3CR1,TNF-α,IL-6,and IL-10 in the spinal cord tissues were obviously increased,and the expression level of CF was notably decreased in the SNL model group than the Sham group(P<0.01).While,Mino treatment remarkably reversed above phenomena,with decreased total number of microglia and number of M1 microglia as well as expression levels of CatS,CX3CL1,CX3CR1,TNF-αand IL-6,and increased number of M2 microglia as well as CF and IL-10 levels when compared with the SNL group(P<0.05).Conclusion Mino alleviates SNL induced neuropathic pain,probably t

关 键 词：米诺环素 胱抑素F 小胶质细胞 脊神经结扎 神经病理性疼痛 体视学 大鼠 

分 类 号：R322.81[医药卫生—人体解剖和组织胚胎学] R441.1[医药卫生—基础医学] R978.14