摘要
目的观察和探讨鞘内注射miR-125b mimic对大鼠脊髓缺血再灌注损伤(SCIRI)后小胶质细胞活化和神经炎症反应的影响及机制。方法120只SD大鼠随机分为4组:假手术组(Sham组,鞘内注射生理盐水)、损伤组(IR组,鞘内注射生理盐水)、mimic组(IR+鞘内注射miR-125b mimic)和control组(IR+鞘内注射miR-125b control)。Sham组仅开胸暴露主动脉弓而不夹闭,其余各组使用动脉夹夹闭主动脉弓14 min,再移除动脉夹制备SCIRI模型。各组大鼠术前给予连续3 d鞘内注射,每天1次。Iba-1作为小胶质细胞特异性标记物,损伤后48 h采用免疫荧光法观察小胶质细胞的形态并计数活化的细胞数量;双免疫荧光观察Iba-1和NOD样受体pyrin结构域含3(NLRP3)在细胞的分布。Western blot法测定脊髓组织中Iba-1、NLRP3、cleaved caspase-1蛋白含量;ELISA法测定炎症因子IL-1β含量。结果损伤后48 h,Sham组脊髓背角内小胶质细胞多为分支样形态,未见高Iba-1荧光信号表达;IR组和control组可见大量小胶质细胞形态为变形虫样,且高Iba-1荧光信号的细胞数量明显增加;而mimic组中小胶质细胞形态为分支样和变形虫样混杂,高Iba-1荧光信号的细胞数量较IR组明显降低(P<0.05)。双免疫荧光法显示NLRP3主要分布于高Iba-1荧光信号的小胶质细胞上。与Sham组相比,IR组脊髓组织中Iba-1、NLRP3、cleaved caspase-1和IL-1β蛋白表达均明显增加(P<0.05)。与IR组相比,mimic组明显降低脊髓中Iba-1、NLRP3、cleaved caspase-1和IL-1β蛋白表达(P<0.05)。而control组与IR组的上述指标差异均无统计学意义(P>0.05)。结论鞘内注射miR-125b mimic通过抑制SCIRI引起的小胶质细胞活化,进而减少脊髓组织中NLRP3炎症体表达,发挥有效的抗炎作用。
Objective To observe and investigate the effect and mechanism of intrathecal injecting miR-125b mimic on regulating microglial and its-mediated NOD-like receptor protein 3(NLRP3)inflammasome activation after spinal cord ischemia reperfusion injury(SCIRI)in rats.Methods 120 Sprague-Dawley(SD)rats were randomly divided into four groups:sham group(sham group,intrathecal injection of normal saline),injury group(IR group,intrathecal injection of normal saline),mimic group(IR+intrathecal injection of miR-125b mimic)and control group(IR+intrathecal injection of miR-125b control).In sham group,the aortic arch was exposed without clamping.In other groups,the aortic arch was clamped for 14 minutes,and then the artery clamp was removed to prepare SCIRI model.Rats in each group were given intrathecal injection for 3 consecutive days before operation,once a day.Immunofluorescence was used to observe the morphology of microglia and count the number of activated cells 48 hours after injury;double immunofluorescence was used to observe the distribution of Iba-1 and NLRP3 in cells.The protein expression of Iba-1,NLRP3 and cleaved caspase-1 in spinal cord were determined by Western blot,and the inflammatory factor IL-1βwas determined by enzyme linked immunosorbent assay(ELISA).Results The activated microglias exhibited the amoeba morphology and increased Iba-1 fluorescence signal in immunofluorescence.The microglias in spinal dorsal horn of Sham group were all branched-like morphology and absent of the increased Iba-1 signal at 48 h post-IR.And the microglias in IR and control groups was all amoeba-like morphology with obviously increased number and intensity of Iba-1 signal,whereas in mimic group,the microglias were mixed morphology with the less increased number and intensity of Iba-1 signal(P<0.05).And NLRP3 signals were mainly distributed in microglias with increased Iba-1 signal.Meantime,compared with Sham group,the protein expression of Iba-1,NLRP3,cleaved caspase-1 and IL-1βwere significantly increased in IR group(P<0.05)
作者
王赫
陈凤收
马虹
Wang He;Chen Fengshou;Ma Hong(Department of Anesthesiology,the First Affiliated Hospital of China Medical University,Shenyang 110001,China)
出处
《中国医师杂志》
CAS
2021年第4期553-557,共5页
Journal of Chinese Physician
