脂肪酸结合蛋白4促进创伤性脑损伤后小胶质细胞诱导的炎症反应  

Fatty Acid Binding Protein 4 Promotes Microglia-induced Inflammatory Response After Traumatic Brain Injury

作　　者：张晓亚[1] 王昆鹏[2] 李爽[3] 郑保平 孙震 王海均 Zhang Xiaoya;Wang Kunpeng;Li Shuang(Department of Magnetic Resonance Imaging Diagnosis,Henan Province,Nanyang 473000,China;Department of Neurosurgery,Nanyang Central Hospital,Henan Province,Nanyang 473000,China;Department of Neurosurgery,Unrion Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,China)

机构地区：[1]河南省南阳市中心医院磁共振影像诊断科,南阳473000 [2]河南省南阳市中心医院神经外科,南阳473000 [3]华中科技大学同济医学院附属协和医院神经外科,武汉430022

出　　处：《华中科技大学学报(医学版)》2025年第2期180-189,共10页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：湖北省自然科学基金面上项目(No.2021CFB549)。

摘　　要：目的探讨脂肪酸结合蛋白4(Fabp4)在调控创伤性脑损伤(TBI)后神经炎症中的作用机制。方法将成年野生型C57雄性小鼠按随机数字表法随机分为假手术组和TBI组,Fabp4基因敲除(Fabp4^(-/-))雄性小鼠随机分为假手术组和TBI组,每组6只。通过Western blot检测各组脑组织Fabp4、IL-1β、TNF-α、NLRP3、Caspase-1和ASC蛋白的表达;采用干湿重量比法测定脑含水量;尼氏染色评估神经损伤;组织免疫荧光双染检测小胶质细胞M1极化标志物(CD86和iNOS)、M2极化标志物(CD206)以及星形胶质细胞极化标志物(C3)的表达;酶联免疫吸附实验(ELISA)检测BV2细胞培养上清中促炎因子IL-1β和TNF-α的水平;MitoSOX Red荧光染料评估各组BV2细胞线粒体ROS水平;ELISA检测TBI后脑组织中丙二醛和8-羟基脱氧鸟苷含量;免疫荧光检测BV2细胞中NLRP3蛋白表达情况。结果在野生型小鼠中,与假手术组相比,TBI组损伤部位脑组织中Fabp4蛋白表达在损伤后12 h开始显著升高(P<0.01),并在3 d时达到高峰(P<0.01)。与野生型TBI组相比,Fabp4^(-/-)小鼠TBI组的IL-1β和TNF-α蛋白水平显著降低(均P<0.01),脑含水量明显减少(P<0.05)。尼氏染色结果显示,Fabp4敲除显著减小了TBI后的脑损伤体积(P<0.05)。免疫荧光实验显示,Fabp4敲除抑制TBI后小胶质细胞M1极化,促进M2极化,但对星形胶质细胞极化无显著影响。细胞实验表明,Fabp4过表达显著增加BV2细胞培养上清中IL-1β和TNF-α的含量(均P<0.01)及线粒体ROS生成(P<0.01)。LPS刺激BV2细胞后Fabp4蛋白水平显著升高(P<0.01),而Fabp4抑制剂BMS309403干预可显著降低IL-1β和TNF-α水平(均P<0.05)及线粒体ROS生成(P<0.01)。在小鼠模型中,Fabp4^(-/-)小鼠TBI组的丙二醛和8-羟基脱氧鸟苷含量显著低于野生型TBI组(均P<0.01)。此外,Fabp4过表达导致BV2细胞中NLRP3、ASC和Caspase-1蛋白水平显著增加(均P<0.01),ROS清除剂可显著抑制上述蛋白的表达(P<0.05,P<0.01)。在小鼠�Objective To investigate the mechanism by which fatty acid-binding protein 4(Fabp4)regulates neuroinflammation after traumatic brain injury(TBI).Methods Adult wild-type C57 male mice were randomly divided into sham group and TBI group, with 6 mice in each group.Fabp4 knockout(Fabp4^(-/-))male mice were randomly divided into sham group and TBI group, with 6 mice in each group.Western blot was used to detect the expression levels of Fabp4,IL-1β,TNF-α,NLRP3,Caspase-1,and ASC proteins in brain tissue from each group.Brain water content was assessed using the wet-to-dry weight method, and neuronal damage was evaluated by Nissl staining.Immunofluorescence double staining was performed to detect the expression of microglial M1 polarization markers(CD86 and iNOS),M2 polarization marker(CD206),and astrocytic polarization marker(C3).Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of pro-inflammatory factors IL-1β and TNF-α in the culture supernatant of BV2 cells.MitoSOX red fluorescence staining was used to assess mitochondrial ROS levels in BV2 cells, and ELISA was employed to determine the levels of malondialdehyde(MDA)and 8-hydroxy-2′-deoxyguanosine(8-OHdG)in brain tissue after TBI.Immunofluorescence was performed to detect NLRP3 protein expression in BV2 cells.Results In wild-type mice, the expression of Fabp4 protein in the injured brain region was significantly increased at 12 hours post-TBI compared to the sham group(P<0.01)and peaked at 3 days post-injury(P<0.01).Compared to the wild-type TBI group, protein expression levels of IL-1β and TNF-α were reduced(P<0.01),and brain water content was decreased(P<0.05)in Fabp4^(-/-) mice from TBI group.Nissl staining revealed that the volume of brain damage following TBI was reduced after Fabp4 deletion(P<0.05).Immunofluorescence experiments showed that M1 polarization of microglia was inhibited after Fabp4 deletion(P<0.01),and M2 polarization was promoted(P<0.01).Fabp4 deletion had no significant effect on astrocytic polarization.BV2 cell expe

关 键 词：脂肪酸结合蛋白4 创伤性脑损伤 小胶质细胞 炎症反应 线粒体ROS NLRP3炎症小体 

分 类 号：R651.15[医药卫生—外科学]