摘要
背景:吲哚丙酸被证明可减轻糖尿病所致的中枢神经系统炎症,但其能否抑制小胶质细胞M1极化治疗脊髓损伤,目前仍缺乏相关研究。目的:通过细胞实验及动物实验探究吲哚丙酸抑制小胶质细胞M1极化治疗脊髓损伤的机制。方法:①体外实验:CCK8法检测BV2细胞活性并筛选最佳的吲哚丙酸使用浓度;然后将BV2细胞分为对照组、单纯给药组(50μmol/L吲哚丙酸)、脂多糖组(100 ng/mL脂多糖)、治疗组(100 ng/mL脂多糖+50μmol/L吲哚丙酸),采用Griess法检测一氧化氮含量;实时荧光定量PCR、Western Blot检测促炎相关因子的mRNA和蛋白的表达;细胞免疫荧光染色检测诱导型一氧化氮合酶的表达;Seahorse实验检测BV2细胞糖酵解压力水平。②体内实验:将30只SD大鼠随机分成3组:假手术组、脊髓损伤组、吲哚丙酸组。采用BBB评分与斜板实验评估大鼠脊髓损伤后功能恢复情况;免疫荧光染色检测脊髓组织中小胶质细胞诱导型一氧化氮合酶的表达情况;ELISA检测脊髓组织中促炎因子白细胞介素1β及肿瘤坏死因子α蛋白表达水平。结果与结论:①体外实验:当吲哚丙酸浓度>50μmol/L时明显抑制BV2细胞活性;吲哚丙酸可通过抑制核因子κB信号通路的激活,进而抑制促炎因子(白细胞介素1β、肿瘤坏死因子α)及BV2细胞M1极化标志物诱导型一氧化氮合酶的mRNA及蛋白表达水平;吲哚丙酸能明显降低脂多糖诱导BV2细胞的糖酵解水平。②体内实验:脊髓损伤大鼠给予吲哚丙酸干预后,BBB评分与斜板实验角度明显增加;脊髓组织中M1极化小胶质细胞的数量显著下降及促炎因子(白细胞介素1β、肿瘤坏死因子α)蛋白表达水平明显降低。③结果说明,吲哚丙酸可通过抑制小胶质细胞M1极化改善炎症微环境促进脊髓损伤后功能恢复。
BACKGROUND:Indolepropionic acid has been shown to reduce diabetes-induced central nervous system inflammation.However,there is a lack of research on whether to inhibit microglia M1 polarization for the treatment of spinal cord injury.OBJECTIVE:To investigate the mechanism of indolepropionic acid inhibition of microglial cell M1 polarization for the treatment of spinal cord injury through cell and animal experiments.METHODS:(1)In vitro experiments:BV2 cell viability was assessed using the CCK-8 assay to determine optimal concentrations of indolepropionic acid.Subsequently,BV2 cells were categorized into control group,administration group(50μmol/L indolepropionic acid),lipopolysaccharide group(100 ng/mL lipopolysaccharide),and treatment group(100 ng/mL lipopolysaccharide+50μmol/L indolepropionic acid).Nitric oxide content was quantified using the Griess method.Real-time quantitative PCR and western blot assay were employed to measure mRNA and protein levels of pro-inflammatory factors.Cell immunofluorescence staining was conducted to assess inducible nitric oxide synthase expression.The Seahorse assay was employed to assess glycolytic stress levels in BV2 cells.(2)In vivo experiments:30 SD rats were randomly divided into three groups:sham surgery group,spinal cord injury group,and indolepropionic acid group.Motor function recovery in rats after spinal cord injury was assessed using BBB scoring and the inclined plane test.Immunofluorescence staining of spinal cord tissue was conducted to evaluate the expression of inducible nitric oxide synthase in microglial cells.ELISA was employed to measure protein expression levels of the pro-inflammatory cytokines interleukin-1βand tumor necrosis factor-αin spinal cord tissue.RESULTS AND CONCLUSION:(1)In vitro experiments:Indolepropionic acid exhibited significant suppression of BV2 cell viability when its concentration exceeded 50μmol/L.Indolepropionic acid achieved this by inhibiting the activation of the nuclear factorκB signaling pathway,thereby suppressing the mRNA and protein expression levels of pro-inflammatory cytokines(interleukin-1βand tumor necrosis factor-α),as well as the M1 polarization marker,inducible nitric oxide synthase,in BV2 cells.Additionally,indolepropionic acid notably reduced the glycolytic level in BV2 cells induced by lipopolysaccharides.(2)In vivo experiments:Following indolepropionic acid intervention in spinal cord injury rats,there was a noticeable increase in BBB scores and the inclined plane test angle.There was also a significant decrease in the number of M1-polarized microglial cells in spinal cord tissue,accompanied by a marked reduction in the protein expression levels of pro-inflammatory cytokines(interleukin-1βand tumor necrosis factor-α).(3)These results conclude that indolepropionic acid promotes functional recovery after spinal cord injury by improving the inflammatory microenvironment through inhibition of microglia M1 polarization.
作者
滕益霖
席德双
冯彦斌
梁宇
邓豪
曾高峰
宗少晖
Teng Yilin;Xi Deshuang;Feng Yanbin;Liang Yu;Deng Hao;Zeng Gaofeng;Zong Shaohui(Department of Spine Surgery,First Affiliated Hospital of Guangxi Medical University,Nanning 530000,Guangxi Zhuang Autonomous Region,China;Department of Spine Surgery,Third Affiliated Hospital of Guangxi Medical University,Nanning 530000,Guangxi Zhuang Autonomous Region,China;Collaborative Innovation Center of Regenerative Medicine and Medical Bioresource Development and Application Co-constructed by the Province and Ministry,Guangxi Medical University,Nanning 530000,Guangxi Zhuang Autonomous Region,China;School of Public Health,Guangxi Medical University,Nanning 530000,Guangxi Zhuang Autonomous Region,China)
出处
《中国组织工程研究》
CAS
北大核心
2024年第31期5010-5016,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金项目(82060399),项目负责人:宗少晖
广西自然科学基金项目(2023GXNSFDA026050),项目负责人:宗少晖
广西医学高层次骨干人才培养“139”计划,项目负责人:宗少晖。
关键词
吲哚丙酸
脊髓损伤
小胶质细胞
M1极化
促炎因子
信号通路
indolepropionic acid
spinal cord injury
microglial cell
M1 polarization
pro-inflammatory factor
signaling pathway
