红景天苷调控lncRNA HOTAIR/NF-κB促进脊髓损伤后运动功能机制研究
Study on the mechanism of salidroside regulating lncRNA HOTAIR/NF-κB to promote motor function after spinal cord injury
谭秀伟 1巫艳岚 1李俸鑫 1卢军良 1杨佰辉 1苏义基1
作者信息
- 1. 广西医科大学第一附属医院,南宁 530021
- 折叠
摘要
目的:探讨红景天苷(SAL)调节长链非编码核糖核酸(lncRNA)HOX转录反义RNA(HOTAIR)对脊髓损伤(SCI)后运动功能的影响.方法:将27只SD大鼠随机分为假手术组、SCI组和SCI+SAL组.采用改良Allen法建立SCI大鼠模型,BBB评分检测大鼠后肢运动功能变化,苏木精—伊红染色(HE)及尼氏染色观察脊髓组织结构及神经元数量,实时荧光定量PCR(RT-qPCR)法测定HOTAIR和相关炎症因子表达情况.在体外构建lncRNA HOTAIR过表达星形胶质细胞,随后将构建的细胞和原代星形胶质细胞各分为8组,即control组、不同浓度SAL组(6.25 μg/mL、12.5 μg/mL、25 μg/mL、50 μg/mL、100 μg/mL、200 μg/mL、400 μg/mL),采用细胞计数试剂盒(CCK-8法)检测细胞存活率.利用脂多糖(LPS)构建继发性SCI炎症模型并给予不同浓度SAL进行干预,RT-qPCR法检测lncRNA HOTAIR、IL-6和TNF-α基因表达,蛋白质免疫印迹法(western blotting)以及酶联免疫吸附试验(ELISA)分别测定NF-κB通路和炎症因子蛋白表达情况.结果:SAL干预明显提高了SCI大鼠的BBB评分,减少组织结构破坏以及神经元损伤并下调SCI后HOTAIR和炎症因子(TNF-α、IL-6)的表达.在体外实验中与control组相比,SAL各剂量组星形胶质细胞的生存率均无显著变化;进一步研究发现SAL能够调控lncRNA HOTAIR降低LPS刺激的星形胶质细胞炎症因子的表达;同时SAL可以靶向lncRNA HOTAIR下调p-NF-κB p65、p-IκB-α、IKKβ蛋白和炎症因子的表达.结论:SAL能够促进SCI大鼠运动功能恢复,其机制可能与减轻炎症反应,抑制lncRNA HOTAIR的表达有关.
Abstract
Objective:To investigate the effect of salidroside(SAL)regulating long non-coding RNA HOX tran-script antisense RNA(lncRNA HOTAIR)on motor function after spinal cord injury(SCI).Methods:Twenty-sev-en SD rats were randomly divided into sham surgery group,SCI group,and SCI+SAL group.A rat model of SCI was established using the modified Allen's method.BBB scoring was used to assess the motor function of the rats'hind limbs.Hematoxylin-eosin(HE)staining and Nissl staining were used to observe the spinal cord tissue structure and the number of neurons.The expression of HOTAIRand related inflammatory factors was deter-mined by reverse transcription-quantitative PCR(RT-qPCR).The lncRNA HOTAIR overexpressed astrocytes were cultured in vitro,and then divided into 8 groups along with primary astrocytes:control group and SAL group with different concentrations(6.25 μg/mL,12.5 μg/mL,25 μg/mL,50 μg/mL,100 μg/mL,200 μg/mL and 400 μg/mL).The cell survival rate was assessed using the cell counting kit-8(CCK-8)method.The model of sec-ondary SCI inflammation was established using Lipopolysaccharide(LPS),and SAL was administered at varying concentrations.The gene expression of lncRNA HOTAIR,IL-6,TNF-α was detected by RT-qPCR,and the pro-tein expression of NF-κB pathway and inflammatory factors was determined by western blotting and enzyme-linked immunosorbent assay(ELISA),respectively.Results:The intervention of SAL significantly enhanced the BBB score in SCI rats,mitigated tissue structure destruction and neuronal damage,and down-regulated the ex-pression of HOTAIRand inflammatory factors(TNF-α,IL-6)following SCI.In vitrostudies revealed no signifi-cant alteration in astrocyte survival rates across different doses groups of SAL compared with the control group.Further investigations demonstrated that SAL could modulate lncRNA HOTAIR to reduce the expression of in-flammatory factors in LPS-stimulated astrocytes,while also targeting lncRNA HOTAIR to down-regulate the ex-pression of p-NF-κB p65,p-IκB-α,IKKβ proteins and inflammatory factors.Conclusion:SAL can promote the recovery of motor function in SCI rats,and its mechanism may be related to reducing inflammation and inhibit-ing the expression of lncRNA HOTAIR.
关键词
红景天苷/HOX转录反义RNA/炎症/脊髓损伤Key words
salidroside/HOX transcript antisense RNA/inflammation/spinal cord injury引用本文复制引用
基金项目
国家自然科学基金资助项目(81960773)
广西自然科学基金资助项目(2023GXNSFAA026149)
广西医科大学"四新"立项项目(SX202228)
广西研究生教育创新计划资助项目(JGY2022075)
出版年
2024
国家科技期刊平台
NSTL
万方数据