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

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(6.25μg/mL、12.5μg/mL、25μg/mL、50μg/mL、100μg/mL、200μg/mL、400μg/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。

Objective:To investigate the effect of salidroside(SAL)regulating long non-coding RNA HOX transcript antisense RNA(lncRNA HOTAIR)on motor function after spinal cord injury(SCI).Methods:Twenty-seven 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 HOTAIR and related inflammatory factors was determined 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(6.25μg/mL,12.5μg/mL,25μg/mL,50μg/mL,100μg/mL,200μg/mL and 400μg/mL).The cell survival rate was assessed using the cell counting kit-8(CCK-8)method.The model of secondary SCI inflammation was established using Lipopolysaccharide(LPS),and SAL was administered at varying concentrations.The gene expression of lncRNA HOTAIR,IL-6,TNF-αwas detected by RT-qPCR,and the protein 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 expression of HOTAIR and inflammatory factors(TNF-α,IL-6)following SCI.In vitro studies revealed no significant 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 inflammatory factors in LPS-stimulated astrocytes,while also targeting lncRNA HOTAIR to down-regulate the expression of p-NF-κB p65,p-IκB‐α,IKKβproteins 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 inhibiting the expression of lncRNA HOTAIR.