电针对骶上脊髓损伤后大鼠神经源性膀胱及血清外泌体miRNA差异表达的影响

关键词：电针  神经源性膀胱  外泌体  微小核糖核酸  生物信息学分析

Keywords:electroacupuncture  neurogenic bladder  exosomes  microrna  bioinformatic analysis

摘要：

      摘要 目的：观察电针对骶上脊髓损伤致尿潴留型神经源性膀胱大鼠的效果，并观察电针对血清外泌体miRNA差异表达的影响。 方法：从48只成年雌性SD大鼠中随机抽取12只为假手术组，剩余36只大鼠进行T8椎体下脊髓完全横断手术造模，制备尿潴留型神经源性膀胱模型，将评估成模大鼠二次随机分为模型组与电针组，各12只。电针组从术后第19天开始干预，共10天。随后，各组行尿流动力学检测后取膀胱组织行HE染色，应用miRNA-seq技术筛选三组血清外泌体中共同差异表达miRNA后，对其靶基因进行生信分析，并行qRT-PCR验证。 结果：与假手术组比，肉眼观下的模型组大鼠膀胱明显肥大，电针组较模型组减轻；与假手术组比，模型组大鼠漏尿点压、灌注时间、膀胱最大容量、膀胱最大压力显著升高（P＜0.01），而电针组较模型组显著降低（P＜0.01）；HE染色见假手术组膀胱组织基本正常，模型组膀胱病理改变明显，电针组病理改变部分减轻；筛选出三组共同差异表达miRNA共11个，6个上调，5个下调，电针治疗作用主要富集在调节细胞代谢、细胞通讯与信号转导、RNA调控、神经发育及损伤等方面，并与轴突导向、肌动蛋白细胞骨架、自噬、缝隙链接、胆碱能代谢等信号通路相关；qRT-PCR结果表明本测序结果可靠性较高。 结论：本研究初步证实了电针对T8椎体下脊髓损伤致尿潴留型神经源性膀胱的效果，并发现其作用的发挥与影响血清外泌体miRNA差异表达密切相关。此外，筛选出了电针在治疗该病机制中的关键miRNA，并对其靶基因进行了全面的基因功能和通路富集分析，为深入研究电针治疗该病的效应机制提供了科学基础。

Abstract:

      Abstract Objective： To observe the therapeutic effect of electroacupuncture in treatment for urinary retention type neurogenic bladder after suprasacral spinal cord injury and to observe the effect of electroacupuncture on the differential expression of serum exosomal miRNA. Method： Twelve rats were randomly selected from 48 adult female SD rats as the sham-operated group， and the remaining 36 rats were surgically modelled by complete transection of the spinal cord under the 8th thoracic vertebra to prepare a neurogenic bladder model with urinary retention. The adult rats with successful model were randomized into a model group and an electroacupuncture group， 12 rats each. The electroacupuncture group was given electroacupuncture treatment for 10 days from the 19th day postoperation. Subsequently， the bladder tissues were taken for HE staining after urodynamic testing. The miRNA-seq technique was applied to screen common differentially expressed miRNAs of serum exosomes in the three groups， and their target genes were analyzed by bioinformat and validated by qRT-PCR. Result： Compared with the sham-operated group， the bladder in the model group was significantly hypertrophied under visual observation， while the bladder in the electro-acupuncture group was less hypertrophied than that in the model group； compared with the sham-operated group， the leakage point pressure， perfusion time， maximum bladder capacity and maximum bladder pressure in the model group were significantly higher （P<0.01）， while the electro-acupuncture group was significantly lower （P<0.01） than that in the model group； HE staining showed that the bladder tissue in the sham-operated group was basically normal， while the bladder tissue in the model group had obvious pathological changes which were partially alleviated in the electroacupuncture group. The miRNA-seq technique revealed a total of 11 differentially expressed miRNAs in the three groups， in which 6 were up-regulated and 5 were down-regulated， and the therapeutic effects of electroacupuncture were mainly enriched in the regulation of cell metabolism， cell communication and signal transduction， RNA regulation， neurological development & injury， and were associated with axon guidance， actin backbone， autophagy， gap linkage and cholinergic metabolism pathways. In addition, the qRT-PCR indicated that the sequencing results were highly reliable. Conclusion： This study initially confirmed the promising therapeutic effect of electroacupuncture on neurogenic bladder caused by spinal cord injury under the 8th thoracic vertebra， and found that its therapeutic effect was closely related to the differential expression of serum exosomal miRNA. In addition， the key miRNAs were screened and comprehensive gene function and pathway enrichment analyses of their target genes were performed. The present study provided a scientific basis for in-depth research of electroacupuncture in the treatment of neurogenic bladder.