College of Acupuncture, Massage and Rehabilitation, Hunan University of Chinese Medcine, 410208 Changsha, Hunan, China.
Department of Rehabilitation Medicine, Chenzhou First People's Hospital, 423000 Chenzhou, Hunan, China.
J Integr Neurosci. 2022 Aug 26;21(5):147. doi: 10.31083/j.jin2105147.
This study aimed to conduct proteomic analysis of the sphincter in a neurogenic bladder caused by T10 spinal cord injury. The differentially expressed proteins (DEPs) of the sphincters (internal urethral sphincter) in the neurogenic bladders (NBs) of rats after complete transection of the T10 spinal cord segment were screened using tandem mass tag (TMT)-based quantitative labeling, and their biological information was analyzed.
Twelve adult Sprague Dawley rats out of 40 were randomly assigned to the blank group ( = 12), while the remaining 28 were placed in the T10 spinal cord injury model via modified Hassan Shaker spinal cord transection; 12 of these rats were then randomly selected as the model group. The rats in both groups underwent urodynamics detection and hematoxylin and eosin (H&E) staining. The proteins expressed in the bladder sphincter were detected using TMT-based quantitative proteomics. DEPs were defined as proteins with fold change >1.5 or <1/1.5, < 0.05, and unique peptide ≥2. The DEPs were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using KOBAS 3.0., and gene ontology functional annotation analysis was performed using the Cytoscape 3.7.1. BiNGO plug-in. The protein-protein interaction network was then constructed using the interactive gene-retrieval tool STRING and Cytoscape software.
The leak-point pressure and maximum cystometric volume in the model group were significantly higher than those in the blank group ( < 0.01), and H&E staining showed continuous interruption of the bladder sphincter fibers in the model group. A total of 250 DEPs were screened in the bladder sphincter, 83 of which were up-regulated and 167 of which were down-regulated. KEGG analysis of the DEPs was used to screen 15 pathways, including metabolic pathways, extracellular matrix (ECM)-receptor interaction, adhesion spots, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, the cytochalasin signaling pathway, and the advanced glycation end-products (AGE)/receptor for AGEs (RAGE) signaling pathway in diabetic complications and vascular smooth muscle contraction.
It is of great significance to explore the pathological mechanism of non-inhibitory contraction of the bladder sphincter caused by spinal cord injury above the T10 segment from the perspective of ECM-receptor interaction, focal adhesion-activated PI3K/Akt signaling pathway, and cell relaxation signaling pathways. Synaptic vesicle glycoprotein (Sv2A) involved in the release of neurotransmitters from synaptic vesicles, arrestin β2 inhibitory proteins involved in α-adrenergic receptors and G-protein-coupled receptor internalization, and calmodulin and calmodulin binding protein involved in calcium-sensitive signaling pathways may be potential targets for developing new ways to treat bladder sphincter overactivity caused by T10 spinal cord injury.
本研究旨在对 T10 脊髓损伤所致神经源性膀胱逼尿肌进行蛋白质组学分析。采用串联质量标签（TMT）定量标记法筛选完全横断 T10 脊髓节段后大鼠神经源性膀胱（NB）逼尿肌中差异表达蛋白（DEPs），并对其生物学信息进行分析。
40 只成年 Sprague Dawley 大鼠随机分为空白组（n=12），其余 28 只大鼠采用改良 Hassan Shaker 脊髓横断法建立 T10 脊髓损伤模型；其中 12 只大鼠随机分为模型组。两组大鼠均行尿动力学检测和苏木精-伊红（H&E）染色。采用 TMT 定量蛋白质组学检测膀胱逼尿肌表达的蛋白质。差异表达蛋白的定义为 fold change >1.5 或 <1/1.5、 < 0.05、unique peptide≥2。采用 KOBAS 3.0 对 DEPs 进行京都基因与基因组百科全书（KEGG）通路富集分析，采用 Cytoscape 3.7.1 的 BiNGO 插件进行基因本体（GO）功能注释分析。然后使用交互基因检索工具 STRING 和 Cytoscape 软件构建蛋白质-蛋白质相互作用网络。
模型组漏点压和最大膀胱容量明显高于空白组（ < 0.01），H&E 染色显示模型组膀胱逼尿肌纤维连续中断。在膀胱逼尿肌中筛选出 250 个 DEPs，其中 83 个上调，167 个下调。对 DEPs 进行 KEGG 分析，筛选出包括代谢途径、细胞外基质（ECM）-受体相互作用、黏附斑、磷酸肌醇 3-激酶（PI3K）/蛋白激酶 B（Akt）信号通路、细胞松弛素信号通路和晚期糖基化终产物（AGE）/AGE 受体（RAGE）信号通路在内的 15 条通路与糖尿病并发症和血管平滑肌收缩有关。
从 ECM-受体相互作用、粘着斑激活的 PI3K/Akt 信号通路和细胞松弛信号通路的角度探讨 T10 以上脊髓损伤导致的膀胱逼尿肌非抑制性收缩的病理机制具有重要意义。突触小泡糖蛋白（Sv2A）参与神经递质从突触小泡中释放，抑制蛋白 arrestin β2 参与 α-肾上腺素能受体和 G 蛋白偶联受体内化，钙调蛋白和钙调蛋白结合蛋白参与钙敏信号通路，这些可能是治疗 T10 脊髓损伤引起的逼尿肌过度活动的新方法的潜在靶点。
