电针治疗骶上脊髓损伤后逼尿肌-膀胱颈协调障碍的蛋白质组学机制

Mechanism of electroacupuncture treating detrusor-bladder neck dyssynergia after suprasacral spinal cord injury by proteomics

    摘要
  • 摘要:
    目的 阐明电针治疗骶上脊髓损伤后逼尿肌-膀胱颈协调障碍（DBND）的潜在机制。
    方法 将52只无特定病原体（SPF）级雌性SD大鼠（10 – 12周龄，250 – 280 g）随机分为假手术组（n = 12）和脊髓损伤造模组（n = 40）。造模组采用Hassan Shaker法在T10水平行脊髓横断术建立DBND模型，符合标准的24只大鼠随机分为DBND组（n = 12）和电针干预组（DBND + EA组，n = 12）。脊髓休克恢复后（造模后第19天），DBND + EA组接受电针治疗，取次髎（BL32）、中极（RN3）、三阴交（SP6）穴位，每次20分钟，频率10/50 Hz，每日1次，连续10天。假手术组和DBND组仅给予麻醉而不进行电针干预。造模后第29天，所有大鼠进行尿动力学检测，随后对逼尿肌和膀胱颈组织进行苏木精-伊红（HE）染色、串联质量标签（TMT）蛋白质组学和蛋白质印迹法（WB）分析。差异表达蛋白（DEPs）定义为P < 0.05、唯一肽段≥2、倍数变化> 1.2或< 0.83的蛋白质。采用KOBAS 3.0进行京都基因与基因组百科全书（KEGG）通路分析（P < 0.01），使用基因/蛋白质相互作用检索工具（STRING） 11.5和Cytoscape 3.9.1构建蛋白质-蛋白质相互作用（PPI）网络。
    结果 与假手术组相比，DBND组的漏点压力（LPP）和最大膀胱测压容量（MCC）均显著升高（均为P < 0.01）。与DBND组相比，电针治疗显著降低了LPP和MCC（分别为P < 0.01和P < 0.05）。HE染色显示电针减少了逼尿肌纤维化并改善了膀胱颈炎症。TMT蛋白质组学在逼尿肌中鉴定出30个重叠DEPs，在膀胱颈中鉴定出59个重叠DEPs（通过比较DBND + EA组与DBND组相对于假手术组的差异）。在逼尿肌组织中，KEGG分析显示10条通路显著富集（P < 0.01），包括丝裂原活化蛋白激酶（MAPK）信号通路。PPI分析显示30个DEPs中有22个相互关联。在膀胱颈组织中，14条通路显著富集（P < 0.01），包括松弛素信号通路，59个DEPs中有51个显示相互关联。TMT和WB验证均表明，与假手术对照相比，DBND大鼠逼尿肌组织中IV型胶原α2链（Col4a2）表达上调，鸟苷酸结合蛋白G(z)亚基α（Gnaz）表达下调，而电针治疗使这两个蛋白表达正常化（均为P < 0.05）。在膀胱颈组织中，与假手术对照相比，DBND大鼠的平滑肌蛋白（Smtn）和钙激活钾通道β1亚基（Kcnmb1）表达下降（均为P < 0.01），电针治疗后两者表达均上调（分别为P < 0.01和P < 0.05）。
    结论 电针通过双靶点机制恢复DBND中的逼尿肌-膀胱颈协调功能。在逼尿肌组织中，电针通过细胞外基质重塑、环磷酸腺苷（cAMP）信号调节以及神经递质介导的三磷酸腺苷（ATP）生物合成增强来调节收缩功能。在膀胱颈组织中，电针通过维持收缩表型、减少纤维化、抑制平滑肌兴奋性以及调节突触前神经递质释放来促进松弛。这些发现为电针治疗DBND的机制提供了新的见解。

     

    Abstract:
    Objectives To elucidate the potential mechanisms of electroacupuncture (EA) in restoring detrusor-bladder neck dyssynergia (DBND) following suprasacral spinal cord injury (SSCI).
    Methods A total of 52 specific pathogen-free (SPF) grade famale Sprague-Dawley (SD) rats (10 – 12 weeks, 250 – 280 g) were randomly assigned to either a sham group (n = 12) or a spinal cord injury model group (n = 40). In the model group, DBND was induced through Hassan Shaker spinal cord transection at T10 level, with 24 rats meeting inclusion criteria and subsequently randomized into DBND group (n = 12) and EA intervention group (DBND + EA group, n = 12). After spinal shock recovery (day 19 after modeling), DBND + EA group received EA treatment at Ciliao (BL32), Zhongji (RN3), and Sanyinjiao (SP6) acupoints for 20 min per session at 10/50 Hz frequencies, once daily for 10 d. Sham and DBND groups received anesthesia only without EA intervention. On day 29 post-modeling, all rats underwent urodynamic assessments, followed by hematoxylin and eosin (HE) staining, tandem mass tag (TMT) proteomics, and Western blot (WB) analysis of detrusor and bladder neck tissues. Differentially expressed proteins (DEPs) were defined as proteins with P < 0.05, unique peptides ≥ 2, and fold change > 1.2 or < 0.83. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using KOBAS 3.0 (P < 0.01), and protein-protein interaction (PPI) networks were analyzed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) 11.5 and Cytoscape 3.9.1.
    Results Compared with sham group, DBND group showed significantly elevated leak point pressure (LPP) and maximum cystometric capacity (MCC) (both P < 0.01). EA treatment significantly reduced both LPP and MCC compared with DBND group (P < 0.01 and P < 0.05, respectively). HE staining revealed that EA reduced detrusor fibrosis and improved bladder neck inflammation. TMT proteomics identified 30 overlapping DEPs in detrusor and 59 overlapping DEPs in bladder neck when comparing DBND + EA/DBND groups with sham group. In detrusor tissue, KEGG analysis revealed 10 significantly enriched pathways (P < 0.01), including mitogen-activated protein kinase (MAPK) signaling pathway. PPI analysis showed 22 of 30 DEPs were interconnected. In bladder neck tissue, 14 pathways were significantly enriched (P < 0.01), including relaxin signaling pathway, with 51 of 59 DEPs showing interconnections. Both TMT and WB validations demonstrated that compared with sham controls, DBND rats exhibited upregulated collagen type IV alpha 2 chain (Col4a2) and downregulated guanine nucleotide-binding protein G(z) subunit alpha (Gnaz) in detrusor tissue, while EA treatment normalized both proteins (both P < 0.05). In bladder neck tissue, DBND rats showed decreased expression of smoothelin (Smtn) and calcium-activated potassium channel subunit beta-1 (Kcnmb1) compared with sham controls (both P < 0.01), which were both upregulated following EA treatment (P < 0.01 and P < 0.05, respectively).
    Conclusion EA restores detrusor-bladder neck coordination in DBND through dual-target mechanisms. In detrusor tissue, EA modulates contraction via extracellular matrix remodeling, cyclic adenosine monophosphate (cAMP) signaling pathway regulation, and enhanced adenosine triphosphate (ATP) biosynthesis mediated by neurotransmitters. In bladder neck tissue, EA promotes relaxation by maintaining contractile phenotypes, reducing fibrosis, suppressing smooth muscle excitation, and regulating presynaptic neurotransmitter release. These findings provide mechanistic insights into EA's therapeutic role in managing DBND.

     

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