去泛素化酶IsoT调控CBS-H2S信号介导内脏痛过敏的机制研究

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by abdominal pain and bloating in association with altered bowel movements. Its pathogenesis and the underlying molecular mechanisms of visceral hyperalgesia remain elusive. Our preliminary data showed that neonatal somatic inflammation produced visceral hypersensitivity and enhanced the endogenous hydrogen sulfide producing enzyme cystathionine β-synthetase (CBS) and TRPV1 expression in dorsal root ganglion (DRG) in adult rats. Through bioinformatics analysis and screening of the ubiquitination enzyme DUBs library, we found that IsoT （Also known as USP5）upregulated CBS protein expression, and showed a dose-dependent effect. IsoT could de ubiquitin CBS. Therefore, we first established a novel IBS model which accords with the clinical features. We aim to clarify how IsoT regulates CBS expression through ubiquitination modification, and then affects neuronal excitability and TRPV1 ion channel and receptor plasticity, and eventually leads to visceral hypersensitivity. Thus, by the mechanism study of IsoT/CBS-H2S/TRPV1 molecular signal axis in adult visceral pain hypersensitivity induced by neonatal somatic inflammation, these studies might shed light on the pathogenesis of IBS and may have important implications for treatment of functional gastrointestinal disorders such as IBS.

肠易激综合征（IBS）是一种常见的功能性胃肠病，IBS腹痛（内脏痛）发生发展的生物学机制仍不清楚。我们前期研究发现：新生期体表炎性刺激可诱导成年个体内脏痛过敏，肠相关背根节DRG神经元兴奋性增加以及硫化氢合成酶CBS及疼痛关键分子辣椒素受体TRPV1表达上调。通过生物信息学分析预测和对去泛素化酶DUBs文库的筛选发现IsoT上调CBS蛋白表达，且呈现剂量依赖效应，且IsoT能对CBS进行去泛素化修饰。因此，本项目通过建立一种新颖的符合临床特征的IBS模型，探讨IsoT是如何通过泛素化修饰调控CBS表达，进而影响神经元兴奋性及TRPV1离子通道和受体可塑性改变，最终引起内脏痛过敏。通过探讨IsoT/CBS-H2S/TRPV1分子轴信号调控新生期体表炎性刺激诱导成年个体内脏痛过敏的分子机制，为慢性内脏痛的发病机理提供理论依据，并可能为临床上寻找功能性胃肠病的疼痛诊断和治疗提供潜在的新靶点。

肠易激综合征（IBS）是一种常见的功能性胃肠病，IBS腹痛（内脏痛）发生发展的生物学机制仍不清楚。本项目按照项目申请书制定的研究计划，通过建立一种新颖的符合临床特征的IBS模型，结合动物实验、电生理、膜片钳技术和多种分子生物学技术实验开展研究。首先，通过完全弗氏佐剂CFA诱导建立新生期起源的功能性胃肠疾病模型，研究新生期体表炎性刺激引起结肠特异性神经元兴奋性变化及离子通道受体的可塑性变化。其次，阐释了CBS-H2S信号如何参与新生期体表炎性刺激引起结肠特异性DRG神经元兴奋性及离子通道受体的可塑性变化。最后，我们还阐释了去泛素化酶IsoT通过去泛素化修饰调控CBS表达及对DRG神经元兴奋性和离子通道受体可塑性变化的影响。我们的结果显示：（1）新生期体表炎性刺激可引起成年大鼠的内脏痛过敏。（2）应用膜片钳记录荧光染料DiI标记的结肠特异性DRG神经元(T13-L2)的兴奋性，显示新生期炎性处理后肠特异性DRG 神经元的兴奋性增加。（3）免疫荧光染色发现：CBS与TRPV1在结肠特异性DRG神经元上存在共表达。结肠特异性DRG神经元(T13-L2)内CBS和TRPV1表达升高。同时，应用硫化氢合酶CBS拮抗剂AOAA可翻转TRPV1的表达水平，翻转新生期CFA诱发的肠特异性DRG神经元兴奋性的增加。（4）新生期CFA刺激诱导的成年个体的肠特异性背根节DRG神经元细胞中，采用慢病毒感染技术过表达Flag-IsoT，发现过表达IsoT能显著稳定CBS的蛋白表达水平。同时，过表达IsoT和Myc-CBS后，发现IsoT能够显著降低CBS的泛素化水平，从而稳定CBS蛋白的表达水平。以上研究结果，揭示了IsoT/CBS-H2S/TRPV1分子轴信号调控新生期体表炎性刺激诱导成年个体内脏痛过敏的分子机制。