疏肝健脾法通过调控组蛋白乙酰化修饰降低肝郁脾虚IBS-D内脏高敏感的机制研究

IBS-D is considered as liver depression and spleen deficiency in traditional Chinese Medicine, which is a functional bowel disease. Clinical manifestations are chronic abdominal pain and diarrhea, and visceral hypersensitivity is the main pathological basis. In recent years, the epigenetic modification mechanism has provided a new target for visceral pain research. The latest study found that histone deacetylase (HDAC) inhibitors were able to reverse the visceral hypersensitivity of IBS-D induced by chronic stress. It is demonstrated that histone acetylation modification can affect the expression of GluR, 5-HTR. Our previous studies have shown visceral sensitivity enhancement in IBS-D model animal with liver depression and spleen deficiency, associated with abnormalities of Glu, 5-HT. Representative formula of soothing liver and strengthening spleen Tong-xie-yao-fang has significant curative effect on IBS-D, associated with the mechanisms that it could decrease visceral hypersensitivity and regulate the expression of 5-HT and so on, but its role on histone acetylation still need to further clarify. In this study, we examined the clinical relationship between histone acetylation and IBS-D visceral hypersensitivity and screen differentially expressed HDACs through protein chip analysis system; Then immunofluorescence and PCR were used for animal model, to further confirm the effect of soothing liver and strengthening spleen method on histone acetylation. The aim of this project is to explore the epigenetic modification mechanism of traditional Chinese Medicine in the treatment of IBS-D, and to provide theoretical basis for new drug development of anti-IBS-D.

IBS-D属中医肝郁脾虚范畴，是一种慢性功能性肠病，表现为腹痛腹泻，内脏高敏感是其主要病理基础。近些年表观遗传机制为内脏疼痛研究提供了新靶标，最新发现组蛋白去乙酰化酶（HDAC）抑制剂能够逆转慢性压力导致的内脏过敏，且已证明组蛋白乙酰化修饰能影响神经递质受体GluR、5-HTR水平。我们前期发现IBS-D模型动物内脏敏感性增强，且和Glu、5-HT异常有关。疏肝健脾中药痛泻要方对IBS-D疗效明显，且能改善其内脏高敏感性、调节5-HT等表达，但对组蛋白乙酰化修饰的调控作用尚需进一步阐明。本研究拟采用蛋白芯片技术，通过临床试验验证组蛋白乙酰化修饰与IBS-D内脏高敏及腹痛的关系，筛选差异表达的HDACs；并采用免疫荧光和PCR等技术，通过动物实验进一步证实疏肝健脾法对组蛋白乙酰化水平的调控作用。本项目旨在探讨中药复方治疗IBS-D的表观遗传机制，为抗IBS-D的新药开发提供理论依据。

背景：IBS-D是一种世界难治性慢性肠病，以腹痛和腹泻为主要的临床表现，目前无明确的诊断标志物，也无令人满意的治疗药物，其诊断主要依据罗马标准。准确定位患者的发病因素，选择合适的治疗策略仍旧是个挑战。.方法：我们通过串联质谱标签（TMT）方法和高通量检测技术对乙状结肠和直肠交界处的活检组织进行蛋白组学分析和微小 RNA测序，筛选出潜在的差异表达的蛋白分子和tRFs & tiRNAs，并进行PRM和q-PCR验证；通过生物信息学分析、相关性分析以及ROC分析，评估差异指标作为IBS-D生物学标志物的临床意义。通过细胞实验验证它们在IBS-D中的作用机制。通过动物实验考察疏肝健脾法对IBS-D相关分子和重要信号通路的影响。 .结果：我们筛选出差异蛋白80个，其中48个上调，32个下调；Go分析和KEGG等分析表明差异蛋白在蛋白质、脂肪的消化和吸收，胆汁和胰腺等的分泌、PPAR信号通路等系统中显著富集；蛋白互作分析表明多种营养素摄取蛋白和免疫分子在IBS-D中的发生相互作用；PRM验证与测序结果一致并具有统计学差异的分子13个；q-PCR检测显示9分子的mRNA发生了改变。其中SELENBP1、VSIG2、HMGB1、DHCR7、BCAP31在基因和蛋白水平均存在显著差异。筛选出差异的tRFs & tiRNAs有28个，上调14个，下调14个。q-PCR实验证明其中3个与测序结果一致：tiRNA-His-GTG-001（上调），tRF-Ser-GCT-113（下调），tRF-Gln-TTG-035（下调）。ROC曲线分析证明3个差异的tRFs & tiRNAs均可作为IBS-D的生物标志物。通过生物信息学分析表明它们可能参与GABA（神经递质Glu相关）、TNF-α、VEGF等多条关键信号通路。细胞实验证明，IBS-D相关的tRFs & tiRNAs能够靶向调控GABBR2、HTR2C（5-HT受体）、SASH1、TLR4、ABARAP-1的表达。.结论：IBS-D肠道存在多种蛋白分子、信号通路以及tRFs & tiRNAs表达异常。研究结果进一步揭示了IBS-D腹痛、腹泻以及麸质饮食等多种食物不耐受等发生的表观遗传机制，也为IBS-D的诊断和治疗提供了潜在的靶标。疏肝健脾法改善IBS-D 腹痛、腹泻的临床症状可能与调控机体异常的蛋白分子和非编码RNA相关。.