益生菌VSL#3对应激诱发的内脏高敏感大鼠中肥大细胞-PAR2-TRPV1通路的调控机制

Irritable bowel syndrome (IBS) is a common functional bowel disorder in clinical, and visceral hypersensitivity is the important feature of IBS. In recent years, many studies found that visceral hypersensitivity was related to mast cell-PAR2-TRPV1 pathway. PAR2-TRPV1 play an important role in pain modulation, but few studies were done about visceral hypersensitivity. Probiotics have significant effects in the treatment of IBS, but the exact mechanism is not clear. In preliminary studies, we found that stress can induce the formation of visceral hypersensitivity, and large numbers of activated mast cells were in the intestinal mucosa. And probiotics VSL#3 intervention can significantly reduce visceral hypersensitivity. In the pre-experiment mast cells and TRPV1 pathway changed, so we speculated that mast cell-PAR2-TRPV1 pathway is the key to the formation of visceral hypersensitivity. And the mechanism of action of probiotics VSL#3 for IBS may be related to the above hypothesis. So far there is no report about the above hypothesis at home and abroad. In order to prove the hypothesis, we observe the change of mast cell-PAR2-TRPV1 pathway in the formation of visceral hypersensitivity and the probiotics VSL#3 intervention of the change by proteomics, mast cell-DRG nerve cell non-contact co-culture and patch clamp technology in vitro and in vivo. Our study will be the new visual clarify the regulatory mechanisms of VSL#3 on the stress-induced visceral hypersensitivity.

肠易激综合征（IBS）是常见的功能性肠病，内脏高敏感是其重要特征，近年来研究发现内脏高敏感可能与肥大细胞-PAR2-TRPV1通路有关，其中PAR2-TRPV1通路在痛觉调制方面发挥着重要作用，但在内脏高敏感中研究较少。益生菌在IBS的治疗上有明显疗效，但具体机制不明。前期研究中我们发现应激能诱发大鼠产生内脏高敏感，且发现肠黏膜存在大量活化的肥大细胞，而益生菌(VSL#3)的干预能明显降低内脏敏感性。预实验中观察到该模型中肥大细胞和TRPV1通路的改变，推测肥大细胞-PAR2-TRPV1通路是内脏高敏感形成的关键，而VSL#3的作用机制可能与此有关，迄今国内外尚无报道。为证明这种假说，本实验拟从体内外两方面应用蛋白组学、肥大细胞-DRG神经元非接触式共培养、膜片钳等技术，观察在内脏高敏感形成中VSL#3干预后对上述通路的影响。本课题将从新的视觉阐明VSL#3对应激诱发内脏高敏感的调控机制。

内脏高敏感是肠易激综合征（IBS）的重要特征，但其具体机制不明。我们采用结直肠黏膜炎症刺激（0.6%醋酸溶液保留灌肠）和束缚应激的方法构建内脏高敏感大鼠（腹部回撤反射评分，AWR评分进行评价），检测到较多活化的肥大细胞存在于内脏高敏感大鼠模型，揭示了肥大细胞在内脏高敏感形成过程中的作用；在体外，通过将肥大细胞与DRG神经元细胞进行非接触共培养，以肥大细胞脱颗粒剂和PAR2拮抗剂进行干预，采用膜片钳技术发现肥大细胞活化后释放的活性物质能激活DRG神经元细胞上的TRPV1通道电流，而PAR2拮抗剂能阻断这一效应，提示肥大细胞通过PAR2调控DRG神经元细胞上的TRPV1通路；以构建好的内脏高敏感大鼠为模型，采用益生菌VSL#3、肥大细胞稳定剂、PAR2拮抗剂和TRPV1通路拮抗剂进行干预，通过免疫组化、免疫荧光、WB等技术检测发现内脏高敏感大鼠PAR2蛋白和TRPV1蛋白的表达明显增加，膜片钳技术检测发现内脏高敏感大鼠TRPV1通路电流明显增加，揭示肥大细胞激活后能进一步作用于DRG神经元的PAR2-TRPV1通路，在内脏高敏感形成中发挥重要作用；益生菌VSL#3干预内脏高敏感大鼠后，活化的肥大细胞明显减少，DRG神经元上的PAR2和TRPV1蛋白表达减少，内脏敏感性降低，揭示益生菌VSL#3能通过肥大细胞及DRG神经元PAR2-TRPV1通路调控内脏敏感性，为临床上治疗IBS提供更多的理论依据。