骶神经刺激对神经生长因子介导的内脏高敏感的作用机制研究

Visceral hypersensitivity is the important reason for the pathogenesis of irritable bowel syndrome (IBS). Recent studies found that nerve growth factor (NGF) is involved in visceral sensitization process, PI3K/Akt and phospholipase C (PLC) signals play import roles on visceral hypersensitivity in visceral hypersensitivity rats, and these two pathways are the downstream signaling pathways regulated by NGF combining with TrkA receptor. Our previous study found that sacral nerve stimulation (SNS) could decrease the visceral sensitivity of sensitized rats induced by acetic acid (AA) enema, and that expression of nerve growth factor (NGF) in serum, colon, and dorsal root ganglion (DRG) of sensitized rats increased significantly, while SNS could reduce the expression of NGF. On the basis of our previous study, this project intends to: on visceral hypersensitivity rats induced by AA, ①test expression of NGF, TrkA receptor, PI3K/Akt, PLC and other signaling molecules by western blot and RT-PCR before and after SNS, and measure effect of SNS on visceral hypersensitivity and test same content after NGF antagonist was used, ②measure effect of SNS on visceral hypersensitivity after PI3K/Akt, PLC and other signaling pathway or/and NGF was blocked to verify whether SNS improves visceral hypersensitivity by downstream pathways mediated via NGF/ TrkA receptor. Study on the mechanisms of SNS promoting visceral hypersensitivity is needed, as it provides a new theoretical basis for neural modulation treating IBS.

内脏高敏感是肠易激综合征（IBS）的重要原因。研究发现神经生长因子（NGF）参与了内脏高敏感；PI3K/Akt及PLC信号对内脏高敏感大鼠的内脏高敏发挥重要作用；这两条通路是NGF与酪氨酸激酶A（TrkA）受体结合调控的下游信号。我们前期研究发现：醋酸灌肠致敏大鼠的血清、结肠及背根神经节（DRG）的NGF明显升高，骶神经刺激（SNS）可以降低大鼠内脏高敏感、并降低NGF的表达。本项目拟：在醋酸灌肠致敏模型大鼠上，①免疫印迹和RT-PCR检测SNS前后结肠和DRG中NGF、TrkA受体及PI3K/Akt、PLC等信号的表达，拮抗NGF后检测以上内容并评估SNS对大鼠内脏敏感性的影响，②单独阻滞各信号通路、及联合拮抗NGF，评估SNS对大鼠内脏敏感性的影响；探讨SNS是否通过NGF/TrkA受体介导的下游信号通路降低模型大鼠的内脏高敏感，为神经调控治疗IBS的内脏高敏感提供新的治疗思路。

背景：肠易激综合征（IBS）是常见的消化道疾病，严重影响患者生活质量并消耗大量医疗资源。IBS机制尚不明确，内脏高敏感是其重要原因。神经生长因子（NGF）参与了内脏高敏感；PI3K/Akt及PLC信号对内脏高敏感大鼠的内脏高敏发挥重要作用。骶神经刺激（SNS）是外周神经调控的一种，对IBS或内脏高敏感的作用鲜有报导。我们前期研究发现，醋酸灌肠致敏大鼠的结肠及背根神经节（DRG）的NGF升高，SNS可以降低大鼠内脏高敏感并降低NGF的表达。方法：在醋酸灌肠模型大鼠上，给予拮抗/激动NGF、抑制PI3K、抑制 PLC-γ，通过免疫印迹和RT-PCR检测SNS前后结肠和DRG中NGF、TrKA、PI3K、AKT、PLC-γ、NF-κB、TRPV1和pTRPV1的表达，荧光分光光度计法检测结肠和DRG中的Ca2+含量，CO-IP法分析NGF和TrKA在结肠和DRG的交互作用。结果：内脏超敏组TRPV1、pTRPV1蛋白表达升高，Ca2+升高；NGF拮抗组NGF、TrKA，PI3K抑制组PI3K、AKT、NF -κB，PLC -γ抑制组PLC -γ几乎未表达；NGF拮抗组NGF、TrKA、PI3K、AKT、PLC‐γ和NF‐κB的表达低于内脏超敏组和NGF激活组；NGF拮抗组NGF、TrKA、PI3K、AKT mRNA表达低于正常组；PLC‐γ和NF‐κB mRNA表达差异无统计学意义；内脏超敏组中MAPK、ERK1、ERK2的表达高于PI3K抑制组和PLC‐γ抑制组；正常组Ca2+曲线平缓，NGF激动组Ca2+曲线峰值最高；内脏超敏组Ca2+高于PI3K抑制组，PLC‐γ抑制组高于NGF拮抗组。结论：TrkA受体与NGF的结合激活MAPK/ERK通路、PI3K/Akt通路和PLC‐γ通路，引起细胞内和细胞外Ca2+流动性变化，导致内脏组织和器官的敏感性增加。