目的 探讨酸敏感离子通道 3 特异性拮抗剂(APETx2)对感染后肠易激综合征(PI-IBS)小鼠内脏敏感性的调控作用及其机制.方法 采用旋毛虫感染NIH小鼠建立PI-IBS模型.通过测量首次排黑便的时间和 6h内收集的粪便颗粒数评估胃肠道运输功能;腹壁回撤反射(AWR)评分评估小鼠内脏敏感性;免疫组织化学法检测结肠组织中降钙素基因相关肽(CGRP)蛋白表达;通过实时定量 PCR(qRT-PCR)法检测结肠组织中脑源性神经营养因子(BD-NF)、CGRP mRNA表达.蛋白质印迹法(Western blot)检测脑组织中酸敏感离子通道 3(ASIC3)、CGRP、瞬时受体电位香草素 1(TRPV1)蛋白表达.结果 与对照组比较,PI-IBS组首次排黑便时间显著降低,6 h内的粪便颗粒数,AWR评分均显著升高,结肠组织中 CGRP蛋白表达显著升高,BD-NF、CGRP mRNA 显著升高,脑组织中CGRP、ASIC3、TRPV1的蛋白表达显著升高;与 PI-IBS组比较,APETx2 组首次排黑便时间显著延长,6 h内的粪便颗粒数,AWR评分均显著降低,结肠组织中 CGRP 蛋白表达显著降低,BDNF、CGRP mRNA表达显著降低,脑组织中CGRP、ASIC3、TRPV1 的蛋白表达显著降低,差异均有统计学意义(P<0.05).结论 APETx2 可通过下调BDNF、CGRP、ASIC3、TRPV1 的表达,减轻PI-IBS小鼠的内脏敏感性并调节胃肠道运动.APETx2可能为治疗IBS提供一个新的治疗选择.

Endothelin-1 (ET-1),an endogenous vasoactive peptide,has been found to play an important role in peripheral pain signaling.Acidsensing ion channels (ASlCs) are key sensors for extracellular protons and contribute to pain caused by tissue acidosis.It remains unclear whether an interaction exists between ET-1 and ASICs in primary sensory neurons.In this study,we reported that ET-1 enhanced the activity of ASICs in rat dorsal root ganglia (DRG) neurons.In whole-cell voltage-clamp recording,ASIC currents were evoked by brief local application of pH 6.0 external solution in the presence of TRPV1 channel blocker AMG9810.Pre-application with ET-1 (1-100 nM) dose-dependently increased the proton-evoked ASIC currents with an ECso value of 7A2 ± 0.21 nM.Pre-application with ET-1 (30 nM) shifted the concentration-response curve of proton upwards with a maximal current response increase of 61.11％ ± 4.33％.We showed that ET-1 enhanced ASIC currents through endothelin-A receptor (ETAR),but not endothelin-B receptor (ETBR) in both DRG neurons and CHO cells co-expressing ASIC3 and ETAR.ET-1 enhancement was inhibited by blockade of G-protein or protein kinase C signaling.In current-clamp recording,pre-application with ET-1 (30 nM) significantly increased acid-evoked firing in rat DRG neurons.Finally,we showed that pharmacological blockade of ASICs by amiloride or APETx2 significantly alleviated ET-1-induced flinching and mechanical hyperalgesia in rats.These results suggest that ET-1 sensitizes ASICs in primary sensory neurons via ETAR and PKC signaling pathway,which may contribute to peripheral ET-1-induced nociceptive behavior in rats.

The locus coeruleus(LC)is one of the essential chemoregulatory and sleep-wake(S-W)modulating cen-ters in the brain.LC neurons remain highly active during wakefulness,and some implicitly become silent during rapid eye movement(REM)sleep.LC neurons are also involved in CO2-dependent modulation of the respiratory drive.Acid-sensing ion channels(ASICs)are highly expressed in some brainstem chemosensory breathing regulatory areas,but their localization and functions in the LC remain unknown.Mild hypercapnia increases the amount of non-REM(NREM)sleep and the number of REM sleep episodes,but whether ASICs in the LC modulate S-W is unclear.Here,we investigated the presence of ASICs in the LC and their role in S-W modulation and the state transition from NREM to REM sleep.Male Wistar rats were surgically prepared for chronic polysomnographic recordings and drug microin-jections into the LC.The presence of ASIC-2 and ASIC-3 in the LC was immunohistochemically characterized.Microinjections of amiloride(an ASIC blocker)and APETx2(a blocker of ASIC-2 and-3)into the LC significantly decreased wakefulness and REM sleep,but significantly increased NREM sleep.Mild hypercapnia increased the amount of NREM and the number of REM episodes.However,APETx2 microinjection inhibited this increase in REM frequency.These results suggest that the ASICs of LC neurons modulate S-W,indicating that ASICs could play an important role in vigilance-state transition.A mild increase in CO2 level during NREM sleep sensed by ASICs could be one of the determinants of state transition from NREM to REM sleep.

目的 探讨粪菌移植对母婴分离诱导的肠易激综合征(IBS)大鼠内脏敏感性的影响并观察酸敏感离子通道3(ASIC3)抑制剂APETx2对其作用及可能机制.方法 取SD孕鼠10只,待其生产后取仔鼠,采用母婴分离法构建IBS模型,收集建模成功的大鼠粪便制成粪菌液.18只健康雄性SD大鼠按照随机数字表法分为正常对照(Con)组、母婴分离(NMS)组、NMS+APETx2处理组,每组6只.NMS组、NMS+APETx2组均予以含有抗生素的鸡尾酒(ABX-water)连续灌胃5 d,构建伪无菌鼠模型,之后给予NMS大鼠粪菌液(1.6 mL/kg)灌胃;Con组给予等体积生理盐水灌胃,每天1次,连续灌胃5 d.灌胃结束后,NMS+APETx2组给予100μg/kg APETx2连续腹腔注射7 d,每天1次.采用墨汁推进实验测定肠道推进率,结直肠扩张刺激后评估内脏敏感性.免疫组化染色法检测结肠组织中ASIC3、c-kit蛋白表达.分离3组大鼠结肠cajal间质细胞(ICC),显微镜观察细胞形态学和数量变化.结果 与Con组比较,NMS组肠道推进率减慢,腹部回撤反射(AWR)评分升高,内脏敏感性增加,结肠组织ASIC3、c-kit表达上调,ICC形态改变.与NMS组相比,NMS+APETx2组肠道推进率加快,AWR评分下降,内脏敏感性降低,结肠组织ASIC3、c-kit表达下调,ICC形态趋于正常,数量明显减少.结论 IBS粪菌移植可以诱导内脏高敏感发生和降低肠道传输速率,APETx2可改善其内脏敏感性和肠道传输速率,其机制可能与APETx2下调ASIC3表达、抑制与ICC相关的c-kit信号有关.