电针“足三里”对功能性消化不良内脏高敏感大鼠肥大细胞/瞬时受体电位香草酸亚型1通路的影响

Influence of electroacupuncture of“Zusanli”(ST36)on mast cells/TRPV1signaling pathway in visceral hypersensitivity rats with functional dyspepsia

目的:基于肥大细胞/瞬时受体电位香草酸亚型1(TRPV1)调控功能性消化不良(FD)内脏高敏感,探讨电针“足三里”对FD的干预机制。方法:新生10d的SD大鼠随机分为正常对照组、模型组、酮替芬组、足三里组,每组15只。采用碘乙酰胺联合夹鼠尾法制备FD大鼠模型。酮替芬组予腹腔注射酮替芬,1mg·kg^(-1)·d^(-1),注射14d;足三里组予电针双侧“足三里”穴,1次/d,连续14d。采用胃内球囊容积/压力(mL/mm Hg)的变化、腹部撤退反射(AWR)评分作为大鼠内脏高敏感评价指标;甲苯胺蓝染色法观察胃黏膜肥大细胞数量及脱颗粒情况;免疫荧光染色法观察胃组织TRPV1的阳性表达;Western blot法检测胃组织TRPV1和蛋白酶激活受体2(PAR2)蛋白表达;ELISA法检测胃组织P物质(SP)、降钙素基因相关肽(CGRP)的含量。结果:与正常对照组比较,模型组胃内球囊压力在50、60、70mm Hg时胃内球囊容积/压力降低(P<0.01),胃内球囊压力为40、50、60、70mm Hg时AWR评分升高(P<0.05,P<0.01);与模型组比较,足三里组胃内球囊压力在50、60、70mm Hg时胃内球囊容积/压力升高(P<0.01,P<0.05)、AWR评分降低(P<0.05,P<0.01),酮替芬组胃内球囊压力在60、70mm Hg时AWR评分降低(P<0.01,P<0.05);与酮替芬组比较,足三里组胃内球囊压力在70mm Hg时胃内球囊容积/压力升高(P<0.05)。正常对照组胃黏膜肥大细胞形态规则;模型组胃黏膜肥大细胞明显增多且脱颗粒状态明显;足三里组和酮替芬组胃黏膜肥大细胞明显减少且脱颗粒情况不明显。与正常对照组比较,模型组胃组织PAR2、TRPV1蛋白表达水平和SP、CGRP含量均升高(P<0.01);与模型组比较,足三里组和酮替芬组胃组织PAR2、TRPV1蛋白表达水平和SP、CGRP含量均降低(P<0.01,P<0.05);与酮替芬组比较,足三里组胃组织SP、CGRP含量降低(P<0.05)。结论:电针“足三里”可能是通过降低胃黏膜肥大细胞活化和脱颗粒程度,调控胃组织PAR2、TRPV1蛋白的表达,降低传递伤害性痛觉信息的神经肽SP、CGRP的释放,起到对FD大鼠内脏高敏感的干预作用。

Objective To explore the interventional mechanism of electroacupuncture(EA)of“Zusanli”(ST36)based on the involvement of mast cells/transient receptor potential vanilloid type1(TRPV1)signaling pathway in relieving visceral hypersensitivity in functional dyspepsia(FD)rats.Methods Sixty SD rats(half male and half female,10days in age)were randomly divided into normal control,model,medication(ketotifen)and EA groups,with 15rats in each group.The FD model was established by gavage of iodoacetamide combined with tail clamping(stress stimulation).Rats of the medication group received intraperitoneal injection of ketotifen(1mg·kg^(-1)·d^(-1))for 14d,and those of the EA group received EA of ST36for 20min,once a day for 14d.An air-balloon was inserted into the rat’s stomach for recording changes of the intragastric pressure(mL/mm Hg)via a pressure transducer.The visceral hypersensitivity was assessed using abdominal withdrawal reflex(AWR)score and the number and degranulation of mast cells of gastric mucosa were observed using toluidine blue staining.The expression levels of TRPV1and proteinase activated receptor 2(PAR2)in the stomach were observed using immunofluorescence histochemistry and Western blot,separately,and the contents of SP and CGRP in the stomach detected using ELISA.Results When the intragastric pressure was at 50,60and 70mm Hg,the gastric compliance was significantly decreased(P<0.01),and the levels of visceral sensitivity increased in the model group(P<0.01)。TRPV1immunofluorescence tensity,expression of PAR2and TRPV1proteins,and contents of SP and CGRP in the stomach were considerably up-regulated in the model group compared with the normal control group(P<0.01).In comparison with the model group,under intragastric pressure of 50,60and 70mm Hg,the gastric compliance was obviously increased,and the visceral hypersensitivity decreased in the EA group(P<0.01,P<0.05).TRPV1immunofluorescence intensity,expression levels of PAR2and TRPV1proteins,and the contents of SP and CGRP in the stomach were considerably down-regulated in both medication and EA groups compared with the model group(P<0.01,P<0.05).The therapeutic effect of EA was significantly superior to that of medication in up-regulating the gastric compliance(at 70mm Hg),and down-regulating the contents of SP and CGRP(P<0.05).No significant differences were found between the EA and medication groups in up-regulating gastric compliance at intragastric pressure of 50and 60mm Hg,and in down-regulating the visceral sensitivity,TRPV1fluorescence intensity,and expression of PAR2and TRPV1proteins(P>0.05).Toluidine blue staining showed an apparent increase of mast cell number with obvious degranulation in the gastric mucosa of rats in the model group,which was milder in the EA and medication groups.Conclusion EA of ST36can suppress visceral hypersensitivity and increase the gastric compliance in FD rats,which may be related with its effects in inhibiting the activation of gastric mast cells,and down-regulating the expression of gastric PAR2and TRPV1proteins and SP and CGRP contents.