肥大细胞和P物质参与急性胃黏膜损伤大鼠体表穴位的敏化过程

Mast Cell and Substance P Are Involved in the Process of Acupoint Sensitization Induced by Acute Gastric Mucosal Injury

目的:观察急性胃黏膜损伤后伊文思蓝(EB)渗出穴区肥大细胞聚集数量和脱颗粒变化及其局部P物质(SP)的表达,以揭示病理状态下穴位的组织细胞特征,确定肥大细胞和SP参与穴位敏化的过程。方法:大鼠随机分为模型组和正常对照组,每组15只。采用空腹稀盐酸灌胃造成急性胃黏膜损伤模型。5 h后尾静脉注射EB,模型组取EB渗出点"脾俞""胃俞"和旁开2 mm处的皮肤和皮下组织,正常对照组取对应点皮肤和皮下组织。采用甲苯胺蓝染色法标记这些部位肥大细胞的聚集分布数量及脱颗粒特征。采用免疫组织化学方法显示局部SP的分布并进行Western blot相对定量测定。结果:急性胃黏膜损伤后EB体表穴区渗出点的皮肤和皮下组织中肥大细胞呈现聚集,其数量和脱颗粒数明显多于正常对照组和"脾俞""胃俞"旁开对照组(P<0.05,P<0.001),脱颗粒率也显著高于旁开点和正常对照组(P<0.01)。在EB渗出点和旁开部位均可见SP蛋白的表达水平显著高于正常组(P<0.01),其阳性纤维多分布于皮下。结论:在急性胃黏膜损伤时,体表出现的敏化穴位中肥大细胞参与了与疾病相关的体表穴位敏化过程,且其局部的SP表达水平增高。

Objective To observe changes of mast cells（MCs） number and morphology,and substance P（SP） expression in Evans blue（EB） extravasated region around acupoint ＂Pishu＂（BL 20） and ＂Weishu＂（BL 21） after acute gastric mucosal injury（AGMI） so as to investigate the mechanism underlying visceral problems-induced acupoint activation.Methods Thirty adult Wistar rats were randomly divided into normal control（n=15） and AGMI groups（n=15）.AGMI model was duplicated by perfusing the rats with 0.5 mol/L HCl（1 mL/100 g） after fasting for 20 h.Five hours after AGMI,the rats were treated by tail-intravenous injection of EB dye（5 mg/100 g,50 mg/mL in normal saline） for inducing dye-plasma extravasation in the skin around BL 20,BL 21 regions,etc.at the back.The rats of the normal control group were treated with tail-intravenous injection of 0.9% NaCl.The skin and subcutaneous tissues（2 mm×2 mm） of extravasated EB dye points（BL 20 or BL 21 region） and those 2 mm lateral to the extravasated EB dye points in the model group and the corresponding points in the normal control group were sampled（followed by fixing them in 4% paraformaldehyde）,sectioned and stained by toluidine blue（for labeling MCs）.The expression of SP in the extravasated EB dye skin and subcutaneous tissues was detected by immunohistochemistry（n=5） and western blot（n=5） respectively.The number of MCs in these samples was counted and the degranulation rate of MCs calculated.Results The total number of MCs and the number of degranulated MCs were significantly more in the EB extravasation points（correspon-ding to BL 20/BL 21 area） of AGMI group than those in the control spots of AGMI group and than those in the normal control group（P0.05,P0.001）.The degranulation rate of MCs was significantly higher in the EB extravasation points of AGMI group than those in the control spots of AGMI group and in the normal control group（P0.01）.In comparison with normal control group,the SP expression level was increased consideraly in the control spots of AGMI group and AGMI group（P0.01）.Conclusion After AGMI,the numbers of MCs and the degranulated MCs,and the SP expression level in BL 20/BL 21 area were increased signi-ficantly,suggesting an involvement of MCs and SP in the process of AGMI-induced activation of acupoints.