慢性应激大鼠模型肠道P物质及其受体表达的变化

The potential effects of chronic stress on the expression of substance P and its related receptors in rat colon

目的建立慢性应激模型，观察慢性应激对P物质（sP）及其受体表达的影响。方法将实验大鼠随机分为反复避水应激（WAS）模型组（WAS组）和对照组（SWAS组），采用连续10d的WAS建立动物模型；腹腔麻醉处死大鼠后取近端结肠，制备纵行肌（LM）和环形肌条（CM），采用浴槽实验观察WAS组和SWAS组结肠平滑肌细胞自发性的收缩活动并比较两组收缩的强度；采用酶联免疫吸附试验（ELISA）检测WAS组和SWAS组血清及结肠黏膜sP水平；采用免疫组织化学法检测神经激肽1型受体（NKlR）和神经激肽2型受体（NK2R）在结肠的分布；采用Westernblot测定NK!R和NK2R在WAS组和SWAS组中结肠肌层的表达。结果反复WAS使大鼠结肠LM和CM自发性收缩幅度增加[LM：（1．14±0．05）g比（0．94±0．04）g；CM：（0．36±0．02）g比（0．25±0．02）g，P〈0．01]，且使模型组于造模第1、3、5、7、10天的大鼠排便增加；与对照组比较，WAS组大鼠m清sP浓度显著升高[WAS组：（212．1±10．6）pg／ml比SWAS组：（105．5±4．4）pg／ml，P〈0．01]；反复避水应激使大鼠结肠黏膜sP水平增加[WAS组：（661．5±20．6）pg／mg蛋白比SWAS组：（250．1±12．1）pgJmg蛋白，P〈0．01]；NK1R主要分布于WAS和SWAS大鼠结肠黏膜上皮、固有层平滑肌细胞及肠神经元；NK2R主要分布于WAS和SWAS大鼠结肠黏膜上皮及肠神经元，固有层平滑肌细胞散在分布；反复WAS使大鼠结肠肌层NKlR表达上调而NK2R表达无明显改变。结论慢性应激诱导大鼠结肠动力增强，SP-NK1R信号系统可能参与该病理生理过程的发生。

Objective To investigate the effects of chronic stress on the expression of substance P （SP） and its related receptors in rat colon. Methods Male Wistar rats were exposed to water - avoidance stress （WAS） or sham WAS （SWAS） （ 1 h/day） for up to 10 consecutive days. The spontaneous contrac- tile activities of longitudinal muscle （LM） strip and circular muscle （CM） strip from rat colon were studied in an organ bath system. Enzyme immunoassay kit was applied to detect the serum SP level and the pres- ence of SP in the colonic mucosa. The distribution and expression of neurokinin- 1 receptor （NKIR） and neurokinin - 2 receptor （NK2R） were investigated by immunohistochemistry and Western blotting. Results The number of fecal pellets expelled per hour by the WAS rats at the day 1,3,5, 7 and 10 was significant- ly greater than that in the SWAS rats. The spontaneous contraction of both LM and CM strips from the WAS rats was significantly increased as compared with tha in the SWAS rats [ （ 1.14 ± 0. 05 ） g vs. （ 0. 94 ± 0. 04） g and （0. 36 ±0. 02） g vs. （0. 25 ±0. 02） g,P 〈0.01 ]. The serum levels of SP and the expres- sion of SP in the colonic mucosa were elevated following WAS. Immunohistochemistry proved the expression of NK1R and NK2R in mucosa, muscularis and myenteric plexus. Western blotting demonstrated stress - induced up -regulation of NK1R in colon devoid of mucosa and submucosa, but not NK2R. Conclusion Chronic stress enhanced the contraction of muscle strips from rat colon. SP - NK1R signal system might be involved in this pathological process.