洛哌丁胺诱导慢传输型便秘小鼠结肠肌间神经元的变化

Loperamide induces neuronal apoptosis in myenteric plexus of mice with slow transit constipation

目的探讨洛哌丁胺诱导慢传输型便秘(STC)小鼠结肠肌间神经元的变化。方法将20只SPF级C57BL/6J小鼠分为对照组(CON组)和慢传输型便秘组(STC组)各10只。STC组给予洛哌丁胺溶液(10 mg/kg)灌胃处理制作STC模型,CON组给予等量生理盐水灌胃,每日2次,持续12 d。造模期间监测小鼠皮毛色泽、精神状态、活动度、体质量及摄食量。造模结束后禁食不禁水16 h,检测肠道传输时间和粪便性状。然后采用颈椎脱臼法处死后取结肠组织,使用改良肠神经全层铺片技术及免疫荧光染色检测结肠肌间神经元形态、数目及凋亡情况。结果造模期间小鼠皮毛开始变暗,精神萎靡,活动减少;进食量减少且在第3天达最低,体质量减低且在第6天达最低。造模结束后STC组与CON相比6 h内粪便粒数、湿重、含水量均减少,肠道传输时间明显延长,差异均有统计学意义(P均<0.05)。免疫荧光显示,与CON组相比,STC组远端结肠肌间HuC/D+神经元数目减少,神经纤维荧光强度减弱(P均<0.05)。免疫荧光双染显示,与CON组相比,STC组Casepase3+HuC/D+神经元数目增加(P<0.05)。结论洛哌丁胺诱导的STC小鼠结肠肌间神经丛神经元减少、凋亡程度增加,这可能是结肠慢传输的关键因素。

Objective To investigate the changes of colonic myenteric neurons in mice with loperamide-induced slow transit constipation(STC).Methods Twenty SPF C57BL/6J mice were divided into the control(CON)and STC groups.Loperamide(10 mg/kg)was administered by gavage in the STC group,and the equal volume of saline was given to mice in the control group,twice a day,for 12 days.The mice were monitored for fur color,mental status,activity,weight,and food intake during the period.After the end of study,the mice were fasted for 16 h,and the bowel transit time was detected.The feces were collected to observe the fecal properties.After the mice were executed,the colonic tissues were taken.Modified whole mount stretch preparation and immunofluorescence staining were used to detect the morphology,number,and apoptosis of neurons of the myenteric plexus.Results During the modeling period,mice of the STC group gradually appeared to have darkened fur,depression,and less activity;food intake decreased and reached its lowest on day 3;body mass was reduced at the same time and was the lowest on day 6.Compared with the CON group,the number,wet weight and water content of fecal pellets were reduced in the STC group within 6 h and the bowel transit time was significantly delayed at the end of modeling,with statistically significant differences(all P<0.05).Immunofluorescence showed that compared with the CON group,the number of HuC/D+neurons decreased,and nerve fiber fluorescence intensity was reduced in the colonic myenteric plexus of the STC group(both P<0.05).Immunofluorescence double-staining results showed that the number of Casepase3+HuC/D+neurons from the colonic myenteric plexus in the STC group increased as compared with that of the CON group(P<0.05).Conclusion The colonic myenteric neurons decrease and the neuronal apoptosis increases in the loperamide-induced STC mice,which may be the key factors for STC.