基于慢传输型便秘大鼠结肠ICC及AQP3变化的温阳益气方改善便秘的作用机制探讨

From the changes of colonic ICC and AQP3 in rats with slow transit constipation to explore the mechanism of Wenyang Yiqi prescription in improving constipation

目的探讨温阳益气方改善便秘的作用机制及其对模型大鼠结肠ICC及AQP3的影响。方法采用洛哌丁胺灌胃制备便秘大鼠模型,造模组大鼠50只,随机分为:模型组,温阳益气方低、中、高剂量组和莫沙必利组(每组各10只)。另有10只作为正常对照组。记录大鼠一般情况、粪便含水量,肠道传输功能,同时测定结肠肌电,ELISA法测定各组大鼠血清SP、VIP的变化,免疫组化检测结肠AQP3、c-kit蛋白的变化,Real-time PCR检测结肠PKA mRNA、AQP3 mRNA、NK-1mRNA,c-kit mRNA的表达情况。结果与模型组相比,温阳益气方高剂量组可显著增加粪便含水量(P<0.05)。温阳益气方低、中、高剂量组大鼠肠道活性炭推进率随着剂量变化而不同程度增加,以中、高剂量组明显增高(P<0.05)。结肠肌电检测结果提示温阳益气汤中、高剂量组在振幅、频率变异系数、振幅变异系数水平明显降低(P<0.05)。免疫组化结果显示温阳益气方中、高剂量组能抑制AQP3表达,提升c-kit蛋白表达(P<0.05),同时与莫沙必利组比较,温阳益气方高剂量组c-kit蛋白表达有统计学意义(P<0.05)。与模型组相比,温阳益气方中、高剂量组血清SP、VIP表达量均明显增高(P<0.05)。RT-PCR结果提示治疗组(中药中、高剂量组及莫沙必利组)大鼠结肠AQP3、PKA mRNA表达量明显降低(P<0.05),治疗组(中药中、高剂量组及莫沙必利组)大鼠结肠c-kit、NK-1 mRNA表达量明显升高(P<0.05)。结论温阳益气方治疗便秘的机制可能是通过增加血清中SP和VIP的含量,调节PKA mRNA和NK-l mRNA的表达,从而调节肠道AQP3及c-kit的表达,通过增加肠道粪便含水量,同时加快肠道蠕动,达到缓解和治疗便秘的作用。

Objective To explore the mechanism of Wenyang Yiqi prescription in improving constipation and its effect on colonic ICC and AQP3 in constipation model rats. Methods A rat model of constipation was prepared by loperamide gavaged. 50 rats were randomly divided into: model group, Wenyang Yiqi prescription low, medium and high dose group and mosapride group(10 mice each). And another 10 were used as normal controls. The general condition of rats, water content of feces, intestinal transit function, and determination of colonic myoelectricity were measured. The changes of serum SP and VIP in each group were detected by ELISA. The changes of AQP3 and c-kit protein in colon were detected by immunohistochemistry. Real-time PCR to detect the expression of PKA mRNA, AQP3 mRNA, NK-1 mRNA and c-kit mRNA in colon. Results Compared with the model group, Wenyang Yiqi prescription high dose group can significantly increase fecal water content(P<0.05). The intestinal propellant propellant rate of rats in the low, middle and high dose groups of Wenyang Yiqi prescription increased with the dose change, and increased significantly in the middle and high dose groups(P<0.05). The results of colonic myoelectric examination showed that the amplitude, frequency coefficient of variation and amplitude coefficient of variation were significantly lower in the middle and high dose groups of Wenyang Yiqi prescription(P<0.05). The results of immunohistochemistry showed that the middle and high doses of Wenyang Yiqi prescription could inhibit the expression of AQP3 and increase the expression of c-kit protein(P<0.05). At the same time, compared with the mosapride group, the expression of c-kit protein in the high dose group of Wenyang Yiqi prescription Statistically significant(P< 0.05). Compared with the model group, the expression levels of serum SP and VIP in Wenyang Yiqi Recipe and high dose group were significantly increased(P<0.05). The results of RT-PCR indicated that the expression of AQP3 and PKA mRNA in the colon of the treatment group(high dose group and mosapride group) was significantly decreased(P<0.05). The treatment group(high dose group and Mosha) The expression of c-kit and NK-1 mRNA in the colon of rats was significantly increased(P<0.05). Conclusion The mechanism of Wenyang Yiqi prescription in treating constipation may be through regulating the content of SP and VIP in serum, regulating the expression of PKA mRNA and NK-l mRNA, thereby regulating the expression of AQP3 and c-kit in the intestine, and increasing the intestinal fecal content. The amount of water, while speeding up the intestinal peristalsis, to relieve and treat constipation.