消痔丸对复方地芬诺酯致小鼠便秘模型的药效评价及作用机制

目的 探究消痔丸对复方地芬诺酯致小鼠便秘模型的药效作用及机制。方法 ICR小鼠随机分为对照组、模型组、溴化甲基纳曲酮（阳性对照，58.5 mg·kg^-1）组和消痔丸低、中、高剂量（1.17、2.34、4.68 g·kg^-1）组，连续ig给药7 d，给药同时除对照组外，其余各组按照7.5 mg·kg^-1 ig复方地芬诺酯混悬液制备小鼠便秘模型。观察小鼠一般情况；末次给药结束后，各实验组禁食不禁水16 h，模型组和各给药组ig给予复方地芬诺酯，30 min后给药组ig含相应受试药的活性炭悬液，对照组、模型组ig活性炭悬液。第1批小鼠观察首次排黑便时间、6 h内排便粒数、6 h和24 h内排便质量及粪便含水率。第2批小鼠检测小肠推进率，酶联免疫吸附实验（ELISA）检测结肠组织和血清中5-羟色胺（5-HT）、P物质（SP）、血管活性肠多肽（VIP）水平变化，实时荧光定量PCR（qRT-PCR）法检测结肠组织水通道蛋白3（AQP3）、AQP9 mRNA水平，Westernblotting法检测AQP3、AQP9蛋白表达水平。结果 与模型组相比，各给药组小鼠粪便干结情况有所好转，排便量均有不同程度的增加，体质量有所增加，精神状态逐渐好转。与模型组相比，各给药组均能显著缩短排首粒黑便时间，提高6 h排便粒数及粪便含水率、6 h及24 h排便质量（P＜0.05、0.01、0.001）；除消痔丸低剂量组，其余各给药组小鼠24 h粪便含水率均显著增加（P＜0.01、0.001）；各给药组小肠炭末推进距离及炭末推进率均明显提高（P＜0.05、0.01、0.001）；各给药组均能显著降低结肠组织中VIP水平（P＜0.01、0.001），除消痔丸低剂量组，其余各给药组均能显著提高结肠组织中5-HT、SP水平（P＜0.01、0.001）；各给药组均能显著增加血清中5-HT、SP水平并降低VIP的表达水平（P＜0.05、0.01、0.001）；各给药组AQP3蛋白表达量均显著下降（P＜0.01、0.001），AQP9蛋白表达量均显著上升（P＜0.05、0.01、0.001）；各给药组AQP3 mRNA水平均显著下降（P＜0.01、0.001），AQP9 mRNA水平均显著上升（P＜0.05、0.01、0.001）。结论 消痔丸对复方地芬诺酯诱导的小鼠便秘模型有显著的治疗效果，可以显著缩短便秘小鼠排首粒黑便时间、增加排便粒数并提高粪便含水率、显著促进小肠推进作用，作用机制与调节肠液分泌相关因子5-HT、SP、VIP等的释放以及AQP3、AQP9表达相关。

Efficacy and mechanism of Xiaozhi Pills on constipation mice induced by diphenoxylate

Objective To investigate the therapeutic effect of Xiaozhi Pills (XZP) on constipation induced by diphenoxylate in mice and its mechanism. Methods ICR mice were randomly divided into control group, model group, methylnaltrexone bromide group (positive control, 58.5 mg·kg^-1) and XZP low, medium, and high doses group (1.17, 2.34, and 4.68 g·kg^-1). Each group was administered by gavage of the corresponding drug once a day for seven days. Except for the control group, the other groups were given diphenoxylate suspension at 7.5 mg·kg^-1 body weight by gavage on the same day. Observe the general situation of mice. After the end of the last administration, each experimental group fasted for 16 hours and couldn''t help but water. The model group and each administration group were given compound diphenoxylate by ig. After 30 minutes, the administration group ig contained activated carbon suspension of the corresponding test drug, while the control group and model group ig contained activated carbon suspension. The first batch of mice were observed for the first time of black stools, the number of stools within six hours, the quality of stools within 6 and 24 hours, and the fecal moisture content. The second batch of mice were tested for intestinal propulsion rate, enzyme-linked immunosorbent assay (ELISA) was used to detect changes in levels of serotonin (5-HT), substance P (SP), and vasoactive intestinal polypeptide (VIP) in colon tissue and serum, real-time fluorescence quantitative PCR (qRT-PCR) was used to detect levels of aquaporin 3 (AQP3) and AQP9 mRNA in colon tissue, and Western blotting was used to detect protein expression levels of AQP3 and AQP9. Results Compared with the model group, the fecal dryness of mice in each treatment group improved, with varying degrees of increase in fecal volume, increased body mass, and gradually improved mental state. Compared with the model group, each medication group significantly shortened the first black stool discharge time, improved the number of fecal particles and fecal moisture content at six hours, and improved the quality of feces at 6 and 24 hours (P < 0.05, 0.01, 0.001); Except for the low-dose group of XZP, the 24-hour fecal moisture content of mice in all other treatment groups significantly increased (P < 0.01, 0.001). The distance and rate of small intestine charcoal powder propulsion in each administration group were significantly increased (P < 0.05, 0.01, 0.001). All treatment groups were able to significantly reduce VIP levels in colon tissue (P < 0.01, 0.001). Except for the low-dose group of XZW, all other treatment groups were able to significantly increase the levels of 5-HT and SP in colon tissue (P < 0.01, 0.001). Each administration group significantly increased the levels of 5-HT and SP in serum and decreased the expression level of VIP (P < 0.05, 0.01, 0.001). The expression of AQP3 protein significantly decreased in each treatment group (P < 0.01, 0.001), while the expression of AQP9 protein significantly increased (P < 0.05, 0.01, 0.001). The levels of AQP3 mRNA was significantly decreased in each treatment group (P < 0.01, 0.001), while the levels of AQP9 mRNA was significantly increased (P < 0.05, 0.01, 0.001). Conclusion XZP had a significant therapeutic effect on the constipation model induced by compound diphenoxylate in mice. It can significantly shorten the first black stool time, increase the number of stool particles and increase the fecal water content, and significantly promote small intestine propulsion. The mechanism of action is related to regulating the release of intestinal secretion related factors such as 5-HT, SP, VIP, and the expression of AQP3 and AQP9.