不同剂量环磷酰胺联合复方地芬诺酯诱导血虚型便秘大鼠模型的优化研究

Optimization of a rat model of blood-deficiency constipation induced by cyclophosphamide combined with compound diphenoxylate at different Doses

关键词:  血虚型便秘  大鼠  复方地芬诺酯  环磷酰胺  剂量优化

Key words:  blood-deficiency constipation  rats  compound diphenoxylate  cyclophosphamide  dose optimization

摘要:

[目的] 本研究基于前期实验基础，旨在优化探索符合中医“血虚便秘”证候的大鼠模型，并通过实验验证筛选最佳实验条件。[方法] 本研究采用44只SPF级SD大鼠，随机分为正常对照组和血虚便秘模型组（n=6），模型组根据环磷酰胺（CTX）剂量梯度分为高（80 mg/kg）、中（70 mg/kg）、低（60 mg/kg） 3个剂量组，每组6只，各组均给予复方地芬诺酯10 mg/kg灌胃处理，连续造模14 d。通过监测大鼠一般状态、存活率、体质量、摄食量、排便功能（6 h/24 h粪便参数、首粒红便排出时间及小肠推进率）和血常规指标评估造模效果；采用HE染色和AB-PAS染色观察结肠组织病理学变化，并运用蛋白免疫印迹（Western Blot，WB）技术检测结肠组织中胆碱乙酰转移酶（ChAT）、神经元型一氧化氮合酶（nNOS）、干细胞因子（SCF）及水通道蛋白3（AQP3）的表达水平。基于上述指标筛选最佳剂量后，通过增大样本量（n=10）重复实验并延长观察期7 d进行模型稳定性验证。[结果] 采用复方地芬诺酯（10 mg/kg）联合不同剂量CTX（高剂量80 mg/kg、中剂量70 mg/kg、低剂量60 mg/kg）造模14 d，实验结果显示，高、中剂量组分别出现67%和84%的存活率，并伴有虚弱、口鼻出血及尿血等不良反应，而低剂量组存活率达100%。通过扩大样本量（n=10）重复实验并延长观察期至7 d验证模型稳定性。实验证实，低剂量组仍保持100%存活率，表明该剂量方案具有最佳稳定性。与正常组相比，血虚便秘高、中、低剂量组体质量、摄食量、6 h及24 h粪便颗粒数、湿质量和含水率显著下降（P<0.05或P<0.01），首粒红便排出时间显著延长（P<0.01），小肠推进率显著降低（P<0.05或P<0.01）。血常规结果显示，血虚便秘高、中、低组大鼠的白细胞（WBC）、红细胞（RBC）、血红蛋白（HGB）和红细胞压积（HCT）均显著下降（P<0.05或P<0.01），表明大鼠出现血虚症状。HE和AB-PAS染色结果显示，血虚便秘高、中、低组大鼠的结肠肌层变薄，黏膜层分离，隐窝萎缩，排列紊乱，杯状细胞和粘液分泌减少（P<0.05或P<0.01）。WB结果显示，血虚型便秘大鼠的SCF、ChAT蛋白及ChAT/nNOS比值显著降低，nNOS和AQP3表达显著升高（P<0.05），证实血虚型便秘大鼠肠动力显著下降。复方地芬诺酯联合不同剂量的CTX造模14天，能成功复制血虚型便秘模型，其中以低剂量组为优。[结论] 采用60 mg/kg的CTX联合10 mg/kg的复方地芬诺酯连续造模14天，可成功复制符合中医“血虚便秘”证候的大鼠模型。该优化方法具有较好的可控性、简便性，并且死亡率较低，适用于血虚型便秘的相关药效和机制研究。

Abstract:

[Objective] Based on preliminary experimental findings，this study aims to optimize and establish a rat model that accurately replicates the“blood-deficiency constipation”syndrome in traditional Chinese medicine （TCM），while identifying the optimal experimental parameters through systematic validation. [Methods] A total of 44 SPF-grade SD rats were randomly allocated into a normal control group and model groups（n=6 per group）. The model groups were further stratified into three subgroups based on cyclophosphamide（CTX）dosage gradients：high- dose（80 mg/kg），medium-dose（70 mg/kg），and low-dose（60 mg/kg）. All model subgroups received intragastric administration of compound diphenoxylate（10 mg / kg）for 14 consecutive days. Model efficacy was assessed by monitoring general status，survival rate，body weight，food intake，defecation function（6 h/24 h fecal parameters， first red stool excretion time，and small intestinal propulsion rate），and hematological indices. Histopathological alterations in colonic tissues were evaluated via hematoxylin-eosin（HE）and Alcian blue-periodic acid-Schiff（AB- PAS）staining，while protein expression levels of choline acetyltransferase（ChAT），neuronal nitric oxide synthase （nNOS），stem cell factor（SCF），and aquaporin-3（AQP3）were quantified by Western blot analysis. Following optimization of the dosage regimen based on these parameters，model stability was verified through replicate experiments with an expanded sample size（n=10）and an extended observation period. [Results] The high- and medium-dose CTX groups showed survival rates of 67% and 84%，respectively，accompanied by adverse reactions including weakness，epistaxis，and hematuria，whereas the low-dose group maintained 100% survival. The low-dose regimen demonstrated optimal stability，confirmed by replicate experiments（n=10）with 100% survival. Compared with the normal control group，all model groups showed significant reductions in body weight，food intake，6 h/24 h fecal pellet count，fecal wet weight，and fecal water content（P<0.05 or P<0.01），along with prolonged first red stool excretion time（P<0.01）and decreased small intestinal propulsion rate（P<0.05 or P<0.01）. Hematological analysis revealed significant decreases in white blood cells（WBC），red blood cells（RBC），hemoglobin（HGB），and hematocrit （HCT）levels（P<0.05 or P<0.01），confirming characteristic blood-deficiency symptoms. Histopathological examination via HE and AB-PAS staining demonstrated colonic alterations including thinning of the muscular layer， mucosal detachment，crypt atrophy，disorganized arrangement，and reduced goblet cells with diminished mucus secretion（P<0.05 or P<0.01）. Western blot analysis showed significantly decreased expression of SCF and ChAT proteins with a reduced ChAT/nNOS ratio，while nNOS and AQP3 expressions were markedly elevated（P<0.05）， indicating impaired intestinal motility in the blood-deficiency constipation model. [Conclusion] Administration of 60 mg/kg CTX combined with 10 mg/kg compound diphenoxylate for 14 consecutive days successfully establishes a rat model that accurately replicates the“blood-deficiency constipation”syndrome as defined in TCM. This optimized protocol demonstrates favorable controllability，operational simplicity，and low mortality，making it highly suitable for investigating pharmacological efficacy and underlying mechanisms related to blood-deficiency constipation.