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OA

[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.