摘要
目的构建并评价符合“虚-瘀-毒”病机的冠心病急性心肌梗死(AMI)病证结合小鼠模型。方法将48只ICR小鼠按随机数字表法分为假手术组、正常饮食组、高胆碱组、氧化三甲胺(TMAO)组,1~8周各组给予相应的饮食、饮水干预处理;第9周开始时,正常饮食组、高胆碱组及TMAO组均给予冠状动脉(LAD)结扎术处理,假手术组只穿线不结扎,饮食饮水处理保持不变;从宏、微观表征不同层面,对小鼠的一般体征、体质量、饮食饮水量、二便、耳廓爪脚状态、行为学等数据进行采集比较,判断高胆碱干预AMI小鼠模型“虚-瘀-毒”的证候类型及指标变化;12周后,采用超声心动图、病理组织苏木精-伊红(HE)染色和Masson染色分别评估小鼠心功能、心肌组织细胞形态变化和心肌纤维化水平;最后,通过免疫荧光法观察各组小鼠心肌组织炎症因子荧光强度,评估模型稳定性和可靠性。结果采集宏观体征发现,心肌梗死术后的各组小鼠体型消瘦、毛色无泽、精神萎靡、活动度降低,其中,高胆碱组和TMAO组小鼠对刺激反应更为迟钝。高胆碱组和TMAO组在1~4周呈现饮食量增加但体质量增长缓慢的特征,5~8周发展为“多饮、多食、体质量下降”趋势,并伴有小便黄赤、大便干燥等变化(P<0.01),9~12周术后体质量明显降低(高胆碱组最明显);高胆碱组和TMAO组表现出耳廓及爪脚红绿蓝值异常改变(P<0.01);行为学检测显示其旷场活动能力明显下降(P<0.01)。心功能及病理学检测发现,相较于假手术组及正常饮食组,高胆碱组和TMAO组小鼠心脏左心室舒张期容积和收缩期容积均升高(P<0.01)、射血分数和短轴缩短率均降低(P<0.01)、心脏指数升高(P<0.05);心肌组织HE染色表明,高胆碱组和TMAO组有较多心肌细胞核固缩、炎细胞浸润;Masson染色显示,高胆碱组和TMAO组梗死边缘区有大量蓝染胶原纤维分布,纤维化程度加重(P<0.05)。免疫荧光检测发现,相较于假手术组及正常饮食组,高胆碱组和TMAO组小鼠白细胞介导(IL)-1β、肿瘤坏死因子(TNF)-α荧光水平升高(P<0.01)。结论高胆碱饮食结合LAD结扎能够成功构建“虚-瘀-毒”病机的冠心病AMI病证结合动物模型。该模型不仅体现了中医理论对冠心病“虚-瘀-毒”病机的认识,同时,为评价中药复方的干预效应及开展中医证候研究提供参考依据。
Objective To establish and evaluate a mouse model of acute myocardial infarction(AMI)with coronary heart disease(CHD)that integrates syndrome differentiation with disease diagnosis,based on the"deficiency-stagnation-toxicity"pathogenesis.Methods Forty-eight ICR mice were randomly divided into four groups using a random number table:sham-operated,normal diet,high-choline,and trimethylamine N-oxide(TMAO).From weeks 1 to 8,each group received corresponding dietary and water interventions.From the 9th week,the normal diet,high-choline,and TMAO groups underwent coronary artery ligation(left anterior descending artery,LAD).In contrast,the sham-operated group only had suture placement without ligation,maintaining the same dietary and water interventions.Data on general signs,body weight,food and water intake,urine and feces,auricle and paw conditions,and behavioral patterns were collected and compared macroscopically and microscopically to determine the syndrome type of the high-choline-induced AMI mouse model and observe changes in the"deficiencystagnation-toxicity"syndrome indicators.After 12 weeks,echocardiography,hematoxylin-eosin(HE)staining,and Masson′s trichrome staining were used to assess cardiac function,myocardial tissue cellular morphology changes,and myocardial fibrosis levels,respectively.The stability and reliability of the model were evaluated by observing the fluorescence intensity of inflammatory cytokines in the myocardial tissues of each group using immunofluorescence.Results Mice in all groups post-AMI surgery exhibited significant weight loss,dull fur,lethargy,and reduced activity.Mice in the high-choline and TMAO groups showed more sluggish responses to stimuli.The high-choline and TMAO groups displayed increased food intake but slow weight gain from weeks 1 to 4,developing into a trend of"increased food and water intake with weight loss"from 5 to 8 weeks,accompanied by yellowish urine and dry stools(P<0.01).Postoperatively(9-12 weeks),body weight significantly decreased,with the most prominent weight loss observed in the high-choline group.The high-choline and TMAO groups exhibited abnormal RGB values in auricles and paws(P<0.01),and behavioral tests showed a significant decline in openfield activity(P<0.01).Cardiac function and pathological examinations revealed that,compared with the sham-operated and normal diet groups,mice in the high-choline and TMAO groups had increased left ventricular end-diastolic and end-systolic volumes(P<0.01),decreased left ventricular ejection fraction and fractional shortening(P<0.01),and elevated heart indices(P<0.05).HE staining of myocardial tissues indicated more pyknotic nuclei and inflammatory cell infiltration in the high-choline and TMAO groups.Masson′s trichrome staining showed extensive blue-stained collagen fiber distribution in the infarct border zones of the high-choline and TMAO groups,with aggravated fibrosis(P<0.05).Immunofluorescence revealed elevated interleukin-1 beta and tumor necrosis factor-alpha levels in the high-choline and TMAO groups compared with the sham-operated and normal diet groups(P<0.01).Conclusion A high-choline diet combined with LAD ligation successfully established an animal model of AMI with CHD that integrates syndrome differentiation with disease diagnosis,based on the"deficiencystagnation-toxicity"pathogenesis.This model not only embodies the traditional Chinese medicine theory′s understanding of the pathogenic features of"deficiency-stagnation-toxicity",but also serves as a reference for assessing the interventional effects of Chinese herbal compound prescriptions and facilitating research on syndrome patterns in traditional Chinese medicine.
作者
钱向毅
郭淑贞
范昕怡
崔伶雯
何奥龙
高阔
李芳赫
于雪
王伟
QIAN Xiangyi;GUO Shuzhen;FAN Xinyi;CUI Lingwen;HE Aolong;GAO Kuo;LI Fanghe;YU Xue;WANG Wei(School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《北京中医药大学学报》
北大核心
2025年第7期919-932,共14页
Journal of Beijing University of Traditional Chinese Medicine
基金
国家重点研发计划项目(No.2022YFC3500104)。
关键词
冠心病
急性心肌梗死
虚-瘀-毒
证候
病证结合
动物模型
小鼠
coronary heart disease
acute myocardial infarction
deficiency-stasis-toxicity
syndrome
disease-syndrome combination
animal model
mice
