心阳片通过抑制骨桥蛋白介导的心脏纤维化信号改善尿毒症心肌病小鼠的作用和机制  

Effects and mechanisms of Xinyang Tablets improving uremic cardiomyopathy mice by inhibiting osteopontin-mediated cardiac fibrosis signaling

作　　者：倪世豪 何星灵 欧阳小露 张小娇 李锦 陈星伶[1,2,3,4] 刘东华 孙术宁 李姿儒 李思静 龙文杰 廖慧丽 王陵军[1,2,3,4,5] 冼绍祥[1,2,3,4] 鲁路 杨忠奇[1,2,3,4,5] NI Shihao;HE Xingling;OUYANG Xiaolu;ZHANG Xiaojiao;LI Jin;CHEN Xingling;LIU Donghua;SUN Shuning;LI Ziru;LI Sijing;LONG Wenjie;LIAO Huili;WANG Lingjun;XIAN Shaoxiang;LU Lu;YANG Zhongqi(State Key Laboratory of Traditional Chinese Medicine Syndrome,The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,China;Lingnan Medical Research Center,Guangzhou University of Chinese Medicine,Guangzhou 510405,China;Guangdong Provincial University Key Laboratory of Traditional Chinese Medicine Prevention and Treatment of Chronic Heart Failure,Guangzhou 510405,China;Guangzhou Key Laboratory for Chinese Medicine Prevention and Treatment of Chronic Heart Failure,Guangzhou 510405,China;Guangdong Provincial Clinical Research Academy of Chinese Medicine,Guangzhou 510405,China)

机构地区：[1]中医证候全国重点实验室,广州中医药大学第一附属医院,广州510405 [2]广州中医药大学岭南医学研究中心,广州510405 [3]广东省普通高校慢性心力衰竭中医药防治重点实验室,广州510405 [4]广州市慢性心力衰竭中医药防治重点实验室,广州510405 [5]广东省中医临床研究院,广州510405

出　　处：《中华中医药杂志》2024年第5期2273-2280,共8页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：2023年度广州中医药大学第一临床医学院优秀博士学位论文培育项目(No.YB202301);国家中医临床研究基地建设项目(No.国中医药科技函[2018]131号),国家自然科学基金项目(No.82374406);青年人才托举工程项目(No.2021-QNRC2-B30);广东省自然科学基金项目(No.2021A1515011457);广州市科技计划项目重点研发计划(No.202206080015);广州中医药大学青年创新拔尖人才团队培育揭榜挂帅项目。

摘　　要：目的:探究心阳片通过调控骨桥蛋白(OPN)介导的心脏纤维化信号改善尿毒症心肌病(UCM)小鼠的作用和机制。方法:采用5/6肾切除(NPM)术构建UCM小鼠模型,并随机分为模型组、心阳片组、美托洛尔组,每组15只,另外15只假手术小鼠作为假手术组。心阳片组、美托洛尔组术后分别给予心阳片(0.34 g/kg)、美托洛尔(0.35g/kg)灌胃,假手术组及模型组给予等量0.9%氯化钠溶液灌胃,治疗8周。给药1、4、8周使用小动物超声观察小鼠心功能变化;8周后观察、检测小鼠毛发光泽、精神状态、抓力、自主活动次数和肛温等阳虚证候表现;检测小鼠血肌酐、尿素氮水平;WGA染色法、TUNEL染色法、天狼猩红染色法观察小鼠心脏组织形态学变化及胶原纤维沉积情况;qPCR法检测小鼠心脏纤维化相关基因mRNA表达;免疫荧光法观察小鼠心脏α-SMA表达;通过心脏转录组数据联合实验手段探究心阳片改善UCM心脏纤维化的作用和机制。结果:与模型组比较,心阳片改善UCM小鼠毛发光泽和精神状态,改善抓力、自主活动次数和肛温(P<0.05);改善心脏收缩和舒张功能(P<0.05);改善心肌细胞面积(P<0.05);减少凋亡心肌细胞数量(P<0.05);减少心脏纤维化面积(P<0.05);降低纤维化相关基因(acta2、col1a1、col2a1、mmp3、mmp9)mRNA表达和α-SMA蛋白表达(P<0.05);而对血肌酐和尿素氮水平无明显影响。基因集富集(GSEA)分析结果显示心阳片抑制OPN信号。免疫荧光结果显示心阳片降低UCM小鼠心脏心肌细胞、巨噬细胞与成纤维细胞OPN蛋白表达(P<0.05);qPCR结果表明心阳片降低心脏巨噬细胞spp1 mRNA表达(P<0.05);ELISA检测结果表明心阳片降低血清OPN水平(P<0.05)。然而,OPN表达升高能够抵消心阳片对心脏纤维化的改善作用(P<0.05)。结论:心阳片能有效改善UCM,其作用机制可能是通过抑制OPN介导的心脏纤维化信号实现。Objective:To explore the effect and mechanism of Xinyang Tablets(XYP)on improving mice with uremic cardiomyopathy(UCM)by regulating osteopontin(OPN)-mediated cardiac fibrosis signaling.Methods:A UCM mouse model was established by 5/6 nephrectomy(NPM)and randomly divided into model group,XYP group,metoprolol group,each group with 15 mice,and another 15 sham-operated mice as sham group.XYP group and metoprolol group were given XYP(0.34 g/kg)and metoprolol(0.35 g/kg)by gavage respectively after surgery,while sham group and model group were given equal amount of 0.9%sodium chloride solution by gavage for 8 weeks.The cardiac function of mice was observed by animal ultrasound at 1,4,and 8 weeks after administration;the yang deficiency syndrome manifestations such as hair luster,mental state,grip strength,spontaneous activity times and anal temperature were observed and measured in mice after 8 weeks;the blood creatinine and urea nitrogen levels of mice were measured;WGA staining,TUNEL staining,Sirius red staining were used to observe the morphological changes and collagen fiber deposition of cardiac tissue in mice;qPCR was used to detect the mRNA expression of cardiac fibrosis-related genes in mice;immunofluorescence was used to observe the expression ofα-SMA protein in cardiac tissue of mice;the effect and mechanism of XYP on improving cardiac fibrosis in UCM were explored by combining cardiac transcriptome data with experimental methods.Results:Compared with the model group,XYP improved the hair luster,mental state,grip strength,spontaneous activity times and anal temperature(P<0.05)of UCM mice;improved cardiac systolic and diastolic function(P<0.05);improved cardiomyocyte area(P<0.05);reduced the number of apoptotic cardiomyocytes(P<0.05);reduced the area of myocardial fibrosis(P<0.05);decreased the mRNA expression of fibrosis-related genes(acta2,col1a1,col2a1,mmp3,mmp9)and expression ofα-SMA protein(P<0.05);but had no obvious effect on blood creatinine and urea nitrogen levels.Gene Set Enrichment Analysis(GSEA)re

关 键 词：尿毒症心肌病 温阳利水法 心阳片 骨桥蛋白 纤维化 

分 类 号：R285.5[医药卫生—中药学]