Klotho蛋白对急性心肌梗死小鼠肠道菌群及心功能的影响.pdf

基金项目:国家自然科学基金资助项目(81871359)作者单位:210029南京医科大学第一附属医院老年心血管科通信作者:许迪,电子信箱:xudi Klotho 蛋白对急性心肌梗死小鼠肠道菌群及心功能的影响李岩松王凯李钟鸣刘先玲丁胤彰孙燕马文杰许迪摘要目的研究 Klotho(KL)蛋白对急性心肌梗死(以下简称心梗)小鼠肠道菌群及心功能的影响。方法将 24 只 6 周龄雄性 C57BL/6 小鼠按照随机数字表法分为 3 组,即心梗组、KL 组和对照组。心梗组和 KL 组小鼠接受急性心肌梗死手术,对照组小鼠接受假手术,术后每组均存活 7 只小鼠。术后第 2 天起对 KL 组小鼠腹腔注射 KL(20ng/kg),每两天注射 1 次,心梗组和对照组小鼠腹腔注射 0.9%氯化钠溶液(15ml/kg),30 天后对小鼠进行超声心动图检查;取小鼠肠道内粪便进行 16S rDNA 基因测序,分析菌群的 多样性、多样性和菌群结构;采集小鼠心肌组织,采用实时荧光定量聚合酶链反应(real-time quantitativepolymerase chain reaction,RT-qPCR)技术检测小鼠心肌 B 型脑钠肽(B-type natriuretic peptide,BNP)、肿瘤坏死因子-(tumornecrosis factor-,TNF-)和转化生长因子-1(trarisforming growth factor-1,TGF-1)的 mRNA 的表达水平。结果主成分分析(principal component analysis,PCA)表明,KL 组与心梗组小鼠肠道菌群构成上存在明显差异。门水平上,心梗组较对照组小鼠拟杆菌门相对丰度增高,厚壁菌门相对丰度降低;属水平上,心梗组小鼠 Muri 菌增加,毛螺菌科 NK4A136 组菌群减少。经过KL 治疗后,上述菌群相对丰度均得到一定的改善。KL 组较其他两组 Epsilonbacteraeota 菌门、杜氏杆菌和幽门螺杆菌相对丰度均明显升高。在 BNP、TNF-和 TGF-1mRNA 的表达上,KL 组较心梗组有明显下降。超声心动图结果显示,KL 组左心室射血分数和缩短分数均较心梗组明显改善。结论KL 具有改善急性心肌梗死小鼠肠道菌群紊乱和心功能的作用,为 KL 的实际应用提供了新的思路和依据。关键词Klotho 蛋白急性心肌梗死肠道菌群心功能中图分类号R541.4文献标识码ADOI 10.11969/j.issn.1673-548X.2023.07.007Effects of Klotho Protein on Intestinal Flora and Cardiac Function in Mice with Acute Myocardial Infarction.LI Yansong,WANG Kai,LIZhongming,et al.Department of Geriatrics,The First Affiliated Hospital of Nanjing Medical University,Jiangsu 210029,ChinaAbstractObjectiveTo evaluate the effects of Klotho(KL)protein on intestinal flora and cardiac function in mice with acute my-ocardial infarction(AMI).MethodsTwenty-four 6-week male C57BL/6mice were randomly divided into three groups:MI group,KLgroup and control group.Mice in MI group and KL group received AMI operation,while control group received sham operation.Sevenmice survived in each group after operation.KL(20ng/kg)was injected intraperitoneally in KL group from the second day after opera-tion,once every two days,and 0.9%sodium chloride solution(15ml/kg)was injected intraperitoneally in the other two groups.After30days,the mice were examined by echocardiography and the feces of mice were taken for 16S rDNA gene sequencing,analyzed the di-versity,diversity and structural features of gut microbiota.The myocardial tissue of mice was collected and the mRNA expression of B-type natriuretic peptide(BNP),tumor necrosis factor-(TNF-)and trarisforming growth factor-1(TGF-1)in mice myocardi-um was detected by real-time quantitative polymerase chain reaction(RT-qPCR)technique.ResultsPrincipal component analysis(PCA)showed that there were significant differences in the composition of intestinal flora between KL group and MI group.At the phylumlevel,the relative abundance of bacteroidetes in MI group was higher than that in control group,and the relative abundance of firmicuteswas lower.At the genus level,mice in MI group had more muribaculaceae and less lachnospiraceae_NK4A136.After KL treatment,therelative abundance of the above flora was improved to some extent.At the same time,we found that the relative abundance of epsilon-bacteraeota,dubosiella and helicobacter in KL group was significantly higher than that in the other two groups.The mRNA expression ofBNP,TNF-and TGF-1 in KL group was significantly lower than that in MI group.The results of echocardiography showed that theleft ventricular ejection fraction and fractional shortening in KL group were significantly improved compared with those in MI group.92医学研究杂志 2023 年 7 月第 52 卷第 7 期论著ConclusionKL can improve the disturbance of intestinal flora and cardiac function in mice with AMI,which provides a new idea and ba-sis for the practical application of KL.Key wordsKlotho;Acute myocardial infarction;Intestinal flora;Cardiac function急性心肌梗死作为一种缺血性心脏病,具有极高的致残率和病死率。每年全球大约有 1600 万人死于急性心肌梗死引起的心力衰竭1。目前,我国心血管病发生率正处于持续上升阶段。据推算,全国约有3.3 亿人患有心脏病,其中冠心病患病人数达到了1139 万。自 2005 年以来,我国急性心肌梗死病死率呈快速上升趋势,2005 年我国城乡地区急性心肌梗死病死率分别为 42.1/10 万和 22.2/10 万,而 2018年我国 城 乡 地 区 急 性 心 肌 梗 死 病 死 率 则 达 到 了120.18/10 万和 128.24/10 万2。吸烟、肥胖、高血压、高血脂、糖尿病等作为急性心肌梗死的传统危险因素,已被广大群众所重视。近些年,越来越多的研究表明,肠道菌群在人类心血管疾病的发生和发展中发挥着重要的作用3。良好的肠道菌群环境不仅可以通过调节体内脂质代谢、抑制炎性反应和氧化应激、调控免疫应答等途径缓解动脉粥样硬化,有效降低急性心肌梗死风险,减少急性心肌梗死后不良事件的发生,还可以通过改善血压、抑制心肌纤维化、改善心脏重构发挥心脏保护作用3 5。Klotho(KL)是一种抗衰老基因,最早于 1997 年在转基因小鼠体内发现6。研究表明,小鼠体内 KL水平升高可以延长小鼠寿命,改善认知功能,同时能有效预防心脏病、肾脏病、血管钙化、氧化应激,而 KL缺乏的小鼠则表现出明显的衰老体征,如寿命短、生长发育迟缓、脊柱后凸、血管钙化、骨质疏松、认知障碍、多器官萎缩等6,7。人类的 KL 基因位于 13 号染色体上,长约 50kb,由 5 个外显子组成,在预防心血管疾病,特别是维持心脏和血管功能方面发挥着重要作用。KL 能预防心肌肥大,改善心肌重塑,防止血管钙化和动脉粥样硬化。因此调节血浆中 KL 水平及其在心肌细胞中的表达对于改善细胞代谢、维持心功能和预防某些疾病至关重要8。但 KL 对急性心肌梗死后小鼠肠道菌群及心功能的影响目前鲜有报道。本研究通过建立小鼠急性心肌梗死模型,应用 16SrDNA 测 序 技 术 和 实 时 荧 光 定 量 聚 合 酶 链 反 应(real-time quantitative polymerase chain reaction,RT-qPCR)技术,比较分析了 KL 治疗的急性心肌梗死后小鼠肠道菌群的结构变化及相关心力衰竭指标的差异。材料与方法1.动物和实验设计:本实验方案按照美国国家卫生研究院(National Institutes of Health,NIH)指南进行,并经南京医科大学实验动物伦理学委员会批准(许可证号:IACUC-2102002)。24 只体质量为 20 25g 的 6 周龄雄性 C57BL/6 小鼠购自北京维通利华实验动物技术有限公司。小鼠重组 Klotho 蛋白(货号:1819-KL-050)购自美国 R&D Systems 公司。所有动物均在 22 24 温度下饲养,每天光暗循环12h,能自由饮食饮水。适应 1 周后,7 只小鼠接受假急性心肌梗死手术,17 只小鼠接受急性心肌梗死手术。急性心肌梗死手术按如下方法进行:用戊巴比妥钠(45mg/kg)麻醉小鼠,在第 2、3 肋间打开胸腔,暴露心脏,用 7-0 缝 合 线 结 扎 左 前 降 支(