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吕李飞, 朱婷婷, 丁帆, 路迎冬, 崔向宁.慢性心力衰竭气虚血瘀证模型大鼠肠道菌群诱发心肌炎症的特征变化[J].湖南中医药大学学报英文版,2024,44(10):1771-1780.[Click to copy
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| 慢性心力衰竭气虚血瘀证模型大鼠肠道菌群诱发心肌炎症的特征变化 |
| 吕李飞,朱婷婷,丁帆,路迎冬,崔向宁 |
| (中国中医科学院广安门医院, 北京 100053) |
| 摘要: |
| 目的 探究慢性心力衰竭气虚血瘀证模型大鼠肠道菌群失调诱发心肌炎症的特征变化。方法 采用皮下多点注射异丙肾上腺素复合力竭及控食方法制备慢性心力衰竭模型大鼠。造模成功后，基于心力衰竭模型大鼠的病证结合研究方法验证证型。从造模成功的18只大鼠中随机选取7只作为模型组，另设空白组7只。经胸心脏超声检测心功能变化；根据旷场实验评价大鼠行为学变化；通过HE染色观察心肌组织病理变化；采用ELISA检测脑钠素（brain natriuretic peptide, BNP）和脂多糖（lipopolysaccharide, LPS）水平；全自动生化分析仪检测肌酸激酶（creatine kinase, CK）、乳酸脱氢酶（lactate dehy drogenase, LDH）和肌酸激酶同工酶（MB isoenzyme of creatine kinase, CK-MB）含量；采用血液流变仪测定全血黏度的低、高切值；RT-qPCR及Western blot检测心肌NOD样受体蛋白3（NOD-like receptor protein 3, NLRP3）、凋亡相关斑点样蛋白（apoptosis related spot like protein, ASC）、胱天蛋白酶-1（cysteine aspartic acid specific protease-1, Caspase-1）和白细胞介素-1β（interleukin-1β, IL-1β）的mRNA和蛋白表达；同时Western blot检测结肠紧密连接蛋白（zonula occluden-1, ZO-1）、闭合蛋白（Occludin）和密封蛋白5（Claudin5）的表达；收集两组大鼠新鲜粪便，利用16S rDNA高通量测序技术测定大鼠肠道微生物群的差异。结果 与空白组比较，模型组大鼠左室射血分数（left ventricular ejection fraction, LVEF）和左室短轴缩短率（left ventricular fraction shortening, LVFS）显著降低（P＜0.01）；在旷场的运动速度和总路程降低（P＜0.05，P＜0.01）；血清BNP和LPS水平显著增加（P＜0.01），CK、LDH和CK-MB含量显著升高（P＜0.01），表现出心功能减退的“心气虚”症状；心肌组织可见大量纤维水肿，细胞质疏松，组织边缘可见心肌纤维溶解，伴有少量炎症细胞浸润；全血黏度的低、高切值显著升高（P＜0.01），表现出“血瘀”症状；NLRP3、ASC、Caspase-1、IL-1β的mRNA和蛋白表达均升高（P＜0.05，P＜0.01），ZO-1、Occludin、Claudin5表达均明显降低（P＜0.01）。肠道菌群测定结果证实：与空白组比较，模型组大鼠肠道菌群物种发生改变，菌群的ɑ和β多样性也存在明显差异，物种差异在门水平上，拟杆菌门（Bacteroidota）和螺旋菌门（Spirochaetota）的丰度上调，厚壁菌门（Bacillota）的丰度下调；种水平上，普雷沃氏菌属（Segatella copri）和琥珀酸密螺旋体菌（Treponema succinifaciens）的丰度上调，Kineothrix alysoides（P＜0.05）、伶俐瘤胃球菌（Ruminococcus callidus）和普雷沃氏菌（Prevotellamassilia timonensis）的丰度下调。结论 慢性心力衰竭气虚血瘀证模型大鼠存在肠道菌群紊乱和心肌炎症反应，并且伴随心功能和微循环障碍，这可能由肠道共生菌Kineothrix alysoides的丰度下调后LPS上调所介导。 |
| 关键词: 慢性心力衰竭 气虚血瘀证 肠道菌群 心肌炎症 Kineothrix alysoides |
| DOI：10.3969/j.issn.1674-070X.2024.10.008 |
| Received:May 21, 2024 |
| 基金项目:国家自然科学基金项目(81973842)。 |
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| Characteristic changes of gut microbiota-induced myocarditis in a rat model of chronic heart failure with qi deficiency and blood stasis pattern |
| LYU Lifei, ZHU Tingting, DING Fan, LU Yingdong, CUI Xiangning |
| (Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China) |
| Abstract: |
| Objective To explore the characteristic changes of gut microbiota dysbiosis-induced myocardial inflammation in a rat model of chronic heart failure (CHF) with qi deficiency and blood stasis pattern. Methods CHF model rats were prepared by subcutaneous multi-point injection of isoprenaline combined with exhaustive exercise and controlled feeding. After the successful modeling, the pattern of qi deficiency and blood stasis in the rats was verified based on the combined research method of disease and pattern in heart failure model rats. Seven rats were randomly selected from the 18 successfully modeled rats to form the model group, and a blank group of 7 rats was set up additionally. Changes in cardiac function were checked by transthoracic echocardiography; behavioral changes in rats were evaluated through the open-field test; pathological changes in myocardial tissue were observed by HE staining; brain natriuretic peptide (BNP) and lipopolysaccharide (LPS) levels were checked by ELISA; content of creatine kinase (CK), lactate dehydrogenase (LDH) and MB isoenzyme of creatine kinase (CK-MB) was determined by automatic biochemical analyzers. Whole blood viscosity at low and high shear rates was measured using a hemorheometer. RT-qPCR and Western blot were used to examine the mRNA and protein expressions of myocardial NOD-like receptor protein 3 (NLRP3), apoptosis related spot like protein (ASC), cysteine aspartic acid specific protease-1 (Caspase-1), and interleukin-1β (IL-1β); Western blot was also used to check the expressions of colonic tight junction proteins such as ZO-1, Occludin, and Claudin-5. Fresh feces were collected from the two groups of rats, and the differences in gut microbiota between the two groups were analyzed by 16S rDNA high-throughput sequencing. Results Compared with the blank group, rats in the model group showed significantly decreased left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) (P<0.01), significantly reduced movement speed and total distance in the open field test (P<0.05, P<0.01), significantly elevated serum BNP and LPS levels (P<0.01), and significantly increased content of CK, LDH, and CK-MB (P<0.01), which were the manifestations of cardiac hypofunction, indicating "heart qi deficiency". In addition, the myocardial tissue of the model group exhibited extensive fibrous edema with loose cytoplasm, and dissolved myocardial fibers observed at the tissue edges, accompanied by a mild inflammatory cell infiltration; the whole blood viscosity at low and high shear rates significantly increased (P＜0.01), indicating symptoms of "blood stasis". The mRNA and protein expressions of NLRP3, ASC, Caspase-1, and IL-1β in the model group were significantly higher (P<0.05, P<0.01), while the expressions of ZO-1, Occludin, and Claudin-5 were significantly lower (P＜0.01). The gut microbiota analysis revealed that the gut microbiota species of rats in the model group had been altered compared with those of the blank group, with significant differences in both ?琢 and β diversity of the microbiota. At the phylum level, the abundances of Bacteroidota and Spirochaetota were up-regulated while the abundance of Bacillota was down-regulated; at the species level, the abundances of Segatella copri and Treponema succinifaciens were up-regulated while the abundances of Kineothrix alysoides (P<0.05), Ruminococcus callidus, and Prevotellamassilia timonensis were down-regulated. Conclusion Rats with CHF of qi deficiency and blood stasis pattern exhibit gut microbiota dysbiosis and myocardial inflammation, accompanied by cardiac dysfunction and microcirculation disturbance, which may be mediated by the upregulation of LPS following the downregulation of the abundance of the intestinal symbiotic Kineothrix alysoides. |
| Key words: chronic heart failure qi deficiency and blood stasis pattern gut microbiota myocardial inflammation Kineothrix alysoides |
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