持续低氧条件下大鼠肠道微生物变化及其与心肌损伤的关联研究

Alterations in gut microbiota and their associations with cardiac injury in rats after exposure to continuous normobaric hypoxia

摘要:

【目的】研究持续性常压低氧对大鼠肠道微生物的影响，并分析其与低氧性心肌肥厚的关联性。【方法】雌性无特异性病原体(specific-pathogen-free, SPF)级SD (Sprague-Dawley)大鼠，按随机数字表法分为2组：常氧组和低氧组。实验开始后，低氧组大鼠置于低氧舱中，氧气浓度设定为10%，持续暴露30 d，常氧组大鼠正常条件饲养。每天记录大鼠体重，并于低氧前(0 d)和低氧后(30 d)分别收集粪便进行16S rRNA基因扩增子测序，测定肠道微生物组的变化。实验结束后进行血常规、血生化和器官指数分析；采用实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qRT-PCR)检测右心室组织中4种分子标志物心房利钠肽基因(atrial natriuretic peptide, ANP)、脑钠肽基因(brain natriuretic peptide, BNP)、心肌肌球蛋白重链6基因(myosin heavy chain 6, Myh6)、心肌肌球蛋白重链7基因(myosin heavy chain 7, Myh7)的mRNA表达；并在肠道微生物组和各类指标之间进行Spearman关联分析。【结果】低氧大鼠体重降低，红细胞数量、红细胞比容、血红蛋白浓度均明显升高。低氧大鼠右心指数显著升高，血清肌酸激酶(creatine kinase, CK)和乳酸脱氢酶(lactate dehydrogenase, LDH)活性增高，BNP和Myh7的表达显著升高而Myh6的表达降低，表明低氧导致大鼠心肌损伤并发生了右心室病理性肥厚。低氧改变了大鼠肠道微生物的α多样性和β多样性；线性判别分析效应大小(linear discriminant analysis effect size, LEfSe)分析结果提示常氧组大鼠的普雷沃氏菌科(Prevotellaceae)和毛螺菌科(Lachnospiraceae)相对丰度较高，而低氧组大鼠的乳酸杆菌科(Lactobacilliaceae)和乳杆菌属(Lactobacillus)相对丰度较高。LDH与摩根菌属(Monoglobus)和帕鲁迪杆菌(Papillibacter)正相关，与苜蓿科(Defluviitaleaceae_UCG-011)负相关；CK与菌株RF39正相关；Myh6的表达量与Prevotellaceae_NK3B31_group和幽门螺杆菌(Helicobacter)正相关；BNP的表达量与瘤胃球菌科(Ruminococcaceae_UCG_009)正相关。【结论】持续性低氧显著改变了大鼠肠道微生物的丰富度、均匀度和菌群结构；该变化与心肌损伤指标之间存在显著相关性，表明肠道菌群可能在低氧性心肌肥厚中发挥重要作用。

关键词:常压低氧;病理性心肌肥厚;器官指数;肠道微生物;脑钠肽;肌球蛋白重链

Abstract:

[Objective] To investigate the alterations in gut microbiota and their associations with the biomarkers of myocardial hypertrophy induced by continuous normobaric hypoxia in rats. [Methods] Sixteen specific-pathogen-free female Sprague-Dawley rats were randomly assigned into two groups: the normoxia group and the hypoxia group. After adaptation for 1 week, the rats in the hypoxia group were immediately placed into the hypoxic chamber with the oxygen concentration at 10% for 30 d, and the rats without hypoxia treatment were set as the control. We monitored the body weight of each rat daily, determined the haematological and biochemical parameters as well as the organ coefficients at the end of the experiments. Further, we collected stools from each rat before (day 0) and after hypoxia challenge (day 30) to test the gut microbiota by using 16S rRNA gene sequencing. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the mRNA levels of gene ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), Myh6 (myosin heavy chain 6), and Myh7 (myosin heavy chain 7), the biomarkers of myocardial hypertrophy in the right ventricular tissue. Spearman correlation analysis was performed to explore the links between intestinal microbiomes and parameters. [Results] Hypoxia reduced the body weight, increased the erythrocyte count, hemoglobin level, and hematocrit, and failed to affect the counts of leukocytes and platelets. Hypoxia increased the right ventricular index, enhanced the activities of creatine kinase (CK) and lactate dehydrogenase (LDH) in the serum, and up-regulated the mRNA levels of BNP and Myh7 in right ventricular tissue, which suggested pathologically myocardial hypertrophy after hypoxia challenge for 30 d. Hypoxia significantly altered the alpha diversity and beta diversity of gut microbiota in rats. The results of linear discriminant analysis effect size (LEfSe) showed that the rats exposed to hypoxia had lower relative abundance of Prevotellaceae and Lachnospiraceae and higher relative abundance of Lactobacilliaceae and Lactobacillus than the control rats. LDH was positively correlated with Monoglobus and Papilibacter and negatively correlated with Defluvialeaceae_UCG-011. CK was positively correlated with strain RF39. The expression of BNP was positively correlated with Ruminocochaceae_UCG_009, and that of Myh6 was positively correlated with Prevotellaceae_NK3B31_group and Helicobacter.[Conclusion] The species richness, evenness, and composition of gut microbiota in rats are greatly affected after exposure to continuous normobaric hypoxia. The changes of gut microbiota have correlations with the biomarkers of myocardial hypertrophy induced by hypoxia, indicating that gut microbiota plays a crucial role in hypoxia-induced myocardial hypertrophy.

Key words:hypoxia;myocardial hypertrophy;organ coefficient;gut microbiota;brain natriuretic peptide (BNP);myosin heavy chain (Myh)