基于肠道菌群探讨运动改善2型糖尿病小鼠骨髓内皮组细胞功能的影响研究

To investigate the effect of exercise on the function of bone marrow endothelial cells in type 2 diabetic mice based on intestinal flora

摘要

目的 探讨有氧联合抗阻运动是否通过改善肠道有益菌群的多样性、丰度、结构及其对2型糖尿病小鼠骨髓内皮祖细胞（endothelial progenitor cells，EPCs）功能的影响。 方法 将40只8周龄雄性db/db小鼠随机分为空白对照组（DZ组）糖尿病模型+有氧联合抗阻运动组（联合运动）（L组）、糖尿病模型+粪便移植组（SY组）、糖尿病模型组（TJ组），每组10只。L组进行8周有氧联合抗阻运动干预，于周一、周三和周五进行有氧运动，周二、周四和周六进行抗阻运动，6 d/周。SY组和TJ组分别采用L组运动8周后的小鼠粪便和DZ组小鼠粪便制成悬浊液予灌肠，2次/d，共14 d。灌肠结束后检测各组小鼠骨髓EPCs增殖、迁移、黏附和体外血管生成能力，采用流式细胞仪检测鉴定EPCs表型CD34和CD31，16SrRNA检测各组小鼠粪便肠道菌群的丰度及多样性ELISA检测各组小鼠血清胰高血糖素样肽-1（glucagon-like peptide1，GLP-1）。 结果 肠道菌群多样性结果显示，在纲水平上，与TJ组比较，SY组小鼠肠道Clostridia（54.60% vs. 32.21%）和Bacteroidia（47.63% vs. 36.39%）的丰度上升，Gammaproteobacteria（0.30% vs. 12.57%）丰度下降，差异有统计学意义（P=0.019、P=0.028、P=0.002）与DZ组比较，SY组小鼠的肠道Bacteroidia（47.63% vs. 38.5%）丰度上升，Bacilli（2.56% vs. 12.36%）丰度下降，差异有统计学意义（P=0.021、P=0.039）。在科水平上，与DZ组比较，SY组小鼠肠道产丁酸盐细菌Lachnospiraceae（43.86% vs. 26.44%）和Oscillospiraceae（7.8% vs. 2.15%）的丰度显著升高（P=0.004、P=0.001）与TJ组比较，SY组小鼠肠道Lachnospiraceae（43.86% vs. 32.89%）、Lactobacillaceae（9.89% vs. 3.8%）和Oscillospiraceae（7.8% vs. 2.21%）菌群丰度显著升高（P=0.045、P=0.006、P=0.001）。SY组小鼠EPCs增殖、迁移、黏附和血管生成能力明显高于DZ组和TJ组（P=0.002、P=0.000、P=0.000、P=0.000、P=0.016、P=0.049、P=0.019、P=0.026）。DZ组SY组和TJ组3组小鼠血清GLP-1（pmmol/L）含量（1.90±0.22，2.82±0.40和2.24±0.49）比较，差异有统计学意义（F=31.082，P=0.000）。与DZ组比较，SY组小鼠血清GLP-1水平明显升高（P=0.000）与TJ组相比，SY组小鼠血清GLP-1水平显著升高（P=0.000）肠道菌群数量与内皮组细胞功能相关性分析结果显示：Clostridia（r=0.696，P=0.041）、Oscillospiraceae（r=0.697，P=0.037）Muribaculaceae与内皮组细胞迁移能力（r=0.753，P=0.019）和血管生成（r=0.810，P=0.008）呈正相关。 结论 有氧联合抗阻运动可能通过提高2型糖尿病小鼠肠道有益菌的丰度、多样性及结构及提高GLP-1表达，从而改善2型糖尿病小鼠骨髓EPCs功能。

Abstract

Objective To investigate whether aerobic exercise combined with resistance exercise can improve the diversity，abundance and structure of intestinal beneficial flora and its effect on the function of bone marrow endothelial progenitor cells（EPCs） in type 2 diabetic mice. Methods Forty 8-week-old male db/db mice were randomly divided into control group（DZ group）. Diabetic model + aerobic and resistance exercise group（combined exercise）（L group），diabetic model + fecal transplantation group（SY group），and diabetic model group（TJ group），with 10 rats in each group. Group L carried out aerobic exercise combined with resistance exercise for 8 weeks，aerobic exercise on Monday，Wednesday and Friday，and resistance exercise on Tuesday，Thursday and Saturday，6 days/W. In SY and TJ groups，the feces of mice in group L and DZ after 8 weeks of exercise were made into suspension，respectively，and then enema was given twice a day for 14 days. After the end of enema，the proliferation，migration，adhesion and angiogenesis ability of bone marrow EPCs in each group were detected. The EPCs phenotypes CD34 and CD31 were identified by flow cytometry，and the abundance and diversity of intestinal flora in feces were detected by 16SrRNA. ELISA was used to detect serum glucagon-like peptide1（GLP-1） in each group. Results At the class level，the abundance of Clostridia（54.60% vs. 32.21%） and Bacteroidia（47.63% vs. 36.39%） in the SY group was higher than that in the TJ group. The abundance of Gammaproteobacteria（0.30% vs. 12.57%） was significantly decreased（P=0.019，P=0.028，P=0.002）. Compared with DZ group，the abundance of Bacteroidia in SY group increased（47.63% vs. 38.5%），and the abundance of Bacilli decreased（2.56% vs. 12.36%），and the difference was statistically significant（P=0.021，P=0.039）. At the family level，the abundance of Lachnospiraceae（43.86% vs. 26.44%） and Oscillospiraceae（7.8% vs. 2.15%） in the SY group was significantly higher than that in DZ group（P=0.004，P=0.001）. Compared with the TJ group，The abundance of Lachnospiraceae（43.86% vs. 32.89%），Lactobacillaceae（9.89% vs. 3.8%） and Oscillospiraceae（7.8% vs. 2.21%） in the intestinal tract of SY group was significantly increased（P=0.045，P=0.006，P=0.001）. The proliferation，migration，adhesion and angiogenesis of EPCs in SY group were significantly higher than those in DZ group and TJ group（P=0.002，P=0.000，P=0.000，P=0.000，P=0.016，P=0.049，P=0.019，P=0.026）. The content of serum glucagon peptide-1（GLP-1）（pmmol/L） in DZ group SY and TJ group（1.90±0.22，2.82±0.40 and 2.24±0.49） was significantly different（F=31.082，P=0.000）. Compared with DZ group，the serum GLP-1 level in SY group was significantly increased（P=0.000）. Compared with TJ group，the serum GLP-1 level in SY group was significantly increased（P=0.000）. The correlation analysis between the number of intestinal flora and the function of endothelial cells showed that：Clostridia（r=0.696，P=0.041），Oscillospiraceae（r=0.697，P=0.037） Muribaculaceae was positively correlated with cell migration（r=0.753，P=0.019） and angiogenesis（r=0.810，P=0.008） in the endothelial group. Conclusion Aerobic exercise combined with resistance exercise may improve the function of EPCs in bone marrow of type 2 diabetic mice by increasing the abundance，diversity and structure of beneficial intestinal bacteria and increasing the expression of GLP-1.

关键词

糖尿病 / 运动 / 胰高血糖素样肽-1 / 内皮组细胞 / 肠道菌群 / 粪便移植

Key words

diabetes mellitus / Exercise / GLP-1 / Endothelial progenitor cells / Gut microbiota / Fecal transplantation

基金资助

国家自然科学基金资助项目(8216090327)

广西自然科学基金资助项目(2018GXNSFAA050091)