Role Of Gut Microbiota In Aerobic Exercise Improving Vascular Endothelial Dysfunction In High-fat Diet-induced Obese Rats

Objective:Obesity threatens human health seriously.The mechanism of exercise in improving cardiovascular function of obese population is still not completely elucidated.Recent studies indicate modifications in the composition of the gut microbiota in obese patients has been linked to cardiovascular diseases.Nevertheless,it is not clear whether exercise intervention ameliorates endothelial function through alteration of gut microbiota composition.In this study,we used a high-fat diet-induced obesity rat model to differentiate the effects of different diets on gut microbiota composition.To observe the effects of 8-week aerobic exercise on vascular endothelial function and intestinal microbes and its metabolite Trimethylamine N-oxide（TMAO）in obese rats,to search for the bacteria taxa related to vascular endothelial function,and to verify the role of Romboutsia lituseburensis JCM 1404 in vascular endothelial dysfunction.Methods:Experiment 1:Establish an obesity model.Sprague-Dawley rats were randomly divided into a standard diet（SD）group and a high-fat diet（HFD）group.Food intake and body weight of the rats in each group were measured at a fixed time.Experiment 2:Exercise intervention.The SD group was randomly divided into a standard diet control group（SD control）and a standard diet trained group（SD trained）;the HFD group was randomly divided into a high-fat diet control group（HF control）and a high-fat diet trained group（HF trained）.SD trained and HF trained groups received 8 weeks of aerobic exercise intervention.Food intake and body weight of the rats in each group were detected at a fixed time.Experiment 3:Transplantation of Romboutsia lituseburensis JCM 1404.The SD group was randomly divided into the SD control group（SD cont）and the strain gavage group（SD romb）;the HFD group was randomly divided into the HF control group（HF cont）and the gavage group（HF romb）.After gavage of antibiotic mixture for 5 days,the treatment groups were orally administered 10⁹cfu/ml every two days for 6 times.Hematoxylin-Eosin staining was performed to observe the morphological changes of the small intestine and abdominal aorta.DMT 620M in vitro microvascular tension was used to measure the endothelial-dependent vasodilation of the secondary mesenteric arteries.The change of tension of vessels was recorded to draw the accumulative concentration-diastolic response curve,and the half maximal response to acetylcholine(EC₅₀)was calculated.16S r RNA technology was used to analyze the differences in intestinal microbes,and the function was predicted based on Tax4Fun.Targeted metabolomics to detect plasma TMAO levels.Based on ultra-high liquid chromatography tandem mass spectrometry detection platform to detect changes of plasma metabolites,and the function was enriched and predicted based on KEGG.Correlation analysis was carried out between different metabolites and different gut microbiota.Results:（1）Compared with the SD group,the body weight,body composition and blood lipids of the HFD group were significantly increased（p<0.05,p<0.01）.Theβ-diversity of intestinal flora was significantly different between the HFD group and SD group（p<0.05）after 20 weeks of HFD intervention.16 species of bacteria（including Romboutsia）were significantly or very significantly up-regulated（p<0.05,p<0.01）.（2）After 8 weeks of aerobic exercise intervention,compared with the HF control group,body weight,fat-free mass,fat mass,and percentage of fat mass decreased significantly in the HF trained group（p<0.05,p<0.01）,and the percentage of fat-free mass was significantly increased（p<0.01）.Blood glucose,insulin,total cholesterol,triglycerides,and low-density lipoprotein decreased significantly（p<0.05）.Compared with HF control group,HF trained significantly decreased CRP concentration（p<0.05）,significantly increased VEGF concentration（p<0.01）,and significantly improved endothelial-dependent relaxation function（p<0.05）.（3）The relative abundance of Bacteroides（p=0.03）,Prevotella＿9（p=0.01）and Hydrogenoanaerobacterium（p=0.009）was higher and the relative abundance of Ruminococcaceae＿NK4A214＿group（p=0.004）,Clostridium＿sensu＿stricto＿1（p=0.008）,Anaeroplasma（p=0.04）and Bifidobacterium（p=0.008）was lower in the SD trained group compared with the SD control group.Compared with the HF control group,the abundance of Ruminiclostridium＿5（p=0.04）and Eisenbergiella（p=0.04）increased and the abundance of Romboutsia（p=0.04）,Clostridium＿sensu＿stricto＿1（p=0.007）,Parabacteroides（p=0.01）and Ruminiclostridium＿1（p=0.006）decreased after exercise intervention.TMAO concentration significantly decreased in HFD group（p<0.01）and betaine significantly decreased in SD group（p<0.01）after aerobic exercise intervention.（4）Twelve genera were significantly correlated with vascular endothelial function indicators,among which Romboutsia was significantly positively correlated with EC₅₀（p<0.05,r=0.478）and significantly negatively correlated with VEGF（p<0.05,r=-0.457）.Ruminococcus＿2 was significantly negatively correlated with body weight（p<0.05,r=-0.802）,fat-free mass（p<0.01,r=-0.885）,and percentage of fat mass（p<0.05,r=-0.793）;Ruminococcus＿gnavus＿group was significantly positively correlated with body weight（p<0.05,r=0.771）,fat mass（p<0.05,r=0.772）,percentage of fat mass（p<0.05,r=0.767）.（5）After 3-week Romboutsia lituseburensis JCM 1404 intervention,HF romb significantly improved endothelial-dependent relaxation function compared with HF cont.HFD significantly decreased intestinal villus height（p<0.05）and increased mean vessel diameter and terminal mean thickness（p<0.05）.（6）Compared with SD cont,Romboutsia lituseburensis JCM 1404 significantly down-regulated Romboutsia（p<0.001）,Clostridium＿sensu＿stricto＿1（p<0.01）,significantly up-regulated Colidextribacter（p<0.05）,Papillibacter（p<0.05）,Intestinimonas（p<0.05）,cofactors and Vitamin metabolism,cardiovascular disease were significantly up-regulated.Compared with HF cont,Romboutsia lituseburensis JCM 1404 significantly down-regulated Romboutsia（p<0.01）,Lactobacillus（p<0.01）,Turicibacter（p<0.05）,Staphylococcus（p<0.05）,Clostridium＿sensu＿stricto＿1（p<0.01）,Bilophila was significantly up-regulated（p<0.05）,and carbohydrate metabolism and endocrine and metabolic diseases were significantly down-regulated.（7）Totally 1537 metabolites were detected after Romboutsia Lituseburensis JCM 1404intervention for 3 weeks.In the SD group,there were 68 significantly different metabolites,including 6 up-regulated metabolites and 62 down-regulated metabolites.Moreover,there were 180 different metabolites in the HFD group,including 6 up-regulated metabolites and 174 down-regulated metabolites.Compared with SD cont,HFD significantly down-regulated triglyceride metabolism,lipid catabolism in adipocytes,cholesterol metabolism,insulin resistance,vitamin digestion and absorption,fat digestion and absorption,and thermogenesis.Compared with the HF cont,these seven metabolism pathways were significantly up-regulated.（8）Romboutsia was significantly positively correlated with bile acids,triglycerides,amino acid derivatives,organic acid derivatives in SD group.In HFD group,Romboutsia was significantly positively correlated with triglycerides and free fatty acids.Conclusion:（1）There were significant differences in gut microbiota composition between HFD group and SD group.Aerobic exercise effectively modulates gut microbiota composition and the relative abundance of Romboutsia was reduced.Furthermore,moderate-intensity aerobic exercise improved the vascular endothelial function of obese rats by reducing the level of gut microbial metabolite TMAO.（2）Romboutsia lituseburensis JCM1404 improved endothelial function by altering gut microbe-associated metabolites in obese rats.（3）Romboutsia Lituseburensis JCM 1404 improved endothelial function in obese rats by up-regulating metabolic pathways,including triglyceride metabolism,lipolysis regulation in adipocytes,cholesterol metabolism,insulin resistance,fat digestion and absorption,and thermogenic.