A Study About The Relation Between The Stroke With Gut Microbiota And Its Metabolite-trimethylamine-N-oxide

BackgroundMicrobial metabolomics was known to more and more people by the development of microbiome and metabonomic technology. Up to now, Gut microbiota has been suggested to play a role in almost all major diseases including obesity, type 2 diabetes, and non-alcoholic fatty liver disease as well as cardio-and cerebrovascular diseases.After 2011, a team lead by Hazen from Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland had showed a newly way which the gut microbiota affect the atherogenesis by using several new technology in this area. Gut microbes transform phosphatidylcholine and L-carnitine from ingested nutrients to trimethylamine (TMA), which is absorbed and further oxidized by hepatic Flavin monooxygenase to trimethylamine-N-oxide (TMAO), a metabolite that could promote atherogenesis partly by promoting the formation of foam cells from macrophages. The blood TMAO level has been reported to be positively correlated with the long-term mortality risk in atherosclerosis, heart failure and chronic kidney disease patients. In addition to the TMAO pathway, a metagenomic study suggested that symptomatic atherosclerosis was associated with an altered gut microbiome.All of thse tell us a new metabolite of gut microbiota which called trimethylamine-N-oxide (TMAO) could be a new target of atherosclerosis prevention and treatment. Nevertheless, for now little is known about gut microbial diversity and its structure changes and blood TMAO levels in stroke and transient ischemia attack (TIA) patients.Purpose and significanceTo premilinary clarify the changes of gut microbiota of the large artery atherosclerosis ischemia stroke/TIA patients compared with asymptomatic people and try to find the relation between LAA/TIA with the gut microbiota; detected the plasma TMAO level of LAA/TIA patients and the asymptomatic and then compared their TMAO level.MethodsWe performed a case-control study of patients with large-artery atherosclerotic ischemic stroke and transient ischemic attack (LAA/TIA). The asymptomatic individuals (not in any acute disease state by the report of physical checking and self-reporting) undergoing physical examinations were recruited as controls. The first fasting blood samples were drawn from all participants within 24 hours after admission, while their first available fecal samples within 48 hours after admission were collected. The fasting blood samples and fecal samples of the controls were collected on the day of their physical examination. After the fasting EDTA-plasma samples, fresh fecal samples were collected, the Plasma aliquots were centrifuged and immediately frozen at-80 ℃ and fecal sample aliquots were frozen at-80 ℃ immediately after collection.The bacterial DNA was extracted from the fecal samples using the PowerSoil DNA Extraction Kit (Mo Bio) according to the manufacturer’s instructions. The DNA product was stored at-20℃ before PCR. The V4 variable regions of the bacterial 16S rRNA gene were PCR amplified. Then all PCR products were mixed together and sent to Beijing Genomic Institute (Shenzhen, China) for sequencing using the Illumina Miseq (PE 150) according to the manufacturer’s protocol. The raw sequences were pre-processed according to the BIPES protocol. We used QIIME (1.8.0) to perform the basic analyses. Random forest classification models were trained on features of the OTU data using the caret R package to classify samples from stroke patients and healthy controls. To determine the significantly differential taxa between stroke patients and healthy controls, we applied linear discriminate analysis size effect (LEfSe) to compare samples between the two groups. The LDA threshold was set to three.Plasma was stored at-80℃ until analysis. The trimethylamine-N-oxide (TMAO) levels in plasma were determined using stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS) (6,460 Series Triple Quadrupole LC/MS, Agilent, USA) as described.Statistical tests were implemented using R (3.0.2). None of the tested indices in the present study passed the Shapiro-Wilk normality test and the Bartlett test of homogeneity of variances at the same time; therefore, we used the Wilcoxon rank sum test or Kruskal Wallis rank sum test (Nemenyi test for multiple pairwise comparisons) to compare two or more groups respectively, in case of false positive statistical significance. The Chi-square test was used for categorical variables. Because the microbiome data is multi-dimensional, we used Adonis test implemented in QIIME 1.8.0. A value of P<0.05 was considered to be statistically significant in the compared groups.ResultsIn the present study,435 patients were screened and 322 of them were recruited according to inclusion criteria. Fasting blood samples were drawn from all participants. Among them,141 patients provided fecal samples within the 48 hours after admission. For the asymptomatic control group,231 provided blood samples and 97 of them collected fresh fecal samplesWithin the asymptomatic control group, participants with and without carotid atherosclerotic plaques (the non-AS, IMT and AS groups) showed similar levels of TMAO without a significant difference in gut microbiota. All of these analyses suggested that atherosclerosis is not related to a significantly different gut microbiome or blood TMAO level within the asymptomatic group.However, the gut microbiome of LAA/TIA patients was clearly different from that of the asymptomatic group. The patient group had a higher diversity than the control group. The difference of the microbiome communities between the two groups could be observed using both traditional principal coordinate analysis (PCoA) and machine learning with the random forest method. According to the unweighted UniFrac distance analyses, the distance between the asymptomatic atherosclerotic group with the LAA/TIA patient group was slightly smaller than the distances of other two groups to the patient group. This result indicated that the gut microbiota of the asymptomatic atherosclerotic individuals exhibited slight dysbiosis, which was similar to that in LAA/TIA patients. Stroke and transient ischemic attack patients had more opportunistic pathogens, such as Enterobacter, Megasphaera, Oscillibacter, and Desulfovibrio, and fewer commensal or beneficial genera including Bacteroides, Prevotella, and Faecalibacterium. This dysbiosis was correlated with the severity of the disease.The TMAO level of the LAA/TIA patients showed a result that was opposite to our initial expectations. Our data clearly showed a significant decrease, rather than an increase, in TMAO levels in LAA/TIA patients.ConclusionsParticipants with asymptomatic atherosclerosis did not exhibit an obvious change in gut microbiota and blood TMAO levels; however, LAA/TIA patients showed significant dysbiosis of the gut microbiota, and their blood TMAO levels were decreased.