Effect And Mechanism Of Aspirin Combined With Atorvastatin On Gut Microbiota And Its Metabolites In Patients With Ischemic Stroke

Objective: The aim is to explore the effect and mechanism of long-term regular oral administration of aspirin and atorvastatin on human gut microbiota and its metabolites including short-chain fatty acids（SCFAs）and trimethylamine oxide（TMAO）.Methods: The patients with ischemic stroke（IS）without obvious sequelae from January 2021 to January 2022 were included in the group who took aspirin and atorvastatin regularly for more than one year as the medication group（group A）.The persons who didn’t take any drugs from the physical examination center were served as control group（group B）.Baseline information was collected.Fresh stool samples were collected for 16 S r RNA gene sequencing analysis of gut microbiota.SCFAs in plasma were detected via gas chromatography mass spectrometrygas and TMAO in plasma was detected by ultra performance liquid chromatography mass spectrometry.Species abundance and diversity of the two groups using Alpha diversity analysis.Similarity in composition of the two groups species by Beta diversity analysis.The relative abundance changes of species at each level shown via the histogram of the relative abundance distribution of species.Wilcoxon rank sum test was used to analyze the microbiota with statistical difference at the genus level between the two groups.Linear discriminant analysis effect size（Lef Se）was used to find the marker microbiota with statistical difference.Principal component analysis（PCA）and orthogonal partial least squares-discriminant analysis（OPLS-DA）were used tocompare SCFAs difference between the two groups.PCA and OPLS-DA were used to compare TMAO difference between the two groups.The SCFAs difference between the two groups were analyzed by boxplot analysis.The TMAO difference between the two groups were analyzed by boxplot analysis.ROC curve showed the diagnostic value of SCFAs.ROC curve showed the diagnostic value of TMAO.P < 0.05 was considered statistically significant.Results: Results of gut microbiota analysis: Group A had 14 males and 6 females,aged（62.25±9.65）years;group B had 12 males and 8 females,aged（61.60±3.57）years.Alpha diversity analysis showed that the Chao1 indexe and Ace indexe of group A were lower than group B（P<0.05）.The Shannon index and Simpson index of group A were higher than group B,but there was no statistical difference.Beta diversity analysis showed that there was significant difference in the species composition of the two groups.The histogram of relative abundance distribution of species showed that the relative abundance of some species in the two groups had obvious differences with the gradual subdivision of phylum,class,order,family and genus.Wilcoxon rank sum test analysis showed that the relative abundances of the 20 species in the two groups were statistically different at the genus level,Uncultured_bacterium_Enterobacteriaceae,UBA1819,Staphylococcus,Reyranella,Parabacteroides,Hungatella,Desulfovibrio,Comamonas,Biliophila,Bifidobacterium were higher than group B（P<0.05）,Uncultured_bacterium_Prevotellaceae,Succinivibrio,Pseudomonas,Prevotella₉,Prevotella₇,Prevotella₂,Mitsuokella,Megasphaera,Lactococcus,Alloprevotella were higher than group A（P < 0.05）.The results of Lef Se analysis showed that the marker microbiota in group A were: Bifidobacterium,Enterobacteriaceae and Parabacteriaceae.The marker microbiota in group B were: Bacteroidales,Prevotella and Lachnospiraceae.Results of SCFAs and TMAO analysis: Group A had 4 males and 4 females,aged（59.00±9.38）years.Group B had 7 males and 6 females,aged（61.23±3.47）years.The results of PCA and OPLS-DA showed significant differences in SCFAs between the two groups.The boxplot showed that the level of acetate in group A was higher than that in group B（P<0.05）,while the levels of butyric acid and valeric acid in group A were lower than that in group B（P<0.05）.There was no statistical difference in the levels of propionic acid,isobutyric acid and isovaleric acid between the two groups.The area under curve（AUC）of the diagnostic prediction model ROC curve of acetic acid,butyric acid and valeric acid were 0.71,0.83 and 0.83 respectively.PCA and OPLS-DA results showed no significant difference in the distribution of TMAO between the two groups.Plasma TMAO in A group was slightly lower than that in group B,but the boxplot showed no statistical difference between the two groups（P = 0.64）.The AUC value of TMAO in ROC curve is 0.52.Conclusions: There were significant differences in the gut microbiota structure and SCFAs levels between people who regularly took aspirin and atorvastatin for a long time and those who did not take it.After taking the two drugs,they may change the level of specific SCFAs by changing the abundance of specific gut microbiota,and affect the preventive effect of IS by regulating inflammation and blood lipids,and there are both positive and negative effects.Long-term oral aspirin and atorvastatin can maintain the plasma TMAO level in healthy people of the same age in patients with previous IS.