| Background:Type 2 diabetes(T2D)is closely related to poor prognosis of ischemic stroke(IS).Inflammation is an important pathophysiological mechanism of T2 D aggravating IS injury.Recent studies have shown that phenylacetylglutamine(PAGln),a metabolite of gut microbiota,is elevated in plasma of T2 D patients,which can promote thrombosis.However,the relationship between gut microbiota and its metabolite PAGln and IS with T2D(IS-T2D)remains unclear.Objective:To determine the change characteristics of gut microbiota and its metabolite PAGln in IS-T2 D patients,and analyze the correlation among the metabolite PAGln,gut microbiota,and inflammatory factor neutrophil extracellular traps(NETs).Methods:85 IS patients and 30 gender-and age-matching healthy controls(HC)were recruited consecutively according to the inclusion and exclusion criteria in Xiangya Hospital,Central South University from December 2019 to December 2020.The IS patients were divided into 35IS-T2 D patients and 50 IS without T2D(IS-NT2D)patients according to the presence of T2 D.The fecal samples were collected from all subjects for 16 S r RNA gene sequences and the plasma samples were collected for targeted metabolomics to detect gut microbiota’s metabolite PAGln concentration.Plasma citrulline histone 3(Cit H3)levels which is the marker of NETs was detected by enzyme-linked immunosorbent assay(ELISA).We further transplanted fecal bacteria from IS-T2 D and IS-NT2 D groups into rats treated with antibiotics(n=5-8).A middle cerebral artery occlusion(MCAO)model was established after transplantation.Neurological deficit score was evaluated by Garcia’s score and infarct volume was evaluated by TTC staining at 24 hours after surgery.ELISA was used to detect the plasma PAGln concentration of rats transplanted by fecal bacteria before and after infarction.Results:(1)Compared with HC group,the plasma PAGln levels in IS-T2 D and IS patients were significantly increased(IS-T2 D vs HC,P<0.05;IS-NT2 D vs HC,P<0.05).The plasma PAGln levels in IS-T2 D patients were also significantly higher than that in IS-NT2 D patients(P<0.05).Multivariate logistic regression analysis showed that plasma PAGln levels were an independent risk factor for IS-T2 D.(2)The community structure of gut microbiota of IS-T2 D and IS groups were significantly different from those of HC group(P<0.05).Further subgroup analysis showed that there were differences in gut microbiota structure between IS-T2 D group and IS group: compared with IS group,the relative abundance of Proteobacteria and Verrucomicrobiota in IS-T2 D patients was significantly increased(P<0.05).LEfse analysis demonstrated that the main different gut microbiota among the three groups included o＿Enterobacterales,f＿Enterobacteriaceae,p＿Verrucomicrobiota,c＿Verrucomicrobiota,s＿Klebsiella＿pneumoniae and g＿Klebsiella(LDA>4).(3)Spearman correlation analysis showed that o＿Enterobacterales,f＿Enterobacteriaceae,p＿Verrucomicrobiota,c＿Verrucomicrobiota,s＿Klebsiella＿pneumoniae,and g＿Klebsiella enriched in IS-T2 D patients were positively correlated with PAGln(r=0.236,P<0.05;r=0.236,P<0.05;r=0.301,P<0.05;r=0.301,P<0.05;r=0.228,P<0.05;r=0.228,P<0.05);NETs was positively correlated with PAGln level(r=0.41,P<0.05),and NETs levels were increased in a dose-dependent manner according to PAGln levels.(4)ROC analysis showed that plasma PAGln level,specific gut microbiota related to IS-T2 D,and plasma NETs level can be used as independent predictors of IS-T2 D,and the combined diagnostic efficiency of the three is higher.(5)Compared with the rats transplanted with fecal bacteria from IS-NT2 D patients,the fecal bacteria from IS-T2 D patients transplanted into rats increased plasma PAGln levels(P<0.05).neurological deficit score decreased(P<0.05),cerebral infarct volume increased(P<0.05).Conclusions:T2D may aggravate cerebral infarction via elevated NETs levels,and this process is mediated in part by gut microbiota and its metabolite PAGln. |
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