Association And Mechanism Of Short Chain Fatty Acids And Stroke Functional Outcome

Objective:To observe the differences in diet quality,short-chain fatty acids（SCFAs）and gut microbiota between stroke patients（Intracerebral Haemorrhage and ischemic stroke）and healthy control,and to explore the influence of microbiota-gut-brain（MGB）axis in stroke.It is intended to discover the association between SCFAs and functional outcome of stroke,and explore the possible mechanism of SCFAs affecting functional outcome of stroke based on the MGB axis theory,to provide a new therapeutic target for the treatment of post-stroke dysfunction.Methods:The population of this study was divided into two parts.The first part is a case-control study.Stroke patients were selected according to sex and age（5years）pairs into the research subjects,which were 49 patients with ischemic stroke（IS）,49 patients with Intracerebral Haemorrhage（ICH）and 49 subjects in healthy controls（HC）.In the second and the third part of the cross-sectional study with stroke patients（n=168）.The basic,clinical data and fecal sample were collected after all subjects signed the informed consent form.After food frequency questionnaires were collectted and calculated Alternative Healthy Eating Index（AHEI）-2010 to obtain the assessment of diet quality.The behavioral assessment defined different functional outcomes were used in stroke patients.The concentration of SCFAs in fecal samples were assayed by gas chromatography.The 16S r RNA V3-V4 amplicon sequencing technique was used to assay the information of gut microbiota in fecal samples.In the case-control study,the differences in diet quality score,SCFAs concertration in fecal samples and gut microbiome in the IS,ICH and HC groups were compared to explore the changes in diet quality,SCFAs concertration in fecal and gut microbiota in stroke patients with two types of stroke and healthy control.In a cross-sectional study of stroke patients,the functional outcomes were divided into high and low groups（Disability in Upper limb and hand,Disability in Low limb,Balance disorder,Ambulation disorder,Dysphagia,and Activities of daily of living）or no group（Cognitive disorder,Aphasia and Severe function disorder）according to different behavioral assessment scales.To compare the differences in diet quality score,SCFAs concertration in fecal samples and relative abundance of gut microbiota in groups with or without different functional outcomes.At the same time,the binary Logistic regression analysis was used to explore the association between dietary quality score,fecal SCFAs concertration and functional outcomes,and to screen out functional outcomes which were associated with the two kinds of independent variables.On this functional outcome,it was to explore that three kinds of independent variables including diet,SCFAs and gut microbita were associated with and predicted the risk of functional outcome with stroke according MGB aix theroy.The correlation analysis was made among the three kinds of variables which were associated with the functional outcome,to find intermediate variables to explore the possible mechanism of the influence of SCFAs on stroke functional outcome.Results:1.The scores of nuts and legumes of AHEI-2010 in IS and ICH groups were lower than those in HC group was,（P<0.05）.The concertration of SCFAs in fecal,acetate,propionate and butyrate in IS and ICH groups were lower than those in HC group（P<0.05）.In LEf Se analysis of gut microbiota,IS compared with HC group,the contribution of gut microbiota in HC group was as follows:f＿＿Prevotellaceae（LDA=5.06,P=2.94E-05）and g＿＿Prevotella（LDA=5.03,P=2.76E-05）,ICH compared with HC group,the contribution of gut microbiota in HC group was as follows:f＿＿Pre-votellaceae（LDA=5.14,P=4.30E-07）and g＿＿Prevotella（LDA=5.12,P=7.55E-04）.The correlation analysis of AHEI-2010 scores for food items,SCFAs and gut microbiota at the genus level showed that the positive correlation was found in acetate,propionate,butyrate,isobutyric acid,valeric acid,nuts and prevotella,（r=0.22,P=0.030）,（r=0.24,P=0.020）,（r=0.38,P=1.02e-04）,（r=0.27,P=0.009）,（r=0.22,P=0.029）,and（r=0.29,P=0.003）,respectively between IS and HC group.The scores of nuts and legumes of AHEI-2010 were negatively correlated with Collinsella,and the correlation coefficient and P value were（r=-0.21,P=0.030）between ICH and HC group,Collinsella was positively correlated with Valeric acid and Isovaleric acid.The correlation coefficient and P value were（r=0.20,P=0.045）and（r=0.22,P=0.030）,respectively.2.Comparison of food items in different functional outcomes of stroke with AHEI-2010 scores:The fruit score was higher in Non-CD and H-ADL/Non-SFD group than that of CD and L-ADL/SFD groups（P<0.05）,and there were no differences in other food items in different functional outcome groups（P>0.05）.Comparison of concertration SCFAs in fecal:The concertration of acetate and propionate in H-DUL group were higher than those in L-DUL group（P<0.05）;The concertration of propionate and butyrate in H-BD,Non-CD and H-DP group were higher than those in L-BD,CD and L-DP group（P<0.05）.The concertration of butyrate in H-DLL,H-ADL/Non-SFD group was higher than that in L-DLL and L-ADL/SFD group（P<0.05）;There was no difference in the outcome of ambulation disorder and aphasia between high and low groups or with or without groups（P>0.05）.Binary Logistic regression analysis:（1）According to no fruit consumption as a reference,there was a negative associated between fruit consumption and cognitive disorder outcome after stroke,OR=0.48（P=0.019）.In the subgroup analysis,it was found that fruit consumption was negatively associated with the risk of cognitive disorder after stroke in overweight group OR=0.15（P=3.91E-04）,P_inter=0.004.（2）According to no fruit consumption as a reference,there was a negative associated between fruit consumption and the severity of functional outcome after stroke（P<0.05）.In the subgroup analysis,it was found that fruit consumption was negatively associated with the risk of severity of functional outcome after stroke in the diabetes group,P_inter=0.004.（3）In the different models adjusted for variables,the level of acetate and propionate was negatively associated with the risk of poststroke disability in upper limb and hand（P<0.05）,showing a linear trend(P_trend<0.05);The level of butyrate was negatively associated with the risk of poststroke disability in low limb and showing a linear trend（P<0.05）;The level of acetate,propionate and butyrate were negatively associated with the risk of poststroke balance disorder in（P<0.05）,showing a linear trend in three models(P_trend<0.05);The level of acetate,propionate and butyrate was negatively associated with the risk of poststroke cognitive disorder,showing a linear trend（P<0.05）;The level of propionate and butyrate was negatively associated with the risk of poststroke dysphagia（P<0.05）;The level of acetate,propionate and butyrate was negatively associated with the risk of severe functional disorder（P<0.05）,showing a linear trend(P_trend<0.05).In subgroup analysis,butyrate was negatively associated with the risk of poststroke cognitive disorder in age≥65 years,OR=0.36（P=0.011）,P_inter=0.019.The level of acetate was negatively associated with the risk of in duration≥30 days group,0R=0.31（P=0.007）,P_inter=0.035;The level of butyrate interaction Pinter=0.047 in the duration of stroke,but intra-group,P>0.05.The level of acetate,propionate and butyrate were all negatively associated with the risk of poststroke cognitive disorder,OR value were 0.38,0.28,and0.41,respectively（P<0.05）,P_inter<0.05,and propionate was negatively associated with the risk of severe function disorder,OR=0.51（P=0.026）,P_inter=0.040,in the ischemic stroke group.The level of acetate was negatively associated with the risk of poststroke dysphagia,0R=0.31（P=0.007）,P_inter=0.035,in duration≥30 days group,and the level of butyrate interaction Pinter=0.047 in the duration of stroke,but intra-group,P>0.05.The level of butyrate was negatively associated with the risk of poststroke disability in low limb in diabetes group,OR=0.08（P=0.017）,P_inter=0.017.It is not found to interact with other subgroup variables,P_inter>0.05.3.The weekly consumption of oranges and the standardized propionate and butyrate in fecal samples in Non-CD group were higher than those in CD group（P<0.05）.The LEf Se analysis of gut microbiota in two groups:Contribution degree of gut microbiota in Non-CD group:g＿＿Prevotella（LDA=4.31,P=0.001）,g＿＿Lachnospiraceae＿NK4A136＿group（LDA=3.45,P=0.001）,g＿＿Dorea（LDA=3.39,P=0.002）,gut microbiota in CD group:g＿＿Klebsiella（LDA=4.06,P=0.038）,c＿＿Bacilli（LDA=3.73,P=0.043）,and g＿＿UBA1819（LDA=3.67,P=0.037）.Binary Logistic regression analysis of the association between fruit,SCFAs and gut microbiota and the risk of poststroke cognitive disorder:（1）Orange consumption was negatively associated with the risk of cognitive disorder,（P<0.05）;Subgroup analysis showed that orange consumption was negatively associated with the risk of poststroke cognitive disorder in the<65 year old and non-DVT group,OR=0.04（P=3.26E-04）,P_inter=0.012;OR=0.09（P=1.62E-04）,P_inter=6.50E-04,respectiv-ely.（2）The genus level of Lachnospiraceae＿NK4A136＿group was negatively associated with the risk of poststroke cognitive disorder（P<0.05）;In subgroup analysis,Lachnospi-raceae＿NK4A136＿group was negatively associated with the risk of poststroke cognitive disorder in non-DVT group,OR=0.36（P=0.005）,P_inter=0.026.In the ROC curve analysis,The AUC vaule of orange,Lachnospiraceae＿NK4A13-6＿group,propionate and butyric acid were 0.654,0.641,0.626,and 0.602,respectively in predicted risk of cognitive disorder after stroke（P<0.05）,and it might be lower when the consumption of orange,the relative abundance of Lachnospiraceae＿NK4A136＿gro-up,the concentration of propionate and butyrate in feces were greater than 17.26g/week,0.04%,11.37umol and 4.96ummol.The concentration of acetate in feces had no significant predictive value（P>0.05）.Orange,acetate,propionate and butyrate were positively correlated with Lachnosp-iraceae＿NK4A136＿group.The correlation coefficient and P value were respectively（r=0.23,P=0.003）,（r=0.19,P=0.016）,（r=0.19,P=0.012）,and（r=0.29,P=2.02E-04）.Lachnospiraceae＿NK4A136＿group had a partial mediating effect on oranges associated with the risk of poststroke cognitive disorder.The standardized effect size was-0.096,95%CI（-0.411,-0.003）,accounting for 23.88%of the total effect.Lachnospiraceae＿NK4A136＿group had a complete mediating effect on acetate,propionate and butyrate associated with the risk of poststroke cognitive disorder.Conclusions:The occurrence of stroke is closely related to diet,gut microbiota and SCFAs.Nuts may prevent stroke by regulating SCFAs levels through Prevotella and Collinsella in gut microbiota.Nuts may be one of the dietary strategies,and Prevotella may be a new target as probiotics for stroke prevention.The level of acetate,propionate and butyrate of SCFAs,especially propionate and butyrate,are negatively associated with the risk of multiple functional outcomes in stroke,and it is possible for a new target to treat post-stroke dysfunction in the future.Cognitive disorder after stroke is closely related to fruit,gut microbiota and SCFAs.Orange may regulate acetate,propionate and butyrate through Lachnospiraceae＿NK4A136＿group to improve cognitive disorder after stroke.Orange may be one of the dietary intervention strategies,and Lachnospiraceae＿NK4A136＿group as probiotic may be a new strategy for the treatment of post-stroke cognitive disorder.The follow-up studies may further use longitudinal large-scale population studies to confirm our findings on the significance of nuts and fruits in stroke development and treatment.Biological experiments can also be used to explore the potential mechanism of SCFAs and gut microbiota in the occurrence and development of stroke.