| Background and Purposes:Ischemic stroke(IS)is the most common type of stroke,accounting for 87% of all types.As a country with one-fifth of the global population,China has experienced explosive growth in disease burden of IS with significant ethnic and geographic disparities,although targeted interventions that reduce these potentially modifiable risk factors,which covered 90% of the population,have been implemented for decades.Hunan is a province in central China,the Global Burden of Disease Study(GBD)2017 data showed an increasing trend for the disease burden of stroke in Hunan province from 1990 to 2017.And the disease burden of IS in Hunan province increased most significantly,with disabilityadjusted life years(DALYs)and standardized DALY rates increased by142.64% and 9.06%,respectively.According to the sixth national population census in 2010,the Miao(9.43 million)and Tujia(8.35 million)minorities were ranked as the fifth and seventh largest minority groups,respectively.Xiangxi Tujia and Miao Autonomous Prefecture(Xiangxi)is the settlement of Tujia and Miao populations,with high incidence of IS.However,few reliable data are available to identify the differences in risk factors for IS among the Tujia,Miao,and Han peoples.Meanwhile,accumulated research evidence suggests that gut microbes can affect gastrointestinal physiology,immune function and even mental health through the microbiota-gut-brain axis,but there were few studies on the gut microbiome of IS patients among the Tujia,Miao,and Han peoples.Therefore,this study intends to make the following explorations in Xiangxi,where the Tujia and Miao people account for 80.84% of the local total population:(1)The ethnic differences of potentially modifiable risk factors for IS among Tujia,Miao,and Han peoples in Xiangxi;(2)The interaction between ethnic categories and potentially modifiable risk factors for IS in Xiangxi;(3)The association between gut microbes and IS among Tujia,Miao,and Han peoples in Xiangxi;(4)The interaction between gut microbes and potentially modifiable clinical indexes for IS in Xiangxi.Methods:A case control study was conducted between May 1,2018,and April30,2019.Structured questionnaires were administered,and physical examinations were performed in the same manner for first-ever IS patients and normal controls(NCs).At the same time the stool and blood samples were collected from the participants of study for 16 S amplicon sequencing and biochemical examination,respectively.(1)On the basis of univariate analysis and multivariate Logistic regression analysis of modifiable risk factors for IS in the whole and ethnicity-specific population,homogeneity analysis was conducted on the correlation effect of modifiable risk factors for IS in different ethnic groups.After finding out the ethnic differences of the correlation effect statistically significant by Breslow-day test,additive interaction analysis was conducted with calculating the indexes,such as relative excess risk of interaction(RERI),attributable proportion due to interaction(API)and synergy index(SI).(2)Species annotation was performed on 16 S amplicon sequencing data to describe the composition,Alpha diversity and Beta diversity of intestinal flora between IS patients and NCs in the whole and ethnicityspecific population.LEf Se analysis and Anosim test were used to explore the differences of gut microbiome in the whole and ethnicity-specific population.And the additive interaction between common modifiable clinical indexes for IS and intestinal microbes,which enriched in IS patients,was analyzed with RERI、API and SI values.(3)Spearman rank correlation analysis was used to explore the correlation between modifiable clinical indexes and intestinal microbes for IS,which ranked top 30 in the whole population.Results:(1)Overview: A total of 324 first-ever IS patients and 394 NCs were enrolled in this study,including 170 patients with mild stroke(50.1% of the total patients)and 115 patients with moderate stroke(33.9%).16 S amplicon sequencing was performed in 82 first-ever IS patients and 82 NCs,respectively.(2)The results of binary multivariate Logistic regression analysis showed that the occupation of manual labor and moderate-intensity physical activity(MIPA)could reduce the risk of IS[odds ratios,ORs with95%CI were 0.48(0.25～0.94)and 0.53(0.30～0.93),respectively).Eating hot pot more than once a week [OR(95%CI)was 2.69(1.15～6.32)],diabetes mellitus(DM)and hypertension[ORs(95%CI)were 2.58(1.51～4.41)and 2.58(1.50～4.44),respectively],apolipoprotein(Apo)B/Apo A1> 0.9 and high-sensitivity C-reactive protein(hs-CRP)≥5.0mg/L[ORs(95% CI)were 2.87(1.60～5.13)and 48.36(29.19～80.13),respectively] could increase the risk of IS.In Tujia people,hypertension[OR(95%CI)was 3.12(1.48～6.57)],DM[OR(95%CI)was2.54(1.22～5.31)],Apo B/Apo A1>0.9 and hs-CRP ≥5.0mg/L still increased the risk of IS[ORs(95%CI)were 4.56(1.93～10.75)and44.82(22.40～89.66),respectively].In Miao people,the occupation of manual labor and MIPA might be the protective factors [ORs(95%CI)were 0.18(0.06～0.61)and 0.38(0.15～0.95),respectively],DM[OR(95%CI)was 3.05(1.22～7.59)],Apo B/Apo A1>0.9 and hs-CRP≥5.0mg/L might be the risk factors of IS[ORs(95%CI)were 3.13(1.21～8.14)and56.58(22.35～143.23),respectively].In the Han people,eating fast food more than once a week [OR(95%CI)was 30.93(2.70～354.28)],eating hot pot more than once a week [OR(95%CI)was 20.91(1.85～235.73)]and hsCRP≥5.0mg/L [OR(95%CI)was 302.25(49.75～1836.31)]increased the risk of IS.(3)Breslow-Day test results showed that only the difference of OR values between Apo B/Apo A1>0.9 and IS in Tujia vs.Miao peoples with P<0.05.Furthermore,the results of additive interaction analysis showed that: compared with Miao people,the influence of Apo B/ Apo A1 ratio on IS increased in Tujia people,indicating a positive interaction between ethnic categories and Apo B/ Apo A1 ratio on IS [RERIs,APIs and SIs with95%CI were 5.75(0.58～10.92)、 0.65(0.38～0.91)and 3.66(1.35～9.93),respectively],there was still a positive interaction[RERIs,APIs and SIs with 95%CI were 6.06(0.50～11.62)、 0.64(0.37～0.91)and3.47(1.32～9.16),respectively] after adjusting for age and sex.(4)By clustering the sequences into OTUs with 97% identity,and all the reads could be annotated and classified into 3536 OTUs in the entire population by the Silva 138.1 Database.LEf Se analysis showed that 13 OTUs played an important role in IS patients and NCs.Eight OTUs enriched in IS patients belong to classes Bacilli and Gammaproteobacteria.Five OTUs enriched in NCs belong to class Clostridia.Family Lactobacillaceae enriched in Tujia,Miao and Han patients with IS;family Lachnospiraceae enriched in Tujia,Miao and Han NCs;family Streptococcaceae enriched in Tujia and Miao patients with IS;family Enterobacteriaceae enriched in Tujia and Han patients with IS;family Enterococcaceae enriched only in Han patients with IS;and family Ruminococcaceae enriched in Tujia and Han NCs.(5)Spearman rank correlation analysis showed that: families Lactobacillaceae,Streptococcaceae,Enterobacteriaceae and Enterococcaceae enriched in IS patients were positively correlated with modifiable clinical indexes,such as systolic blood pressure(SBP),diastolic blood pressure(DBP),Apo B/Apo A1 ratio and hs-CRP;but were negatively correlated with the potential protective clinical index for IS,such as high-density lipoprotein cholesterol(HDL-C).Families Lachnospiraceae and Ruminococcaceae enriched in NCs were negatively correlated with the clinical indexes for IS,such as SBP,DBP,Apo B/Apo A1 ratio,fasting plasma glucose(FPG)and hs-CRP.(6)An additive interaction analysis was performed between all microbiota enriched in IS patients and traditional modifiable clinical indexes for IS.The results showed that family Enterococcaceae had positive interactions with SBP,DBP and hs-CRP,with APIs(95%CI)were0.74(0.51～0.97),0.71(0.41～1.00)and 0.85(0.66～1.05),respectively;and SIs(95%CI)were 4.45(1.57～12.60),3.78(1.27～11.20)and7.01(1.79～27.40),respectively.Conclusions:(1)In Xiangxi,the occupation of manual labor and MIPA might be the protective factors for IS,while eating hot pot more than once a week,hypertension,DM,Apo B/Apo A1>0.9 and hs-CRP ≥5.0mg/L might be risk factors for IS.In Tujia residents hypertension,DM,Apo B/Apo A1>0.9 and hs-CRP ≥5.0mg/L might be risk factors for IS.In Miao residents,the occupation of manual labor and MIPA might be the protective factors for IS;DM,Apo B/Apo A1>0.9 and hs-CRP≥5.0mg/L might be the risk factors.In Han residents,MIPA might be the protective factor for IS;eating hot pot more than once a week,hs-CRP≥5.0mg/L and eating fast food more than once a week might be risk factors for IS.It showed that there were ethnic differences in the risk factors for IS.(2)Compared with Miao people,the influence of Apo B/ Apo A1 ratio on IS increased in Tujia people,indicating a positive interaction between ethnic categories and Apo B/ Apo A1 ratio on IS.(3)There were differences in composition of gut microbes between IS patients and NCs in ethinicity-specific population in Xiangxi,such as family Lactobacillaceae enriched in Tujia,Miao and Han patients with IS;family Lachnospiraceae enriched in Tujia,Miao and Han NCs;family Streptococcaceae only enriched in Tujia and Miao patients with IS;family Enterobacteriaceae only enriched in Tujia and Han patients with IS;family Enterococcaceae only enriched in Han patients with IS;and family Ruminococcaceae only enriched in Tujia and Han NCs.(4)Families Lactobacillaceae,Streptococcaceae,Enterococcaceae and Enterobacteriaceae enriched in IS patients were positively correlated with the clinical indexes for IS(SBP,DBP,Apo B/Apo A1 ratio and hsCRP),while negatively correlated with the protective clinical index for IS(HDL-C).However,families Ruminococcaceae and Lachnospiraceae enriched in NCs were negatively correlated with the clinical indexes for IS(SBP,DBP,Apo B/Apo A1 ratio,FPG and hs-CRP).(5)Family Enterococcaceae enriched in IS patients had positive interactions with SBP,DBP,and hs-CRP on the risk of IS,respectively. |
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