| Part Ⅰ Correlation analysis of gut microbiota and inflammation in ACS patients based on AS lesions.Objective Although substantial progress has been made in the diagnosis and treatment of acute coronary syndromes(ACS),it still remains the leading cause of death globally.Atherosclerosis(AS)is the major cause of ACS which is a significant contributor to both morbidity and mortality in the world.AS is closely associated with chronic low-grade inflammation and gut microbiota composition.However,the relationships among the ACS,inflammation and gut microbiota have not been properly validated.Methods stools and plasma were collected from 57 individuals with ACS and 44 healthy controls(HC),and then compared gut microbial composition using 16 S RNA gene sequencing in fecal samples.The levels of tumor necrosis factor(TNF)-α,interleukin(IL)-6,IL-1β,IL-10 and lipopolysaccharide(LPS)in plasma were measured with enzyme linked immunosorbent assay(ELISA)kit.Results Altered composition of gut microbiota was demosntrated to be associated with ACS and exacerbated inflammatory status.Moreover,parameters in ACS including body mass index(BMI),aspartate aminotransferase(AST),alanine aminotransferase(ALT),triglycerides(TG),C reactive protein(CRP),creatinine(Cre),white blood cell(WBC),serum glucose(Glc),homocysteine(Hcy)and cardiac troponin(c Tn)were elevated.Furthermore,pro-inflammatory IL-1β,IL-6,TNF-α,and LPS in ACS were increased respectively.The results of 16 S r RNA sequencing and analysis displayed that the overall community of gut microbiota in ACS was notably changed mainly through decreasing butyrate-producing bacteria(Subdoligranulum,Roseburia,Faecalibacterium).Further analysis showed that there was a significant correlation among the above differences in butyrate-producing bacteria and inflammatory indicators.Conclusion ACS was demonstrated to be associated with gut dysbiosis and increased inflammation,which may potentially represent comprehensive targets for promoting or preventing ACS.Part Ⅱ Elucidation of the molecular mechanism of butyrate-GPR43/ HDAC3-mi RNAinflammation based on AS animal modelObjective Chronic low-grade inflammation is regarded to an important signature of atherosclerosis(AS).Macrophage(Mψ)and related polarization have been demonstrated to play a crucial role in the occurrence and development of AS inflammation.Butyrate,a bioactive molecule produced by the intestinal flora,has been increasingly demonstrated to exhibit a vital role for regulating the inflammation in chronic metabolic diseases.However,the effectiveness and multiple anti-inflammation mechanisms of butyrate on AS still need to be further understood.Methods The mice were randomly assigned to 4 groups(n = 15/each group): control group(CON),CON treated with Na B group(CON+Na B),atherosclerosis group(AS)and AS treated with Na B group(AS+Na B).C57BL/6J mice in the CON or Apo E-/-mice in AS were respectively fed normal or HFD diet.Meanwhile,mice in CON and AS groups were administered normal saline,as well as mice in CON+Na B and AS+Na B groups were fed with Na B(200mg/kg,dissolved by normal saline)by gavage once daily.After 14 weeks of feeding,the feces of the mice were collected.All mice were euthanized with 4% sodium pentobarbital and associated indications were investigated,including:(1)The pathological changes in AS were measured with en face oil red O staining,HE staining,and Masson’s trichrome staining.(2)Inflammatory indicators: Tumor necrosis factor(TNF)-α,interferon(IFN)-γ,IL-6,IL-1β,IL-17 A,and IL-10 were respectively determined by Ray Biotech(QAM-INT-1-1)chips and quantitative real-time PCR(q RT-PCR).The expression of aortic macrophages(Mψs)in situ were measured by flow cytometry.(3)Lipopolysaccharide(LPS): The plasma LPS level in each group was examined using a Limulus amebocyte lysate kit.(4)Gut microbiota: 16 S r RNA hypervariable sequencing was used to detect the diversity and abundance of gut microbiota among diverse groups.(5)Correlation analysis: Pearson method was used to analyze the correlation among gut microbiota,inflammation and serum lipids.(6)Transcriptional m RNA levels of genes including histone deacetylase(HDAC)1-3,specificity protein 1(Sp1),peroxisome proliferator-activated receptor(PPAR)γ,Gproteincoupledreceptors(GPR)43,β-arrestin2(ARRB2),Toll like receptor(TLR4),NOD like receptor protein 3(NLRP3),nuclear factor(NF)-κB and zonula occludens(ZO-1)were performed by q RT-PCR.(7)Micro RNAs(mi RNAs): Illumina high-throughput sequencing was performed to screen differentially expressed mi RNAs.Results The atherosclerotic lesion in the AS group was dramatically reduced after Na B intervention.Moreover,deteriorated routine parameters of AS including body weights(BWs),low-density lipoprotein(LDL-C),triglyceride(TG),total cholesterol(TC)were significantly reversed by Na B administration.Abnormal elevated plasma and aorta pro-inflammatory indicators including interleukin(IL)-1β,IL-6,IL-17 A,tumor necrosis factor(TNF)-α and lipopolysaccharide(LPS),as well as reduced anti-inflammatory IL-10 in plasma were respectively rectified after Na B administration.Consistently,accumulated Mψ and associated imbalance of polarization in the arota were attenuated with Na B treatment.Importantly,we demonstrated that the suppression of Mψ and associated polarization of Na B was dependent on binding G-protein coupled receptor(GPR)43 and inhibiting histone deacetylase(HDAC)3.Moreover,we found that intestinal butyrate-producing bacteria,anti-inflammatory bacteria and intestinal tight junction protein zonula occludens-1(ZO)-1 may contribute to this effectiveness.Intriguingly,according to transcriptome sequencing of atherosclerotic aorta,29 elevated and 24 reduced mi RNAs were found after Na B treatment,especially mi R-7a-5p,suggesting that noncoding RNA may possess a potential role in the protection of Na B against AS.Correlation analysis showed that there were close complicated interactions among gut microbiota,inflammation and differential mi RNAs.Conclusion Collectively,this study revealed that dietary Na B may ameliorate atherosclerotic inflammation by regulating Mψ polarization via GPR43/HDAC-mi RNAs axis in Apo E-/-mice.Part Ⅲ Identify the molecular mechanism of butyrate-GPR43 / HDAC3-mi R-7a-5pinflammation based on an inflammatory cell modelObjective Buytrate(Na B)was found to exhibit anti-inflammatory effect in in Apo E-/-mice in our study.The animal experiments in this study have basically confirmed the role of macrophages.And we will further expand in subsequent in vitro experiments to confirm the specific mechanism of the signaling pathway.Methods The RAW264.7 cells were stimulated by lipopolysaccharides(LPS).In addition,mi R-7a-5p mimics or mi R-7a-5p inhibitor was transfected into the RAW264.7 cells,respectively.Western blotting and RT-q PCR were used to detect the level of histone deacetylase(HDAC)1-3,specificity protein 1(Sp1),peroxisome proliferator-activated receptor(PPAR)γ,Gprotein-coupledreceptors(GPR)43,β-arrestin2(ARRB2),Toll like receptor(TLR4),NOD like receptor protein 3(NLRP3),nuclear factor(NF)-κB in LPS-stimulated or Na B-treated RAW264.7 cells.The levels of TNF-α,IL-6,IL-1β and IL-10 in LPS-stimulated or Na B-treated RAW264.7 cells were measured with RT-q PCR and enzyme linked immunosorbent assay(ELISA)kit.Results The level of mi R-7a-5p was significantly upregulated in Na B-treated RAW264.7 cells.Meanwhile,the level of HDAC3/Sp1,TLR4,NLRP3 and NF-κB were significantly increased and PPARγ,GPR43 and ARRB2 were decreased in LPS-stimulated RAW264.7 cells,which were markedly reversed by Na B.In addtion,LPS markedly increased the expression of proinflammatory cytokines in RAW264.7 cells,which were reversed in the presence of Na B.Moreover,mi R-7a-5p enhanced the anti-inflammatory effects of Na B in LPS-stimulated RAW264.7 cells via inhibiting HDAC3/GPR43 related pathways and proinflammatory cytokines.Conclusion Taken together,our results suggested that Na B might exert anti-inflammatory effects in LPS-stimulated RAW264.7 cells via upregulation of mi R-7a-5p.Therefore,Na B might serve as a potential agent for the treatment of AS inflammation. |
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