Inhibitory Effect Of Human Placental Fetal Mesenchymal Stem Cell-derived Exosomes On Ventricular Remodeling After Acute Myocardial Infarction By Altering Intestinal Flora And Inflammation

Part Ⅰ Establishment of human placental fetal-derived mesenchymal stem cells(PMSCs)culture system and their exosomes isolationSection Ⅰ Isolation,culture,and identification of mesenchymal stem cells from human placentaObjective Multiple differentiated PMSCs were extracted from human placental tissue for isolation,culture,identification,and establishment of mesenchymal stem cell culture system suitable for this study.Methods The fresh placental tissue on the decidual surface of the placenta base of healthy pregnant women at 37-42 weeks was selected.The PMSCs were isolated and cultured by cell attachment method using serum-free medium.The obtained cells were identified:basic morphological observation;Flow cytometry was used to identify the expression of specific surface marker molecules on PMSCs.The differentiation ability was detected by differentiation kit.Results 1.PMSCs adhered to the wall and grew in the shape of swirling colonies,with typical morphology and uniform distribution.The cells expressed the specific surface molecular markers of typical PMSCs including CD73(100%),CD90(99.98%)and CD105(97.01%),without the expressions of CD34(0.00%),CD45(0.08%),CDllb(0.00%)and HLA-DR(0.75%),demonstrating that PMSCs culture system was successfully established.2.After induced culture in adipoblast induction medium,the cells were stained with oil red O,and fat droplets were found to form in the cells,which were bright red.After induction by osteoblastic induction medium,the formation of intercellular calcium nodules was observed by alizarin red staining,which showed red dense and opaque masses,suggesting the multidirectional differentiation potential of PMSCs.Conclusion The serum-free culture system of PMSCs was established,which had the characteristics of mesenchymal stem cells defined by the International Stem Cell Association,and maintained high cell activity after subculture,providing an experimental basis for the subsequent exosome extraction and intervention studies.Section Ⅱ Extraction and identification of secreted exosomes from human placenta fetal mesenchymal stem cellsObjective Human placental fetal lateral mesenchymal stem cells exosomes(PMSC-Exos)were obtained and identified to lay a foundation for subsequent intervention experiments in acute myocardial infarction.Methods Exosomes were extracted from the supernatant of PMSCs medium the P3 to P6 generations by differential and overspeed centrifugation.1.Morphological characteristics of PMSC-Exos were detected by transmission electron microscopy(TEM).2.Nanoparticle tracking analysis(NTA)was applied to measure the particle size,concentration,and distribution of PMSC-Exos.3.Western blot was used to determine the expression of marker molecules CD9 and TSG101 on the surface of PMSC-Exos.4.Transcriptome sequencing and analysis was adopted to reveal the compositions of mRNA and non-coding RNA in PMSC-Exos.Results Exosomes were extracted by(differential)hypervelocity centrifugal method and special round or oval,uniform vesicles with complete lipid membrane structure were observed by transmission electron microscopy(TEM).Through NTA,the size of exosomes concentrated around 60-200nm,with an average size of 130.6nm,and exosomes accounted for 99%of the total.Western blot analysis showed that PMSC-Exos specifically expressed CD9 and TSG101 molecules,indicating that exosomes were successfully isolated from human PMSCs.Transcriptome sequencing and analysis showed that PMSC-Exos included a series of mRNA and non-coding RNA,top of which were selected and noted via GO and KEGG.Conclusion The hypervelocity(differential)centrifugal method was used to extract classic exosomes with high purity,this method was suitable for the requirements of this experiment.The morphology,characteristics,and surface molecular expression of the extracts were consistent with classical exosomes,basically meeting the requirements of subsequent experiments.Part Ⅱ Changes of immune inflammatory factors in repair period of acute myocardial infarctionObjective Animal models of acute myocardial infarction(AMI)were established to further study whether there were dynamic changes of immune inflammatory factors in the repair period of acute myocardial infarction,and to explore the role of immune inflammatory factors in acute infarction.Methods Male C57BL/6 mice(20-22g)were randomly divided into two groups(15/group):the AMI model group and the Sham group,The Sham group was treated with almost the same procedures as the AMI group,except that no coronary occlusion was performed.In AMI group,noninvasive ventilation was performed,and blunt dissection was performed into the thoracic cavity,and left anterior descending coronary artery was ligated under direct vision.Identification model:Electrocardiogram was performed 2 minutes before and 5 minutes after ligation of coronary arteries.On the day 28 after the surgery,the wall thickness,movement and cardiac function were examined by ultrasound doppler.On the 28th day after surgery,the mice were sacrificed,and the hearts were removed and prepared into frozen sections or paraffin sections.HE staining and Masson staining were performed to examine the range of necrotic myocardial cells,inflammation and fibrosis.Plasma and myocardial tissue were collected after myocardial infarction for determination of tumor necrosis factor-α(TNF-α),interleukin-1β(IL1β),interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1)and lipopolysaccharide(lipopolysaccharide).Myocardial necrosis markers including AST(aspartate transaminase),BNP(brain natriuretic peptide),MYO(myoglobin),and Tn-I(troponin-I)were analyzed for the levels of inflammatory factors and changes in necrosis markers between AMI group and Sham group.Lipid indicators including high-density lipoprotein(HDL)and total cholesterol(TC)in plasma were determined.Results Two mice died during the surgery,4 mice died 1 week after the surgery,and the rest twenty-four mice survived until to 28 days after the surgery.The model of AMI was successfully established in vivo.After a few seconds of LAD occlusion,the myocardium below the occlusion site turned from pink to pale,and the activity of the local ventricular wall weakened or even disappeared,especially at the infarction junction.ECG indicated that ST segment of lead Ⅰ,Ⅱ,Ⅲ and AVF was significantly increased after coronary artery ligation.After 28 days of AMI establishment,cardiac ultrasonic doppler examined and found that compared with Sham,in myocardial infarction model group area ventricular wall motion was decreased or disappeared,room wall thinning,left ventricular end-diastolic wall thickness(LVPWD)were attenuated(P=0.0058),left ventricular ejection fraction was reduced(LVEF,P=0.0003),left ventricular short axis shortening rate(LVFS)was decreased(P=0.0005),left ventricular end-diastolic diameter(LVEDD,P=0.0004)and left ventricular end-systolic volume(LVEDV,P=0.0003)were increased.Hematoxylin-eosin staining showed that the boundary between the infarct area and the non-infarct area were obvious in the AMI group.The myocardial cells in the infarct area were disordered and necrotic,infiltrated by neutrophils and proliferated with collagen fibers.Masson’s trichrome staining showed a large proliferation of blue-stained collagen fibers and only a few pink normal myocardia in AMI group.The myocardial necrosis markers(AST,BNP,MYO,Tn-I),inflammatory factors(TNF-α,IL-1β,IL-6,MCP-1,LPS)and lipid indicators(HDL and TC)were significantly increased in AMI group(P<0.05),suggesting that the myocardial infarction model was successfully constructed with significant changes in inflammatory immune factors after myocardial infarction.Conclusion By noninvasive ventilation,the anterior descending coronary artery ligation of direct environment(LAD)build myocardial infarction animal model was successful with inflammation,which laid the foundation for the next step to explore the effects of PMSCs secreting exosomes(PMSC-Exos)treatment in AMI,as well as further assess whether PMSCExos could improve AMI via anti-inflammation.Part Ⅲ Human placental fetal mesenchymal stem cell exosomes(PMSC-Exos)inhibit ventricular remodeling after acute myocardial infarction by altering inflammation and intestinal floraObjective To explore the mechanism of mesenchymal stem cell exosomes(PMSC-Exos)to improve the immune changes of myocardial infarction inflammation.Methods First of all,PMSC-Exos were successfully extracted and myocardial infarction model was successfully constructed.Mice with MI group were injected with PMSC-Exos through tail vein for 4 weeks(injected at 7,9,11 days after MI),and the ventricular wall motion,wall thickness,ventricular lumen size and cardiac function were detected by doppler ultrasound.After 28 days of intervention,the mice were killed and the hearts were removed and prepared for paraffin section.The myocardial infarction cells,inflammation and myocardial fibrosis were examined by HE staining and Masson staining.Feces were collected for 16 sRNA highthroughput sequencing analysis of intestinal flora composition,gas chromatography-mass spectrometry(GC-MS)analysis was used to measure gut bacteria metabolites short chain fatty acids(SCFAs).The plasma and the myocardial tissue were collected for the determination of lipid metabolism(high density lipoprotein-cholesterol(HDL-C),low density lipoproteincholesterol(LDL-C),total cholesterol(TC)and triglyceride(TG)),myocardial necrosis markers,inflammatory factors(TNF-α,IL-1β,IL-6 and MCP-1)and lipopolysaccharide(LPS)The above indications were compared among analysis of Sham group,the AMI model group and PMSC-Exos treatment group.Results The changes among PMSC-Exos group(intervention group),AMI group(model group)and Sham group were compared and analyzed.Compared with AMI group,cardiac function was significantly improved after PMSC-Exos intervention,which was reflected in reduced ventricular lumen size,increased ejection fraction,reduced left ventricular end diastolic diameter(LVEDD,P=0.0031),reduced left ventricular end diastolic volume(LVEDV,P=0.0026),increased left ventricular ejection fraction(LVEF,P=0.0228)and increased left ventricular short axis shorting rate(LVFS,P=0.042).The proinflammatory IL1β(P=0.009),IL-6(P=0.0181),TNF-α(P=0.0126)and MCP-1(P=0.0415)were decreased,and the plasma LPS level was significantly decreased(P=0.0299)with PMSC-Exos intervention.Plasma BNP(P=0.0149)and Tn-I(P=0.0014)were decreased,AST(P=0.0011),BNP(P=0.0199),MYO(P=0.0265)and Tn-I(P=0.0184)were decreased in myocardial tissue with PMSC-Exos administration.PMSC-Exos decreased the ratio of Firmicutes to Bacteroidetes(F/B ratio,P=0.0311).The relative abundances of Akkermansia(P=0.0489)and Bifidobacterium(P=0.0179)at the genus level were increased,and the concentrations of SCAF including isobutyric acid(P=0.0405),butyric acid(P=0.0018)and valeric acid(P=0.0032)were increased.Increase tendency in HDL-C level without significance difference was observed in PMSC-Exos group(P>0.05),other lipid indicators including LDL-C,TC and TG showed no changes between PMSC-Exos group and AMI group,respectively(P>0.05).Conclusion PMSC-Exos ameliorated myocardial infarction by anti-inflammatory and regulating intestinal microbiota and metabolites SCFAs.Part Ⅳ The role and mechanism of intestinal flora in immune inflammation of patients with acute coronary syndromeObjective To further clarify the role of inflammation and intestinal flora in ACS,we analyzed whether there were differences in intestinal flora composition and inflammatory factors between ACS patients(acute myocardial infarction(AMI)and unstable angina pectoris(UAP)and healthy people(HC).Methods Hospitalized patients with coronary angiography(CAG)diagnosis(AMI and UAP)were recruited in this study from April 2019 to October 2019 in Heart Center,General Hospital of Ningxia Medical University.Stool specimens and blood specimens were collected from ACS patients and HC controls to investigate the feces 16srRNA intestinal microbes sequencing analysis and levels of inflammatory IL-6,IL-1β,TNF-α,IL-10,MCP-1 and LPS.Results The comparison of basic clinical data between the AMI group and the HC healthy group showed that body mass index(BMI),aspartate aminotransferase(AST),troponin I(Tn-I),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),high homocysteine(HCY),C-reactive protein(CRP),IL-1β,IL-6,TNF-α,IL-10 and MCP-1 were significantly increased in AMI(P<0.05).BMI,TC,HDLC,LDL-C and CRP were significantly increased in UAP compared with HC(P<0.05).Gut microbiota analysis showed that there were 789 common bacteria among AMI group and HC group,368 specific bacteria in AMI group,788 common bacteria among UAP group and HC group,414 specific bacteria in UAP,and 596 common bacteria among AMI,UAP and HC healthy groups.Compared with HC group,in ACS(AMI and UAP),at the phylum level,Firmicutes and Bacteroidetes were dominant,the relative abundance of Firmicutes and F/B ratio were significantly decreased,and the relative abundance of Bacteroidetes,Proteobacteria and Verrucomicrobia were significantly increased.At the genus level,compared to HC group,the levels of Bacteroides,Parabacteroide,Unidentified_Enterobacteriaceae,Subdoligranulum,Akkermansia,Alistipes,Streptococcus,Paraprevotella,whereas and Paraprevotella were significantly increased,whereas Blautia,Agathobacter,Bifidobacterium and Anaerostipes were significantly reduced.Conclusion Patients with ACS possessed the dysbiosis of gut flora and inflammation,both of which were closely correlated.