Study On The Relationship Between The Regulation Of S1PR1 And The Circulating Exosomes MiR-155

Background : Rheumatic heart disease(RHD)is an autoimmune disease caused by rheumatic fever after infection with group A hemolytic streptococcus.The disease mainly affects the mitral valve,manifesting as valve stenosis and insufficiency.RHD still has a high prevalence in China,and it is one of the major cardiovascular diseases leading to chronic heart failure and death.How heart valve tissue causes lesions leading to rheumatic heart disease,the exact mechanism is not clear,to study its pathogenic mechanism and further search for new rheumatic valvular disease Intervention targets are of great significance.OBJECTIVE:To establish RHD rat model,examine the expression of miR-155 in exosomes of RHD rat and control group.Simultaneous detection of S1PR1 in both valve tissues was performed.The expression status of circulating exosomes miR-155 and S1PR1 in RHD rats and control rats wasinitially investigated,and the luciferase reporter gene system was used to verify the relationship between S1PR1 and miR-155。METHODS:Thirty Lewis female rats were randomly divided into RHD group rats and control group.Constructing rat model of RHD: On the 1st week,the RHD group was injected subcutaneously with an 0.2 ml antigen I in the footpad(Inactivated GAS Bacteria: Complete Freund’s adjuvant emulsifier =1:1).The dosing interval was 7 days,and 0.5 ml antigen I was subcutaneously injected into the abdomen at 2nd,3rd,4th,and 5th weeks.The control group was injected with sterile saline + complete Freund’s adjuvant emulsifier at the same time,in the same dose,at the same site.From the 6th week onwards,the RHD group was injected subcutaneously with 0.5 ml antigen II(inactivated GAS liquid)into the abdomen for a period of 7 days until the end of the 28 th week.The control group was given the same dose at the same time.The site was injected with sterile saline.The 32-week model build ends.At the end of the model,each rat underwent transthoracic echocardiography.The left ventricular ejection fraction(EF%),left ventricular short axis fraction(FS%),and E/A ratio were compared between the two groups.Heart function of rats in RHD group and control group.At the end of model establishment,rats were sacrificed.Exosomes were extracted from rat plasma and used to identify their morphology.At the same time,total exosomal RNA was extracted and the expression of miR-155 in plasma exosomes was detected by fluorescent quantitative PCR.HE staining and Masson staining were performed on rat myocardium and mitral valve.Part of the valve tissue was taken for immunohistochemistry and fluorescence quantitative PCR to detect the expression of S1PR1 at the gene level and protein level.Bioinformatics methods were used to predict the binding sites of miR-155 and S1PR1,3’UTR mutant plasmid containing S1PR1 containing the binding site was constructed.The recombinant plasmid was co-transfected with miR-155 and miR-NC into 293 T cells.After overexpression of miR-155,detection of changes in luciferase activity can quantitatively reflect the inhibitory effect of miR-155 on S1PR1.RESULTS:1.During the modeling process,there were two rats died in the RHD group and zero deaths in the control group.2.Left ventricular ejection fraction(EF%)in the RHD group was 55.06±6.56 and the EF% in the control group was 77.80±5.97,P value<0.05,the difference was statistically significant;the left ventricular short axis shortening fraction(FS%)in the RHD group was 23.60±3.90,and the FS% in the RHD group was 39.83±5.60 FS,P values.<0.05,the difference was statistically significant.The E/A ratio in the RHD group was 0.72±0.12,and the E/A ratio in the control group was 1.56±0.18.The P value was <0.05,and the difference was statistically significant.3.HE tissue staining results showed that there were 3 rats in the RHD group with myocardial inflammatory cell infiltration.In the RHD group,there were 10 chronic rat mitral valve tissues with chronic lymphocyte and plasma cell infiltration,collagen hyperplasia,calcium deposition,and angiogenesis.The proportion of valvular disease in RHD group was 76.9%(10/13).No inflammatory cell infiltration was found in the mitral valve tissue of the control group.Masson staining showed that the control group showed light blue staining and no obvious proliferation of collagen fibers.The staining of RHD group was significantly deeper,and the proliferation of collagen fibers was obviously disordered.The expression level of miR-155 in circulating exosomes of RHD rats was higher than that of control rats,p<0.05,the difference was statistically significant.6.In S1PR1 mRNA level or protein level,the expression of S1PR1 in the valve tissue of RHD group was lower than that in the control group.7.Compared with the co-transfected S1PR1-3′ UTR and miR-NC groups,the luciferase activity of the co-transfected S1PR1-3′ UTR and miR-155 groups decreased significantly,P<0.05.CONCLUSIONS : Increased expression of miR-155 in circulating exosomes in a rat model of rheumatic heart disease,down-regulation of S1PR1 expression in a rat model of rheumatic heart disease,and expression of miR-155 targeting S1PR1 in the development of rheumatic heart disease process.