The Improvement Effects And Mechanisms Of MSCs Exosomes Of Bone Marrow In Young Rats On The Decline Of Angiogenesis Induced By Aging

Objective:Aging is one of the important factors for the decline of angiogenesis,and MSCs are a kind of stem cells with anti-aging and promoting angiogenesis.The MSCs-derived exosomes can not only reflect the function and characteristics of the parental cells,but also can be used as a replacement therapy to make up for the limitations of MSCs itself.MSCs-derived exosomes carry many aging-related miRNAs,the expression capacity is down-regulated with age,and further lack regulatory functions and disorders.This paper intends to investigate whether MSCs-derived exosomes of young rats can improve the angiogenesis ability of rats under simulated aging conditions,then establishing the differential expression profiles of miRNAs in exosomes of MSCs derived from young rat and aged rat,so as to screen out the key miRNAs and target genes which improve the angiogenesis ability of aged rats,and further explore the mechanism of its enhanced angiogenesis ability.Approaches and results:1.The isolation of MSCs of SD rats and exosome identificationApproaches:SD rats were divided into young SD rat group and aged SD rat group according to the age of 4-6 weeks and older than 48 weeks,and the SD rats were extracted by grouping.Primary bone marrow MSCs cells from rats were isolated,sub-cultured,and finally extracted from SD rats bone marrow MSCs by high-speed centrifugation,and confirmed by electron microscopy,particle size analysis and exosome surface antigen WB identification methods.The extracted substances are exosomes.Results:MSCs were successfully isolated from SD rats.The classical exosome structure of double-layered envelope was observed under electron microscope.The expression of TSG101,CD9,and Alix antigen proteins was positive by method of WB,The results showed that the diameter of extracted exosomes range from 45.09-115.71 nm,the average diameter was69.01nm,and the average concentration was 3.17×10¹⁰ particles/ml,which was in line with the particle size of exosomes,and the extraction of exosomes was successful.2.Differences in the Restoration and improvement of ischemic effects in MSCs-derived exosomes of young and old SD ratsApproaches:1.an animal model of lower extremity ischemia was created by ligating the rat femoral artery,and MSCs-derived exosomes of young and old SD rats were injected in ischemia area respectively.The blood supply of the on the 0day,3 days,7 days,and 14 days was observed with the help of laser Doppler blood perfusion imager,and also the blood perfusion ratio was measured.At the end of above time point,the gastrocnemius muscle in the ischemic area was extracted for HE staining to observe the changes of muscle morphology and detection of angiogenesis-related index proteins in WB.Muscle morphological changes were observed by staining,and angiogenesis-related index proteins were detected.2.Routine in vitro culture of HUVEC cells,immunofluorescence tracing method was used to evaluate whether exosomes entered the cells to participate in biological reactions,and then CCK-8,cell cloning,Transwell migration,angiogenesis,and WB were used to detect growth,proliferation,migration and angiogenesis of HUVEC cells treated with 100μg of MSCs-derived exosomes from young and old SD rats.WB method was used to detect the expression changes of angiogenesis-related proteins after HUVEC cells were treated with two types of exosomes,to evaluated comprehensively the effect and degree of angiogenesis improvement of bone marrow MSCs-derived exosomes of different mouse ages from in vitro and in vivo experiments Results:Compared with the control group,at the animal level,the exosome treatment group is much better in promoting blood flow recovery of the lower extremities and improving the pathological morphology of the gastrocnemius muscle in the ischemic area.At the cellular level,the exosome group is much better in promoting the expression of proteins related to proliferation,cloning,migration,tube formation and angiogenesis of HUVEC（P<0.05）,and the improvement effects of the young exosome group was more significant than that of the old group,and the differences were statistically significant（P<0.05）.3.Molecular screening and target gene prediction of differentially expressed miRNAs in MSCs-derived exosomes from young and old SD rats Approaches:By extracting miRNAs from MSCs-derived exosomes of young and old SD rats respectively,and sequencing them on the Illumina Hi Seq 2500 platform,we have done the identification of the known miRNA and the prediction of the new miRNA,and analysis of miRNA expression,screening of differentially expressed miRNA,the analysis of differentially expressed miRNA and miRNA cluster,to locate the most differentially expressed miRNA molecules,and by GO classification and KEGG annotation methods to search for downstream target genes and possible biological processes involved,and finally to use targetscan prediction software to clarify the relationship between miRNA and downstream target genes.Results:Through high-throughput sequencing,80 differential miRNA molecules in exosomes of young group and old group were screened,of which 32 were up-regulated and 48 were down-regulated.According to the P value and the closeness of involvement in angiogenesis,miR-140-5p is a differentially expressed miRNA molecule,and it is predicted that Bach1 may be a target gene involved in angiogenesis.4.miR-140-5p improves angiogenesis by negatively regulating Bach1gene:Approaches:First,miR-140-5p up-regulated HUVEC cells(HUVEC^{miR-140-5p-mimics})and miR-140-5p down-regulated HUVEC cells(HUVEC ^{miR-140-5p-inhibitor})were obtained by transfection,CCK-8,cell clone,Transwell Migration,angiogenesis,and WB assays were used to observe the effects of miR-140-5p on cell growth,proliferation,migration,and angiogenesis;dual luciferase assay and RIP assay were used to verify whether miR-140-5p and Bach1 genes were targeted Finally,the m RNA expression of the two was quantitatively detected by PCR;HUVEC^{miR-140-5p-mimics} cells overexpressed Bach1 or HUVEC^{miR-140-5p-inhibitor}down-regulated Bach1 expression to verify whether miR-140-5p negatively regulates Bach1 gene and thus Angiogenesis was improved,and the results were statistically significant（P<0.05）.Results:miR-140-5p has the effect of improving angiogenesis,which can be involved in angiogenesis by targeting the Bach1 gene.Conclusions:1.Bone marrow MSCs-derived exosomes in both young and old SD rats can improve angiogenesis,and the young group is better than the old group;2.To construct the differential expression profiles of miRNAs derived from MSCs derived from young and aged SD rats,and to analyze their biological processes and signaling pathways,and obtained miR-140-5p as the differential expression of MSCs derived from bone marrow MSCs of different mouse ages.Expressed key genes;3.miR-140-5p improves angiogenesis through the mechanism of negatively regulating Bach1 gene.88 references,34 figures,4 tables...