ABSTRACT
BACKGROUND: Mesenchymal stem cell-derived exosomes (MSC-EXO) have emerged as novel therapeutic strategies for myocardial infarction (MI). However, many questions remain untouched and unanswered regarding their roles in myocardial fibrosis. This study aimed to probe the therapeutic effects of MSC-EXO on myocardial fibrosis after MI and possible mechanisms. METHODS: Myocardial tissues were obtained from MI rats, and myocardial cell viability, fibrosis, apoptosis, and epithelial-mesenchymal transition (EMT) were detected by immunohistochemistry, Masson's staining, TUNEL, and western blot. Bone marrow-derived MSCs and corresponding EXO were identified, and cardiac function were detected after treatment of MSC-EXO. Bioinformatics analysis and ChIP assay were conducted to detect the downstream genes of EZH2. EZH2 was upregulated alone or with HMGA2 overexpression in myocardial tissues of MI rats upon MSC-EXO treatment, and PI3K/AKT pathway activity in myocardial tissues was detected using western blot. RESULTS: The proliferative activity in myocardial tissues of MI rats was significantly decreased, along with accentuated fibrosis, increased collagen volume and EMT. MSC-EXO treatment resulted in partial restoration of cardiac function and reduced EZH2 expression in the myocardium of rats. EZH2 inhibited HMGA2 expression by increasing the H3K27me3 modification. PI3K/AKT pathway was altered under the influence of the EZH2/HMGA2 axis. EZH2 inhibited the effect of MSC-EXO on the recovery of cardiac function and accelerated fibrosis, while HMGA2 reversed the effect of EZH2 to reduce fibrosis and enhance cardiac function. CONCLUSION: MSC-EXO alleviated fibrosis in MI rats via inhibition of EZH2, whereas EZH2 inhibited HMGA2 expression and impaired the PI3K/AKT pathway.
Subject(s)
Exosomes , Mesenchymal Stem Cells , Myocardial Infarction , Animals , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Enhancer of Zeste Homolog 2 Protein/pharmacology , Exosomes/genetics , Exosomes/metabolism , Fibrosis , Humans , Mesenchymal Stem Cells/metabolism , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardial Infarction/therapy , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , RatsABSTRACT
In order to develop novel immunoadjuvants to boost immune response of conventional vaccines, experiments were conducted to investigate the regulating effects of porcine interleukin-6 gene and CpG motifs as the molecular adjuvants on immune responses of mice that were co-inoculated with trivalent vaccines against Swine fever, the Pasteurellosis and Erysipelas suis. Synthetic oligodeoxynuleotides containing CpG motifs were ligated into pUC18, forming recombinant pUC18-CpG plasmid. Eukaryotic plasmid expressing porcine interleukin-6 (VPIL-6) were also constructed as molecular adjuvants in an attempt to enhance levels of immune responses of mice co-administered with the trivalent vaccines in this paper. The cellular and humoral immune responses of mice were systematically analysed, and the experimental results were observed that the number of white blood cells, monocytes, granuloytes and lymphocytes significantly increased, respectively, in the mice immunized with VPIL-6, compared with those of the control; the IgG content and titre of specific antibodies to the trivalent vaccine mounted remarkably in the sera from the VPIL-6 vaccinated mice; the proliferation of lymphocytes and induced IL-2 activities were significantly increased in the vaccinated groups. The above-mentioned immune responses of mice co-inoculated with pUC18-CpG plasmid were significantly stronger than those of co-inoculated with pUC18 plasmid, suggesting that the immunostimulatory effect of oligodeoxynuleotides CpG is closely connected with the number of CpG motifs. These results suggest that the porcine IL-6 gene and CpG motifs could be employed as effective immunoadjuvants to elevate immunity to conventional vaccines.
Subject(s)
Adjuvants, Immunologic/pharmacology , Gene Expression Regulation/drug effects , Interleukin-6/genetics , Oligodeoxyribonucleotides/pharmacology , Vaccines, Combined/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/analysis , Antibody Formation/physiology , Classical Swine Fever Virus/immunology , Erysipelas/immunology , Female , Immunity, Cellular/physiology , Interleukin-6/immunology , Lymphocyte Activation , Lymphocytes/immunology , Mice , Mice, Inbred BALB C , Pasteurella/immunology , Plasmids , Swine , VaccinationABSTRACT
Porcine IL-6 gene and CpG sequences were used to enhance some indirect indicators of the immune response of mice. An indirect ELISA was used to quantify the amount of IgG in the sera from mice that had been inoculated with VPIL-6, a recombinant VR1020 vector into which had been inserted the porcine IL-6 gene cloned in our laboratory, or with CpG, pUC18 or VR1020. The induced bioactivity of IL-2 of lymphocytes in the spleen was assayed by the MTT method, and the proliferation of lymphocytes stimulated with ConA was tested to identify the immune response of the experimental mice. The amount of IgG in the immunized mice was significantly higher than that in the control group. Among the immunized groups, inoculation with VPIL-6 induced the highest content of IgG (p < 0.05), the greatest bioactivity of IL-2 and the greatest proliferation of lymphocytes from the spleen of the mice. These results suggest that inoculation with porcine IL-6 gene and CpG sequences may enhance the immune response of mice, and might be used as an immunoadjuvant.
