Mechanism Of Bone Marrow Mesenchymal Stem Cell-derived Exsomes Regulating Cardiomyocyte Pyroptosis After Myocardial Infarction

Background:Acute myocardial infarction(AMI)is the leading cause of disability and death worldwide and its morbidity and mortality remain high,threatening the health of all human beings.Stem cell transplantation to repair damaged myocardium and promote cardiomyocyte regeneration has been verified in many animal models,but the mechanism of its distribution,migration,survival and differentiation in vivo is still unclear.Studies have confirmed that the occurrence of inflammation after myocardial infarction,usually in the form of paracrine mediators continue to worsen the injury.NOD-like receptor thermal protein domain associated protein 3(NLRP3),Absent in Melanoma 2(AIM2)and other inflammasomes form in the myocardium within 3-24 hours after AMI participate in inflammatory response and aggravates ischemia-reperfusion injury.Studies suggest that targeted inflammasome therapy may be a viable strategy for reducing AIM size and preventing post-MI heart failure.Bone mesenchymal stem cells(BMSCs)have become the seed cells for myocardial infarction treatment because of their abundant sources,selfrenewal,multi-directional differentiation potential and low immunogenicity,however,the mechanism is still unclear.Researchers are more inclined to study the paracrine effect of stem cells to play their protective role,and these factors include the study of exosomes.Bone marrow-derived mesenchymal stem cell exosomes(BMSCs-Exo)through cell-cell communication are a new cellular immunotherapy,and we speculated that BMSCs-derived exosomes might improve the occurrence of heart failure after myocardial infarction through cell-cell communication.ObjectiveIn this study,we used ligation of the anterior descending artery(LAD)of the rat heart to induce AIM in rats as a model to study the specific mechanism of BMSCs-Exo in regulating pyroptosis after myocardial infarction.This study may provide a theoretical basis for cardiac repair mechanisms after myocardial infarction and provides a new target for exosome vector intervention therapy.Methods and ResultsBMSCs from rat were extracted for isolation and culture,we cultured in DMEM-F12 medium in vitro to observe the growth status of rat BMSCs and describe their growth curves;observe their morphological characteristics and use flow cytometry to analyze the surface markers.The results showed that the cells grew rapidly on the 3rd to the 7th day after they adhered to the wall,and the growth curve showed an"S" shape.Most of the cells are long spindle-shaped or triangular and polygonal fibroblast-like cells,which are arranged in clusters and grow neat and directional.We used flow cytometry to identify surface marker antigens of rat BMSCs,which were positive for CD29,CD90,CD34 and MHC II PE.We used the third-generation rat BMSCs in good condition to extract exosomes from the supernatant of their culture medium to identify exosomes.Under the transmission electron microscope,it was found that the exosomes were round or oval with different diameters and had a "cup holder"-like lipid bilayer structure with a particle size of 20-140 nm.The expression of exosome surface-specific markers CD63 and CD9,TSG101 was significantly increased by Western Blot.After successfully extracting BMSCs-derived exosomes,we did further research,successfully induced a rat model of acute myocardial infarction,and used the extracted exosomes for local injection in the myocardial infarction area.We found that on the 1st and 7th days after myocardial infarction,the cardiac function of the PBS group and the BMSCsExo group showed a significant downward trend.From the 7th day to the 28th day after the operation,the EF and FS of the BMSCs-Exo group gradually increased.Histological examination(HE staining,masson staining)showed that the degree of myocardial cell necrosis,inflammatory cell infiltration and fibrosis in the BMSCs-Exo treatment group was significantly improved compared with the control group,suggesting that BMSCs-Exo can significantly promote the recovery of cardiac function in rats.In order to further explore the mechanism of BMSCs-Exo in improving myocardial infarction,we firstly determined whether pyroptosis occurred in rats after AMI.The results showed that the high expression of cardiomyocyte pyroptosis-related proteins NLRP3,AIM2,and Caspase-1 was detected 3 days after myocardial infarction in rats.We used H2O2 to induce pyroptosis in H9C2 cardiomyocytes,and detected genes related to pyroptosis including NLRP3,Caspase-1,IL-18 and IL-1B.We found that exosomes at an appropriate concentration can significantly reduce the effect of cardiomyocyte pyroptosis by inhibiting related inflammatory factors(AIM2,caspase-1,IL-1β and IL-18).Finally,in order to further explore the mechanism of action of BMSCs-Exo,combined with the results of exosome sequencing analysis in previous studies,we found that miR-1433p was highly expressed in BMSCs-Exo by bioinformatics analysis,which was verified in our experiments.Then,we detected the decreased expression of AIM2 in the exosometreated group and the exosome-overexpressing miR-143-3p group after myocardial infarction by Western blot;24 hours after the exosomes were co-cultured with cardiomyocytes,cardiomyocyte pyroptosis was induced.The total RNA of the cells was extracted,and the inflammatory factors downstream of AIM2 were detected by RT-qPCR.The results showed that the expression levels of Caspase-1,IL-18 and IL-1B in the miR143-mimics treatment group were lower after pyroptosis;The expression levels of Caspase1,IL-18 and IL-1B increased in the miR-143-inhibitor treatment group.These data,combined with previous findings,demonstrate that BMSCs-Exo carry miR-143-3p into MI tissues and cells,targeting the inhibition of the AIM2/caspase1/IL-1β/IL-18 signaling axis and regulating pyroptosis after myocardial infarction.ConclusionOur results found that AMI may lead to cardiomyocyte pyroptosis,and BMSCs-Exo can effectively reduce the cardiomyocyte pyroptosis after AMI and significantly improve cardiac function.The exosomes secreted by BMSCs deliver miR-143-3p into cardiomyocytes,target and inhibit the AIM2/caspase1/IL-1β/IL-18 signaling axis and regulate cell pyroptosis after myocardial infarction,repair damaged myocardium,and improve heart function.This study may provide a theoretical basis for the mechanism of heart failure and cardiac repair after myocardial infarction and provide a new target for exosome vector intervention therapy.