| Cardiovascular disease(CVD),particularly acute myocardial infarction(AMI),is a significant health concern.Despite remarkable progress in risk factor control,pharmacological treatment,and cardiovascular interventions,severely damaged or necrotic myocardium cannot be repaired or regenerated.Therefore,it is essential to investigate the pathogenesis of myocardial injury caused by CVDs like AMI and develop effective prevention and treatment strategies.In the field of cardiovascular diseases,exosomes derived from stem cells have been found to possess therapeutic potential.Specifically,exosomes derived from human umbilical cord mesenchymal stem cells(h UC-MSC-EXOs)have shown promise in treating myocardial injury caused by CVD.However,the specific role and mechanisms of h UC-MSCEXOs in this context have yet to be fully understood.This study aimed to create an in vitro model of myocardial injury by treating rat cardiomyocytes with adriamycin(doxorubicin,DOX).The objective was to investigate the effects of h UC-MSC-EXOs on DOX-induced mitochondrial injury,inflammation and oxidative damage in rat cardiomyocytes.In this study,H9C2 cardiomyocytes were treated with varying concentrations of DOX(0,1,1.5,2,5,and 10 μmol/L)for 24 hours.The cell counting kit 8(CCK8)was used to assess cardiomyocyte viability and determine the optimal modeling conditions.h UC-MSC-Exos were isolated from the supernatant of h UC-MSCs using ultracentrifugation and characterized using transmission electron microscopy,dynamic light scattering,and protein blotting(Western blotting,WB).To evaluate the effectiveness of h UC-MSC-Exos in DOX-treated H9C2 cells,various concentrations(5,10,20 μg/ml)were used and their viability was measured using CCK8 assay.The cells were observed for any morphological changes using ordinary light microscopy.Apoptosis levels between the groups were analyzed using apoptosis flow cytometry.The study utilized the LDH assay kit to quantitatively analyze cytotoxicity by measuring LDH activity in cell culture supernatants of each group.In addition,the intracellular ROS level was measured through cell flow assay,while the intracellular MDA level was assessed using the Malondialdehyde assay kit to determine oxidative damage between the groups.This study utilized transmission electron microscopy and the Next Gen-O2 k multidimensional energy metabolism analysis system to detect and analyze the ultrastructure and oxidative phosphorylation function of mitochondria in H9C2 cells.Additionally,levels of inflammatory factors,specifically tumour necrosis factor-α(TNF-α)and interleukin-6(IL-6),were measured between groups using real-time quantitative fluorescence polymerase chain reaction.The study found that(1)DOX had a significant negative impact on the viability of H9C2 cardiomyocytes,and the extent of its damage increased with higher drug concentrations(p < 0.05).On the other hand,h UC-MSCExos were found to have a protective effect on DOX-induced myocardial injury,significantly improving the decrease in H9C2 cell viability caused by DOX(p < 0.05).These results suggest that h UC-MSC-Exos may be a potential therapeutic option for DOX-induced myocardial injury.(2)Under ordinary light microscopy,the H9C2 cells exhibited good growth with a long shuttle shape.However,DOX treatment resulted in significant damage to the cells,increasing the number of cell deaths and altering the morphology of the surviving cells.Following intervention with h UC-MSC-Exos,the number of H9C2 cell deaths was reduced and the morphology of the surviving cells improved.(3)DOX treatment resulted in a noticeable increase in the rate of apoptosis of H9C2 cells(p < 0.05).However,the use of h UC-MSC-Exos was able to effectively inhibit the DOX-induced apoptosis of myocardial cells(p < 0.05).(4)DOX caused a significant increase in LDH release level of H9C2 cells(p < 0.05).However,the use of h UC-MSC-Exos was found to alleviate the myocardial injury caused by DOX and reduce LDH release.(5)DOX treatment led to a significant increase in ROS and MDA levels in H9C2 cells(p < 0.05).However,treatment with h UC-MSC-Exos significantly reduced the production of ROS and MDA in cardiomyocytes,thereby protecting H9C2 cells from DOXinduced oxidative damage(p < 0.05).(6)DOX has been found to have a detrimental effect on the mitochondrial structure and oxidative phosphorylation function of cardiomyocytes.The use of transmission electron microscopy has revealed that the mitochondrial volume in the DOX group was significantly reduced,with mitochondrial rupture,ridge fracture,and reduction or even disappearance.However,the administration of h UCMSC-Exos has been shown to alleviate the structural changes caused by DOX in the mitochondria.DOX treatment significantly reduced the respiratory capacity of H9C2 cardiomyocytes as measured by cell energy metabolism assay.This reduction was observed in basic respiratory capacity,maximum respiratory capacity,reserve respiratory capacity,proton leakage level,and ATP synthesis capacity(p < 0.05).Following the administration of h UC-MSC-Exos,there was a notable improvement in mitochondrial function and ATP production capacity.Additionally,there was a significant increase observed in the basic respiratory capacity,maximum respiratory capacity,reserve respiratory capacity,and proton water leakage of myocardial cells(p < 0.05).(7)DOX treatment led to a significant increase in intracellular levels of inflammatory factors TNF-α and IL-6 in H9C2 cells(p < 0.05).However,intervention with h UC-MSC-Exos resulted in a significant reduction in intracellular levels of inflammatory factors in cardiac myocytes(p < 0.05).The findings of this study indicate that h UC-MSC-Exos can protect H9C2 cells from DOX-induced toxic damage through multiple mechanisms.These include inhibiting cardiomyocyte apoptosis,reducing ROS and MDA production in cardiomyocytes,regulating respiratory function and ATP synthesis of restored mitochondria,maintaining mitochondrial morphological structure,and reducing inflammatory cytokines levels in cardiomyocytes.These results provide valuable insights for the development of new prevention and treatment strategies for myocardial injury caused by CVD. |
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