The Effects Of HUC-MSCs On The Expression Of Inflammatory Factors Of The Senescent Cardiomyocytes

Objective: The changes of some inflammatory related index were explored to investigate the effects and possible mechanism of hUC-MSCs on the senescent cardiomyocytes induced by H₂O₂.Methods:H9c2 cardiomyocytes were treated with different concentrations of H₂O₂?30,50,100,150,200?mol/L?.Senescence-associated ?-galactosidase staining was used to detect the senescent cells,and Annexin V-FITC/PI Apoptosis Detection kit was used to analyze the degree of cell apoptosis.Eventually we determined the optimal H₂O₂ concentration to establish premature senescence model.H9c2 cardiomyocytes and human umbilical cord-derived mesenchymal stem cells?hUC-MSCs?were cultured in a contact co-culture system by the transwell chambers.The senescence-associated ?-galactosidase staining was also used to detect the senescent cells of H9c2 cardiomyocytes co-cultured with hUC-MSCs.The pro-inflammatory cytokines?TNF-?,IL-1??and anti-inflammatory?IL-10?in the extracellular fluid of H9c2 cardiomyocytes were detected by corresponding ELISA kits.Results: 1.The establishment of the senescent cardiomyocytes induced by H₂O₂.1.1 Compared to the control group,the number of ?-galactosidase staining-positive cells was increased along with the increasing concentration of H₂O₂.the number of ?-galactosidase staining-positive cells was unchanged when the H₂O₂ concentration was 30?mol/L compared to that of the control?P>0.05?.When the H₂O₂ concentration reached 50?mol/L,100?mol/L,150?mol/L,200?mol/L,a significantly increased ?-galactosidase staining-positive cells was oberserved and it was dose-dependent?P<0.05?.1.2 An increasing apoptosis of cardiomyocytes was oberserved after H₂O₂ treatment for 4h?P<0.05?and it was dose-dependently.When treated with H₂O₂ in 30?mol/L,50?mol/L,100?mol/L,the percentage of cardiomyocytes in apoptosis had no difference compared to that of control in respectively?P>0.05?.While the apoptosis of cells treated with H₂O₂ with the concentration of 150 ?mol/L and 200 ?mol/L was increased significantly compared with that of the control?P<0.05?.2.H9c2 cardiomyocytes and hUC-MSCs were cultured in a contact co-culture system.The experiment was devided into four groups including Control group,H₂O₂ group,H₂O₂ + hUC-MSCs medium group and H₂O₂ + hUC-MSCs group.2.1 The ?-galactosidase staining result showed that the number of senescent cardiomyocytes in H₂O₂ group was significantly increased when compared to the Control group?P<0.05?.However,compared to the H₂O₂ group,the number of ?-galactosidase staining-positive cells was significantly reduced in H₂O₂+ hUC-MSCs group?P<0.05?.2.2 ELISA results in the extracellular fluid of H9c2 cardiomyocytes showed that H₂O₂ treatment significantly increased level of the pro-inflammatory factors TNF-? and IL-1?,and decreased the level of the anti-inflammatory factors IL-10 compared to the Control group?P<0.05?.While hUC-MSCs treatment after H₂O₂ lesion led to a decrease in TNF-? and IL-1?,and an increase in IL-10 compared to H₂O₂ group?P<0.05?.Conclusion: H9c2 cardiomyocytes senescence model was established successfully by 100?mol/L H₂O₂ concentration.hUC-MSCs can decreased the senescence degree of H9c2 cardiomyocytes induced by H₂O₂,and decreased the level of pro-inflammatory factors TNF-? and IL-1?,and increased the anti-inflammatory factor IL-10 level,which suggested that hUC-MSCs had protective effects on the senescence of H9c2 cardiomyocytes induced by H₂O₂.The potential mechanisms may be associated with the down-regulation of the pro-inflammatory factors expression and up-regulation of the anti-inflammatory factors expression.