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（30,50,100,150,200μmol/L）.Senescence-associated β-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 β-galactosidase staining was also used to detect the senescent cells of H9c2 cardiomyocytes co-cultured with hUC-MSCs.The pro-inflammatory cytokines（TNF-α,IL-1β）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 β-galactosidase staining-positive cells was increased along with the increasing concentration of H₂O₂.the number of β-galactosidase staining-positive cells was unchanged when the H₂O₂ concentration was 30μmol/L compared to that of the control（P>0.05）.When the H₂O₂ concentration reached 50μmol/L,100μmol/L,150μmol/L,200μmol/L,a significantly increased β-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 30μmol/L,50μmol/L,100μmol/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 μmol/L and 200 μmol/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 β-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 β-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-α and IL-1β,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-α and IL-1β,and an increase in IL-10 compared to H₂O₂ group（P<0.05）.Conclusion: H9c2 cardiomyocytes senescence model was established successfully by 100μmol/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-α and IL-1β,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.