| Objective: Doxorubicin(DOX)is a broad spectrum and high efficient antitumor drug commonly used for malignant tumors.However,adosedependent cardiac toxicity including dilated cardiomyopathy and delayed severe heart failure limited its clinics usage.The main mechanisms of DOX-induced cardiac toxicity are oxidative stress,mitochondrial damage,calcium overload and cell apoptosis.Bradykinin(BK)is an advanced glycation end-product of kallikrein-kinin system(KKS).KKS is a complex endogenous enzyme system,and widely involved in the regulation of inflammation,vascular tension,angiogenesis,blood coagulation,pain and other physiological and pathological functions.Although lots of basic and clinical studies have confirmed that myocardial infarction,diabetes,hypertension,heart failure and left ventricular hypertrophy were associated with the decrease of KKS activity,whether it could affect the myocardial injury induced by DOX has not been reported nevertheless.This study will establish the cytotoxicity model of DOX-induced myocardial injury to explore the role of KKS in cardiac toxicity induced by DOX and its possible intracellular signal transduction pathway in the cellular level.Method:1 Toxic effects of DOX on H9c2 cellsH9c2 cells were treated with different concentrations of DOX(0.1μM,0 3μM,1μM,3μM,10μM,30μM),for different time(12h,24 h,36 h,48h,60 h,72h).The time and concentration-dependent toxicity of DOX on H9c2 cells was observed.2 Effcts of DOX on Cell viabilitys of H9c2 cells and AC16 cellsBoth H9c2 cells and AC16 cells were treated with different concentrations of DOX(0.01μM,0.03μM,0.1μM,0.3μM,1μM,3μM,10μM,30μM,100μM)for 24 h.MTT method was used to detect the cell viabilitys,the concentration of DOX used for decreasing viabilitys by 50% in H9c2 cells and AC16 cells were determined.3 The influence of BK on the decreased viabilitys of myocardial cells induced by DOXH9c2 cells and AC16 cells were treated with BK 10μM,1μM and 0.1μM 30 min respectively before DOX 1 μM(H9c2 cells)or 5 μM(AC16 cells)was added.MTT assay was used to determine the effect of BK on the decreased viabilitys of myocardial cells for 24 h induced by DOX.4 The influence of BK on the increment of MDA,LDH and the decrement of SOD induced by DOXH9c2 cells were treated with BK 0.1μM 30 min before DOX 1 μM was added.Cell lysis solutions were taken from control group,DOX group,BK+DOX group and BK group.Protein concentration was determined by UV spectrophotometer and the activity and content were determined by MDA,LDH and SOD kits.5 The influence of BK on intracellular ROS production induced by DOXBoth H9c2 cells and AC16 cells were treated as above,Reactive oxygen species(ROS)specific fluorescent probe 2,7-dichlorofluorescenin diacetate(DCFH-DA)1 μM was used to stain the cells for 30 min and cell fluorescence intensity was detected by laser scanning confocal microscope.6 The influence of BK on the decrease of mitochondria membrane potential induced by DOX.H9c2 cells were treated above,0.5μM mitochondrial membrane potential specific fluorescent probe Rh 123 was used to stain the cells for 30 min and cell fluorescence intensity was detected by laser scanning confocal microscope.7 The influence of BK on the increment of the apoptosis ratio induced by DOX.H9c2 cells were treated and grouped accordingly,Corresponding protein concentrations were determined by UV spectrophotometer and protein expression levels were detected by Western Blot.8 Investigation of the possible mechnisms of protective effect of BK against DOX-induced myocardial cell injury.H9c2 cells and AC16 cells were incubated with 0.1μM BK 30 min before DOX and B2 receptor inhibitor HOE-140,PI3 K inhibitor LY294002,NOS inhibitor L-NAME,and PLC inhibitor U-73122 were added respectively.Cells were taken from control group,DOX group,BK+DOX group BK+DOX+H group,BK+DOX+L group,BK+DOX+LY group and BK+DOX+U group.Cells viabilitys were detected by MTT assay,the change of ROS and MMP were detected by laser confocal microscope.Results:1 The growth curve of H9c2 cells showed that the toxicity of DOX on H9c2 cells was time-and concentration-dependent.2 The viability of H9c2 cells was decreased by 50% when the cells were treated with 1 μM DOX,the viability of AC16 cells was decreased by 50% when the cells were treated with 5 μM DOX,1 μM DOX and 5 μM DOX were used to established doxorubicin damage model in the subsequent experiments therefore.3 Different concentrations of BK were added 30 min before DOX to H9c2 cells and AC16 cells.0.1 μM、1 μM and 10 μM BK could significantly alleviate the damage of H9c2 cells and AC16 cells induced by DOX(P < 0.01).A decline of viability on AC16 cells were observed in 1 μM and 10 μM BK,0.1 μM BK was used in the subsequent experiments therefore.4 MDA and LDH level were increased in cell lysates or culture medium(P < 0.01)while the activity of SOD was decreased in cell lysates(P < 0.01)after cells were treated with DOX.MDA,LDH,and SOD activity were recovered after cells were pretreated with BK(P < 0.05 or 0.01)compared with DOX treatment.5 Swellen and cracked cells and increased intracellular ROS were found after H9c2 cells and AC16 cells were treated with DOX(P < 0.01).ROS levels were decreased when cells were pretreated with BK(P < 0.01).6 The fluorescence intensity and MMP of H9c2 cells were decreased after the cells were treated with DOX(P < 0.01)which were recovered after cells were pretreated with BK(P < 0.05).7 The ratio of Bax/bcl-2 increased after H9c2 cells were treated with DOX(P < 0.01),which was decreased after the cells were pretreated with BK.(P < 0.05).8 When H9c2 cells and AC16 cells were pretreated with BK combined with B2 receptor inhibitor HOE-140,PI3K inhibitor LY294002,NOS inhibitor L-NAME,and PLC inhibitor U-73122 inhibitor respectively,.The protective effect of BK on the decrement of myocardial cell viability,increment of ROS level and the decrement of MMP induced by DOX was intervented(P < 0.01).Conclusion:BK ameliorates DOX-induced cardiac toxicity in H9c2 cells and AC16 cells by reducing oxidative stress level and apoptosis in myocardial cells via PI3K/Akt/NO signaling pathway. |
PDF Full Text Request