| Objective:(1) To evaluate the protective effect of ZMY on Adriamycin-induced oxidative stress and cardiotoxicity in vitro and in vivo. And explore the mechanisms by which ZMY induced Inhibitor of apoptosis and exerted protective effect of cardiac toxicity. (2) To explore the mechanisms of Sunitinib-induced cytotoxicity in rat H9c2 cells, and discussion the protective effect of ZMY on Sunitinib-induced cardiomyocyte toxicity.Methods:1. Antioxidant activity and anti-tumor activity of ZMY:(1) MTT method was used to detect vitro antioxidant activity of ZMY in vitro. (2) MTT method was used to detect cytotoxicity of Adriamycin combination with ZMY on K562, NB4, U937 leukemia cells. (3) U937 xenografted athymic mice model was established to evaluate anti-tumor activity of Adriamycin combination with ZMY in vivo.2. ZMY on the protective effect of Adriamycin-induced cytotoxicity in rat primary myocardial:(1) MTT method was used to detect inhibition effect of Adriamycin combination with ZMY on primary rat cardiomyocytes. (2) Automatic biochemical analyzer was used to detect the changes in myocardial enzymes in cell supernatant after combination of Adriamycin and ZMY. (3) Carboxy-DCFDA method was used to detect the ROS levels changes in primary rat cardiomyocytes after the combination treatment of Adriamycin and ZMY. (4) DAPI staining was used to evaluate the apoptosis rate of the primary rat cardiomyocytes after the combination treatment of Adriamycin and ZMY. (5) JC-1 method was used todetect the changes in changes in mitochondrial membrane potential after combination of Adriamycin and ZMY. (6) Western-blot assay was used to investigate the expression of the protein related to apoptosis in primary rat cardiomyocytes after the combination treatment of Adriamycin and ZMY.3. ZMY showed the protective effect of Adriamycin-induced cardiac toxicity in vivo:(1) The ICR mice model of cardiac toxicity of Adriamycin was established to observe changes in body weight and survival rate of mice of Adriamycin combination with ZMY in vivo. (2) Automatic biochemical analyzer was used to detect the changes in serum myocardial enzymes after combination of Adriamycin and ZMY.4. The mechanisms of Sunitinib-induced cytotoxicity in rat cardiac H9c2:(1) MTT method was used to detect cytotoxicity of Sunitinib on cardiac H9c2 cells and calculate the median inhibitory concentration (IC50 value). (2) DAPI staining was used to evaluate the apoptosis rate of H9c2 cells after Sunitinib treatment. (3) AO staining was used to evaluat the generation of the autophagic acidic vesicular organelles in H9c2 cells after Sunitinib treatment. (4) Transfection of GFP-LC3 plasmid to evaluat the generation within the autophagic acidic vesicular organelles in H9c2 cells after Sunitinib treatment. (5)Western-blot assay was used to investigate the expression of the protein related to autophagy in H9c2 cells treated with Sunitinib. (6)Western-blot assay was used to investigate the expression of protein related to autophagy in Beclinl-knockdown H9c2 cells.5. ZMY showed the protective effect of Sunitinib-induced cytotoxicity in rat cardiac H9c2:(1) MTT method was used to detect inhibition effect of Sunitinib combination with ZMY on H9c2 cells. (2) Carboxy-DCFDA method was used to detect the ROS levels changes in H9c2 cells after the combination treatment of Sunitinib and ZMY. (3) AO staining was used to evaluat the generation within the autophagic acidic vesicular organelles in H9c2 cells after the combination treatment of Sunitinib and ZMY. (4) Western-blot assay was used to investigate the expression of the protein related to autophagy in H9c2 cells after the combination treatment of Sunitinib and ZMY.Results:1. ZMY showed trong antioxidant activity and effective anti-tumor activity in vitro and in vivo:(1) ZMY showed strong DPPH radical scavenging effect, and its free radical scavenging capacity better than vitamin C. (2) ZMY had a synergistic activity with Adriamycin on suppressing cell proliferation in on K562,NB4, U937 leukemia cells.(3)In U937 xenografts model, the mice were sacrificed after ZMY and Adriamycin administration for 5 days. The tumor inhibition rate (%) of Adriamycin 2 mg/kg, ZMY 500 mg/kg and the combination of Adriamycin and ZMY group were 14.5, 18.0, and 57.1, respectively, and the corresponding T/C (%) values were 69.4,73.5, and 41.3, respectively.2. The mechanisms of ZMY protective effect on Adriamycin-induced cardiomyocyte toxicity:(1) ZMY showed strong protective effect on adriamycin-induced rat primary myocardial cell toxicity, after the treatment of 2μM Adriamycin or the combination of Adriamycin and ZMY for 24 h,the inhibition rates (%) were 30.7 and 14.5, respectively. (2) Myocardial enzyme test results showed that the value of AST, LDH, CKMB in cells pretreated with ZMY than that of treated with Adriamycin alone was significantly decreased. (3) After treated for 3 h, in 2μM Adriamycin alone group, the generation of ROS level as 241.45%; 50μM ZMY pretreatment group, ROS generated as 80.0%, and compared with Adriamycin alone group ROS levels were significantly reduced. (4) With DAPI staining, apoptosis rate induced by Adriamycin decreased in rat primary myocardial cells pretreated with ZMY. (5) Pretreatment with ZMY can reverse the Adriamycin-induced reduction in mitochondrial membrane potential of rat primary cardiac cells. (6)Western-blot assay implied that ZMY can inhibit JNK activation, thereby inhibiting up-regulation of Beclinl induced by the Sunitinib to reduce Suni-induced myocardial autophagic cell death.3. ZMY demonstrated a strong protective effect of cardiac toxicity in vivo:(1) In the ICR mice model of cardiac toxicity induced by Adriamycin, the body weight and survival rate of mice were observed.The results showed that the weight loss of mice pretreated with ZMY was significantly slower than Adriamycin alone group. And the survival rate (%) of Adriamycin 20 mg/kg and the combination of Adriamycin 20 mg/kg and ZMY 500 mg/kg group were 84.62 and 7.69, respectively. (2) Adriamycin 20 mg/kg alone group AST value is 578±84 IU/L, and combined with ZMY 500 mg/kg group is 276±102 IU/L; Adriamycin alone group LDH value is 1603±303 IU/L, and combined with ZMY 500 mg/kg group is 293 ±199IU/L; Adriamycin alone group CKMB value of 922±120 IU/L, and combined with ZMY 500 mg/kg group is 248±270 IU/L.4. Sunitinib-induced cytotoxicity in rat cardiac H9c2 cells is closely related to autophagy:(1) Cytotoxicity assay demonstrated that Sunitinib showed strong cytotoxicity in vitro with IC50 values of 6.53μM in H9c2 cells. (2) With DAPI staining, with the Sunitinib dose concentration increased, H9c2 cell death rate will be increased, but apoptosis rate is not obvious. (3) AO staining showed that the generation of the autophagic acidic vesicular organelles significantly increased in H9c2 cells after Sunitinib treatment. (4) AO staining and GFP-LC3 transfection methods showed that the generation of the autophagic acidic vesicular organelles significantly increased in H9c2 cells after Sunitinib treatment. (5) Western-blot assay showed that the expression of intracellular autophagy-related protein including LC3, Beclinl, was obviously related with Sunitinib treatment in concentration-dependent and time-dependent manner. (6) The autophagy pathways play an important role in Sunitinib-mediated cell death in H9c2 cells, and our results showed that the survival rate was significantly increased in Beclinl-knockdown H9c2 cells after Sunitinib treatment compared with the control group.5. ZMY showed the protective effect on Sunitinib-induced cytotoxicity in rat cardiac H9c2 cells:(1) ZMY showed significant protective effect on Sunitinib-induced H9c2 cell toxicity, after the treatment of 5μM Sunitinib or the combination of Sunitinib and ZMY for 24 h,the inhibition rates (%) were 28.3 and 3.1, respectively. (2) With Sunitinib treatment for 3 h, the generation of ROS level was significantly increased in concentration-dependent manner. (3) With AO staining, the generation of the autophagic acidic vesicular organelles significantly reduced in H9c2 cells pretreated with ZMY. (4) Western-blot assay implied that ZMY can inhibit JNK activation, thereby inhibiting up-regulation of Beclinl induced by the Sunitinib to reduce Suni-induced myocardial autophagic cell death.Conclusion:(1) ZMY demonstrated a strong protective effect on cardiac toxicity in vitro and in vivo.(2) Sunitinib-induced cytotoxicity in rat cardiac H9c2 cells is closely related to autophagy.(3) ZMY showed the protective effect on Sunitinib-induced cytotoxicity in rat cardiac H9c2 cells. |
PDF Full Text Request