| ObjectiveThe purpose is to investigate the pharmacological effects of ASⅣ on diabetic rat cardiomyocytes induced by STZ and H9c2 cell induced by high glucose.To explore the mechanism of diabetic cardiomyopathy on two aspects of mitochondrial energy metabolism and myocardial apoptosis.MethodsIn vivo study,50 six-week-old male SD rats were divided into five groups: control group,model group,ASⅣ10 mg · kg-1 group,ASⅣ20 mg · kg-1 group,ASⅣ40 mg· kg-1 group.Except the control group,other rats were established diabetic rats via the tail vein injection of STZ in the dose of 35 mg · kg-1 and given different doses of ASⅣ for 16 weeks.Blood glucose levels and cardiac hemodynamic parameters were detected on the rats.HE staining was applied to detect myocardial pathology.The quantity of ATP,ADP,AMP in cardiomyocytes were determined by ELISA assay.TUNEL was applied to detect the cardiomyocytes apoptosis index.The amount of protein about PGC-1α and NRF-1 were determined by Western blot.The amount of protein about Cyt C and Cleaved-caspase-3 were assessed by Immunohistochemistry.In vitro study,H9c2 cells were divided into six groups which are control group,mannitol group,high glucose group,HG+ASⅣ 20 μmol·L-1 group,HG+ASⅣ 40 μmol·L-1 group,HG+ASⅣ 80 μmol·L-1 group.And ELISA was applied to detect the quantity of ATP,ADP,AMP.Flow cytometry tested apoptosis rate.The m RNA expression of PGC-1α and NRF-1 were detected by PCR.Cyt C and Cleaved-caspase-3 protein was assessed by Western blot.PGC-1α si RNA transfected cells were divided into four groups: control group,high glucose group,high glucose + PGC-1α si RNA transfection group,high glucose + Scrambled RNA group,western blot tested protein expression of Cleaved-caspase-3 in each group.ResultsIn vivo study: Compared to control group,the results of diabetes group show in the following: blood glucose was rised.LVEDP was increased,but LVSP and ± dp / dtmax were declined remarkably.HE staining show cardiac structural was abnormal.The ATP/ADP ratio were reduced,so was ATP/AMP.TUNEL result showed apoptosis rate was increased significantly.The amount of PGC-1α and NRF-1 protein were decreased.Contrarily,the amount of protein about Cyt C and Cleaved-caspase-3 was increased in cardiomyocytes.Compared to diabetes group,different doses of ASⅣ groups has declined on blood glucose.LVEDP was declined significantly,and LVSP and ± dp / dtmax were rised.HE staining show that myocardial interstitial tissue was reduced and the morphologically of myocardial cells was regulated.ELISA results show the rate of ATP/ADP and ATP/AMP was rised.The apoptosis index in TUNEL staining image was markedly decreased.The amount of PGC-1α and NRF-1 protein were increased.The amount of Cyt C and Cleaved-caspase-3 protein were reduced.In vitro study: Contrasted to control group,the consequent of ATP/ADP,ATP/AMP rate in high glucose group was decreased,and the apoptosis index was increased by flow cytometry.PGC-1α and NRF-1 m RNA was decreased.The amount of protein about Cyt C in cytosol and Cleaved-caspase-3 was increased.Contrasted to high glucose group,the result of ASⅣ treatment groups was in the following: the rate of ATP/ADP and ATP/AMP was rised,the apoptosis index was decreased,the m RNA expression about PGC-1α and NRF-1 was increased,the amount of protein about Cyt C in cytosol and Cleaved-caspase-3 was declined.In addition,contrasted to high glucose group,the amount about Cleaved-caspase-3 protein in high glucose + PGC-1α si RNA transfection group was increased and high glucose + Scrambled RNA group has almost remained unchanged.Conclusions1.ASⅣ can lower blood glucose leve in diabetic rats,improve cardiac structure,and enhance heart function.2.ASⅣ can facilitate mitochondrial biogenesis of myocardial cells in diabetic rats by PGC-1α pathway and improve energy metabolism.3.The apoptosis protein can be reduced by ASⅣ via PGC-1α pathway.ASⅣ effectively inhibit myocardial cell apoptosis in diabetic rats.And we can suggest that ASⅣ have protective effect on diabetic cardiomyopathy. |
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