Based On The Vitamin D System, The Molecular Mechanism Of Saponin Against Cardiac Hypertrophy Was Explored

Objective:With the literature associated tools ARROWSMITH,we found that there is a very valuable implicit rule between Astragalus and vitamin D in treatment of cardiovascular diseases.Therefore we put forward the treatment molecular mechanism of astragaloside on cardiovascular diseases through the vitamin D system regulating of the renin-angiotensin-aldosterone system.In this study,we use endothelin-1 induced cardiomyocyte hypertrophy model,combined with siRNAto explore the effective of Astragalus on myocardial cell vitamin D system,which regulates the expression of renin in cardiomyocyte hypertrophy.We expect to provide new therapeutic targets and new ideas for the traditional Chinese medicine to prevent and treat the congestive heart failure.Methods:1.The primary cell culture:We took newborn 24-72 h SD rats and obtained the primary cardiomyocyte with the method of Trypsin to digest cells,then used a-actin immunohistochemical to identify it.2.The influence of astragaloside on the expression of VDR,CYP27B,CYP24A,renin mRNA and protein in cardiomyocyte hypertrophy model induced by endothelin-1:the cells were divided into control group(Ctl),Model group(endothelin-1,10-8mmol/L,ET-1),vitamin D group(endothelin-1,10-mmol/L,vitamin,10-8mmol/L,VD),astragaloside low dose group(endothelin-1,10-8mmol/L,astragaloside,10?g/ml,Low),astragaloside middle dose group(endothelin-1,10"8mmol/L,astragaloside,20?g/ml,Mid),astragaloside high dose group(endothelin-1,10-8mmol/L,astragaloside,40?g/ml,Hig),collected cells after 24h intervention.To detect the expression of mRNA and protein by qRT-PCR and Western Blot.3.To construct plasmid of LV3-VDR siRNA by the principle of siRNA design.The LV3-VDR siRNA was detected by quantificational real-time polymerase chain reaction(qRT-PCR)and DNA sequencing.After the lentivirus had been packaged,the 293T cells were transfected by LV3-VDR siRNA made virus drops degree to reach 1 x 108TU/ml.Then the vector were transfected cardiomyocyte.The efficiency of gene silencing of VDR gene transfected by LV3-VDR siRNA was detected using reverse transcription-polymerase chain reaction(RT-PCR).4.The influence of astragaloside on the expression of CYP27B,CYP24A Renin mRNA and protein in siRNA VDR cardiomyocyte hypertrophy model induced by endothelin-1:Ctl group,VDR siRNA negative group(Neg),VDR siRNA group(Ko),VDR siRNA+endothelin-1 group(endothelin-1,10-8mmol/L,ET-1),VDRsiRNA + vitamin D group(endothelin-1,10-8mmol/L,vitamin,10-8mmol/L,VD),VDRsiRNA+astragaloside low-dose group(endothelin-1,10-8mmol/L,astragaloside,10?g/ml)(AST-L),VDRsiRNA+astragaloside middle-dose group(endothelin-1,10-8mmol/L,astragaloside,20?g/ml,AST-M),VDRsiRNA+astragaloside high-dose group(endothelin-1,10-8mmol/L,astragaloside,40?g/ml,AST-H),collected cells after 24h intervention.To detect the expression of CYP27B,CYP24A,renin mRNA and protein by qRT-PCR and Western Blot.5.The influence of astragaloside on the apoptosis of cardiomyocyte hypertrophy model which induced by endothelin-1:We detected the influence of astragaloside on the apoptosis of cardiomyocyte before and after VDR siRNA by flow-cytometry.6.The influence of astragaloside on the ultrastructure of cardiomyocyte hypertrophy model which induced by endothelin-1:We detected the influence of astragaloside on the ultrastructural changes of cardiomyocyte before and after cardiomyocyte by electron microscopy.Results:1.The primary cell culture:After dyeing cardiomyocyte cell by a-actin,cell cytoplasm has dense brown granules,that is the characteristic of cardiomyocyte cell staining.2.The influence of astragaloside on the expression of VDR,CYP27B,CYP24A and renin in cardiomyocyte hypertrophy model which induced by endothelin-1:Compared with Ctl,the expression of VDR,CYP27B mRNA in ET-1 group are reduced(P<0.01),but the expression of CYP24A,renin mRNA are increased(P<0.05).Compared with ET-1 group,the expression of VDR,CYP27BmRN A in VD group and AST-H group are increased(P<0.05),meanwhile the expression of CYP24A,renin mRNA in VD,AST-H and AST-Lgroup are decreased(P<0.05).3.To construct plasmid of LV3-VDR siRNA and transfection of cardiomyocytes effectively restrained the expression of VDR,and made the expression rate of VDR in cardiomyocytes down-regulation of gene expression 75%,LV3-VDR siRNA efficiently interfered the expression of VDR in cardiomyocytes.The efficiency of transfection in VDR siRNA negative group are quite with VDR siRNA group,but it did not affect the expression of VDR mRNA.4.The effect of astragaloside on the expression of CYP27B,CYP24A,Renin mRNA and protein in VDR siRNA cardiomyocytes hypertrophy model which induced by endothelin-1:1)Compared with Ctl group,the expression of CYP27B mRNA in Ko group was decreased(P<0.05);CYP24A,renin mRNA was increased(P<0.05).2)Compared with Ko group,the expression of CYP27B mRNA in the ET-1 group is decreased significantly(P<0.05),the expression of CYP24A and renin mRNA is increased(P<0.05).3)Compared with ET-1 group,VD group,AST-H group increased the expression of CYP27B mRNA(P<0.05);down regulated Renin mRNA(P<0.05).4)Compared with Ctl group,the expression of CYP27B protein in Ko group group significantly decreased(P<0.05),while the expression of CYP24A and Renin protein significantly increased(P<0.05).5)Compared with Ko group,the expression of CYP27B protein is decreased,while the expression of CYP24A and Renin protein significantly increased is significantly increased in ET-1 group(P<0.05);VD group and AST-H group significantly increased the expression of CYP27B protein(P<0.05);VD group down regulates CYP24A and Renin protein express(P<0.05).AST groups down regulate CYP24A and renin protein express(P<0.05).5.The effect of astragaloside on the apoptosis of myocardial hypertrophy model which induced by endothelin-1:1)Compared with Ctl group,the apoptosis rate of myocardial cells in the ET-1 group increased significantly(P<0.05).Compared with Ctl group,the apoptosis rate of myocardial cell decreased in VD group(P<0.05).Compared with ET-1 group,the apoptosis rate of myocardial cell decreased in the AST-H and AST-M group(P<0.05),there is no difference between AST-L group and ET-I group(P>0.05).2)Compared with normal group,the apoptosis rate of myocardial cell in VDR knockout group increased.After induced by endothelin-1(ET-1),the apoptosis rate of myocardial cell increased significantly(P<0.05).After intervened by vitamin D and AST groups,the apoptosis rate of myocardial cells can be effectively inhibited,the total rate of apoptosis in each group showed significant difference than ET-1 group(P<0.05).6.The influence of astragaloside on the ultrastructure of myocardial hypertrophy model which induced by endothelin-1:1)Under the electron microscope(EM),we saw that the morphology of myocardial cell is normal,the cytomembrane is integrated,the number of mitochondria and other organelle are normal.The cell nucleus are clear and complate in Ctl group.In the ET-1 group,we saw that the morphology of numerous myocardial cells are irregular,mitochondria increased significantly,cell structure are disordered,the boundary of cell nucleus are disordered and the chromatins are with paramorphia.Compared with ET-1 group,the morphology of numerous myocardial cell are improved,the number and structure of mitochondria are relatively normal,the apoptosis body reduced relatively,nuclear membranes are in integrity and the damage of cell reduces holistic in VD,AST-H and AST-M group of cells,while the improvement in AST-L group is not obvious.2)Under the electron microscope we saw that the morphology of myocardial cells are normal,the cytomembrane is integrated,the number of mitochondria and other organelle are normal,the cell nucleus are clear and complate in Ctl group.In the Ko group,the number of mitochondria increased and their structures are almost normal,the nuclear membranes are almost complete.In ET-1 group,most of the cells are irregular,the number of mitochondria increased.Cells becomes larger and their structure are disordered,the boundary of nucleus are disordered,the chromatin is scattered.Compared with ET-1 group,the morphology of numerous myocardial cells improved in AST groups,the size and structure of mitochondria are relatively normal,the apoptosis body reduced relatively,nuclear membranes are in integrity and clear,the configurations of chromatin are inerratic.Conclusion:1.Vitamin D axis(vitamin D receptor,CYP27B,CYP24A)are involved in the myocyte hypertrophy pathophysiological process inducted by endothelin-1.2.The mechanism of astragaloside protecting the myocyte hypertrophy which induced by endothelin-1 may have two:the one is inhibiting the expression of renin,through the adjustment of vitamin D axis in myocardial cells,the other is blocking the activity of renin-angiotensin aldosterone system(RAAS)from the source.3.Astragaloside can inhibit the apoptosis of myocardial cell which induced by endothelin-1 through the vitamin D axis and protect the ultrastructure of myocardial cells.