Inhibition Of Ang Ⅱ-induced Cardiomyocyte Hypertrophy By Isoproterenol And Its Mechanism

Objective:The hypertrophy model of H9c2 mouse cardiomyocytes induced by Ang Ⅱ was used to observe the effects of different concentrations of propofol on hypertrophic cardiomyocytes,and to explore the possible mechanism of action.Methods:The H9c2 mouse cardiomyocytes were cultured,extracted and seeded in 6-well plates when the cells were in the logarithmic growth phase,and the experiments were divided into 6 groups:（1）Control group（blank group）:cell culture medium with equal volume of Ang Ⅱ;（2）Model group（Ang Ⅱ group）:cell culture medium with 1μmol/L Ang Ⅱ;（3）Experimental control group(circle（4）Propofol low-dose group:0.5mmol/L Propofol and 1μmol/L AngⅡcell culture medium;（5）Propofol medium dose group:1.0mmol/L Propofol and 1μmol/L AngⅡcell culture medium;（6）Propofol high dose group:1.5mmol/L Propofol phenol and 1μmol/L AngⅡcell culture medium.Each group of the experiment was intervened in H9c2 mouse cardiomyocytes for 48hours,and the changes in the area and protein content of the cells in each group were detected,mainly including:1.Rhodamine-phalloidin staining,observing and collecting images under a fluorescence microscope,and Image J software was used to analyze the changes of cell area and bone microfilaments in each group;2.Use Coomassie’s brilliant blue kit to detect the total protein of H9c2 mouse cardiomyocytes,observe the absorbance with a microplate reader,and calculate the total protein concentration of each group of cells according to the standard formula;3.Use q RT-PCR method to detect the content of hypertrophy gene markers ANP m RNA and BNP m RNA in cardiomyocytes of each group;4.By the method of Fura-2/AM fluorescent probe,observe the content of Ca²⁺in cardiomyocytes in each group,observe and collect Ca²⁺fluorescence images under a fluorescence microscope,and use Image J software to process and analyze the fluorescence intensity of Ca²⁺;5.Western blot was used to detect the expression of ANP,BNP and Ca N pathway proteins and AT1R-RAGE pathway proteins;6.Statistical processing The experimental results were expressed by`x±s,and the comparison method of one-way ANOVA was used for statistics Graph Pad software was used to make graphs,and when P<0.05,it indicated that the experimental difference was statistically significant.Results:1.Changes of cardiomyocyte area and cytoskeletal microfilaments:The cell area of Ang Ⅱ group（2386±184.9μm²）was significantly larger than that of blank group（P<0.001）,the density of cytoskeletal microfilaments increased,and the distribution was uneven and arranged.confusion.After the intervention of propofol,the area of hypertrophic cardiomyocytes and the density of cytoskeletal microfilaments were significantly reduced,and the inhibitory effect increased with the increase of the concentration,and the difference was statistically significant（P<0.01）.2.Changes of total protein content in cardiomyocytes:The total protein content of cardiomyocytes in AngⅡgroup（146.0±0.34μg/ml）was significantly higher than that in blank group（P<0.001）;The content decreased with the increase of propofol concentration,and the difference was statistically significant（P<0.001）.3.Changes of ANP m RNA and BNP m RNA content in cardiomyocytes:After Ang Ⅱ treatment for 48h,the expression levels of hypertrophic gene markers ANP m RNA and BNP m RNA were 2.28 times and 1.96 times higher than those of blank group,respectively.After intervention with different concentrations of propofol,the expression of hypertrophy gene marker ANP m RNA decreased（P<0.01）,and after intervention with medium and high concentrations of propofol,the expression of hypertrophy gene marker BNP m RNA decreased（P<0.01）,the difference between the low-concentration propofol treatment group was not statistically significant（P>0.05）.4.Changes of intracellular Ca²⁺content:the relative fluorescence intensity of intracellular Ca²⁺in AngⅡgroup（56.64±3.82）was significantly higher than that in blank group（P<0.001）;after propofol intervention,the intracellular Ca²⁺fluorescence intensity It decreased significantly with the increase of propofol concentration,and the difference was statistically significant（P<0.001）.5.Changes in the expression levels of ANP,BNP and Ca N pathway proteins and AT1R-RAGE pathway proteins in cardiomyocytes:The results of Western Blot showed that compared with blank group,ANP,BNP hypertrophy protein and Ca N,NFAT3 expression levels in Ang Ⅱ group were significantly higher.The expression levels of pathway proteins and AT1R-RAGE pathway proteins were significantly changed（P<0.001）;after the intervention of different concentrations of propofol,compared with the Ang Ⅱ group,the expression levels of ANP,BNP and AT1R-RAGE pathway proteins were higher than those in the Ang Ⅱ group.There was a concentration-dependent decrease,and the difference was statistically significant（P<0.05）;while the protein expressions of Ca N and NFAT3 in cardiomyocytes decreased,but there was no difference between the low-concentration propofol group and the AngⅡgroup Statistical significance（P>0.05）,and the medium and high concentrations of propofol groups were statistically significant（P<0.01）.Conclusion:1.Isoproterenol inhibited Ang Ⅱ-induced cardiomyocyte hypertrophy,and the mechanism may be related to inhibition of the Ca²⁺-Ca N-NFAT3 signal transduction pathway.2.Isoproterenol inhibited Ang Ⅱ-induced cardiomyocyte hypertrophy,and the mechanism may be related to the reduction of the ATIR receptor pathway protein of Ang Ⅱ.