| Objective: The calcineurin(CaN)/nuclear factor of activated T-cell(NFAT)signalling pathway plays an important role in pathological cardiac hypertrophy.Here,we investigated the potential effects of stachydrine hydrochloride(Sta),a bioactive constituent extracted from the Chinese herb Leonurus japonicus Houtt.(Yimucao),on pathological cardiac hypertrophy during chronic α-androgen receptor activation and the underlying mechanisms.Methods: 1.Network pharmacology analysis to predict and screen its effective active components,possible targets and possible signal pathways.m RNA microarray analysis was performed on NRVMs to determine the differentially expressed genes.2.In vitro experiment,PE induced neonatal rat ventricular myocytes(NRVMs)myocardial hypertrophy model was used to predict the possible targets,gene differential expression and possible regulatory signaling pathways.Single adult mouse cardiomyocytes were isolated by Langendoff constant current perfusion system.Calcium transient,calcium spark and calcium leakage in cardiac myocytes were detected by ion imaging system.The sarcomere length measurement system detected the relaxation and contraction function of single cardiomyocytes.Immunofluorescence was used to detect the distribution of cardiomyocyte protein.3.In vivo,PE-induced myocardial hypertrophy model was used.Vevo2100 small animal ultrasound imaging system to detect cardiac function and structural parameters;Pressure volume conductance was used to measure the hemodynamic parameters.HE and WGA staining were used to observe the morphological changes of heart and myocardial cells.Protein and m RNA expression levels in myocardial tissue were detected by Western blotting and Q-PCR.Results: 1.Network pharmacological analysis: a total of 51 active components and 785 corresponding targets of Leonurus were obtained.Among them,the active component Sta can regulate adrenergic receptors and regulate classic signaling pathways related to cardiac hypertrophy,such as HIF-1,MAPK,Ras and calcium.2.MRNA microarray analysis showed that there were 5128 m RNA up-regulated and 5071 m RNA down-regulated in hypertrophic myocardium compared with normal myocardium,13 cardiac hypertrophy signaling pathways,and 20 genes regulating transcriptional modification of cardiac hypertrophy.3.In primary cultured neonatal rat ventricular myocytes,Sta inhibited PE induced cardiac hypertrophy and reduced the m RNA expression of cardiac surface area and biomarkers of hypertrophy(ANP,BNP and-MHC/-MHC).4.In adult mouse cardiomyocytes,Sta can improve pe-induced myoid contraction,calcium transient and calcium spark,block the hyperphosphorylation of Ca MKII,Ry R2 and PLN,and prevent the separation of FKBP12.6 and Ry R2.5.Sta inhibited CaN activation and NFATc3 nuclear translocation in neonatal rat ventricular myocytes induced by PE;6.Sta inhibited myocardial hypertrophy induced by PE and improved cardiac function in PE mice treated with PE.Conclusion: our data show that Sta decreases cardiac hypertrophy in PE-stimulated hearts by inhibiting the CaN/NFAT pathway,which might contribute to alleviation of pathological cardiac hypertrophy and cardiac dysfunction by Sta after PE stimulation This also indicated that governing of CaN/NFAT pathway might serve as a preventive or therapeutic strategy for pathological cardiac hypertrophy. |
