The Effect And Mechanism Of Activating TGR5 Receptor On Endothelin-1 Induced Hypertrophy Of Rat Cardiac Myocytes

bjective: Cardiac hypertrophy is an adaptive hyperplasia of the heart to various stimuli.It is a slow and effective compensatory function,but this compensatory function can increase the oxygen demand of the heart muscle,gradually lead to heart failure,and cardiovascular Increasing accidents and even sudden deaths are important to explore ways to prevent,alleviate or even reverse cardiac hypertrophy.Stimulating factors of cardiac hypertrophy include mechanical stretch,pressure load,ischemia,hypoxia,nitric oxide deficiency and various inflammatory factors.Endothelin-1（ET-1）is one of the causes of cardiac hypertrophy.G protein-binding bile acid receptor 1（TGR5）is a cell surface G-protein coupled receptor regulated by bile acid（BA）,also known as GPBAR1,M-Bar,CPR131 or BG37,which is one of the GPCR families.Members,widely expressed in the liver,small intestine,heart,skeletal muscle,spleen,kidney,placenta and white blood cells and other tissues and organs,participate in the pathophysiological processes such as bile acid,glucose metabolism,lipid metabolism and energy metabolism.In this study,we studied the effects of TGR5 receptor on the ET-1-induced cardiomyocyte hypertrophy on the cell membrane of sensitized cardiomyocytes,and studied the possible mechanisms to provide treatment ideas for improving cardiac hypertrophy.Methods: Primary culture of neonatal rat cardiomyocytes.1.Observe the effects ofdifferent concentrations of ET-1 on cardiomyocytes at different times.Experimental group: blank control group,ET-1 10^-8 mmol/L group,ET-1 10^-7 mmol/L group,ET-110^-6 mmol/L group,each group was treated for 12 h,24h,respectively.At 36 h and 48 h,the surface area and total protein concentration of each group were detected,and a cardiac mast cell model was established.2.siRNA-TGR5 was transfected into cardiomyocytes.Divided into control group,siRNA-NC,siRNA1-TGR5,siRNA2-TGR5 and siRNA3-TGR5.After 6 hours of transfection,the expression levels of TGR5 receptor protein and TGR5 mRNA were detected by Western blot and RT-PCR,and the siRNA-TGR5 with obvious effect was selected.Carry out subsequent experiments.3.To observe the effect of TGR5 activation on cardiomyocyte hypertrophy and explore its mechanism.Experimental group: blank control group: DMEM/F-12 complete culture solution for 48h;ET-1 group: ET-1 10^-6 mmol/L for 48h;ET-1+INT-777 group: ET-1 10^-6 mmol/L and TGR5 receptor-specific agonist INT-777 30umol/L（according to the data from this experimental group,the paper has not been published yet）for 48 hours;ET-1+ INT-777+ TGR5 siRNA1 group: After transfecting cardiomyocytes with TGR5 siRNA1 for 6 hours,ET-1 10^-6 mmol/L and INT-777 30umol/L were co-cultured for 48 hours;ET-1+ INT-777+TGR5 siRNA-NC group: After transfecting cardiomyocytes with TGR5 siRNA empty virus for 6 hours,ET-1 10^-6 mmol/L and INT-777 30 umol/L were co-cultured for 48 hours.The surface area of myocardial cells was determined by image analysis system.The total protein content was determined by BCA method.The mRNA of TGR5 and calcineurin（CaN）wasdetected by RT-PCR.The atrial natriuretic factor（ANF）and β-muscle were detected by Western blot.Changes in protein expression of protein heavy chain（β-MHC）,TGR5,CaN,and activated T cell nuclear factor 3（NFAT3）.Results:1.ET-1 induced cardiomyocyte hypertrophy in a concentration-dependent manner in the concentration range of 10^-8 mmol/L¹0^-6 mmol/L.The longer the culture time in 48 hours,the more the cardiomyocyte hypertrophy obvious.A cell model of cardiac hypertrophy was established by selecting a concentration of 10^-6 mmol/L ET-1 for 48 h.2.In the validation experiment of siRNA-TGR5 transfected cardiomyocytes,TGR5 mRNA was significantly decreased in siRNA1-TGR5 group,TGR5 receptor protein expression was significantly decreased,and siRNA-TGR5 was successfully transfected into cardiomyocytes.siRNA1-TGR5 was selected for subsequent experiments.3.Compared with the control group,the expression of myocardial cell surface area,total protein and protrophic factor in ET-1 group increased,and the expression of CaN and NFAT3 also increased.Compared with the ET-1 group,the expression of myocardial cell surface area,total protein and protrophic factor increased in the ET-1+INT-777 group,and the increased expression of CaN and NFAT3 was inhibited.Compared with the ET-1+INT-777 group,the expression of myocardial cell surface area,total protein and protrophic factor increased,and the expression of CaN and NFAT3 increased in ET-1+ INT-777+TGR5 siRNA1 group;There was no statistical difference.Compared with ET-1+ INT-777+TGR5 siRNA1 group,the expression of myocardial cell surface area,total protein and protrophic factor in ET-1+INT-777+TGR5 siRNA-NC group was inhibited,and the expression of CaN and NFAT3 was increased.Inhibition;no statistical difference compared to the ET-1+INT-777 group.Conclusion: Within 48 h,ET-1 induced cardiomyocyte hypertrophy in a concentration-dependent and time-dependent manner in the concentration range of 10^-8 mmol/L¹0^-6 mmol/L;activation of TGR5 receptor on myocardial cell membrane Inhibition of ET-1 induced mouse cardiomyocyte hypertrophy may be related to inhibition of CaN-NFAT3 signaling pathway.