The Study On The Effect Of Testosterone In Delaying Cardiomyocytes Senescence Through The MIGF-1/SIRT1 Pathway

AimParaquat was used to establish a model of HL-1 cardiomyocyte senescence,to observe the effect of testosterone on senescence of cardiomyocytes,and to explore the role of the mIGF-1 / SIRT1 pathway in the effect of testosterone on senescence of cardiomyocytes.Method(1)Pre-experiments determine the optimal time for the effect of paraquat on the senescence of HL-1 cardiomyocytes and screen the optimal concentration of testosterone intervention.β-galactosidase staining method was used to determine the cell senescence rate.Flow cytometry was used to measure the level of reactive oxygen species(ROS)in cardiomyocytes for cell senescence detection.(2)To observe the effect of testosterone on the expression of mIGF-1 and SIRT1 proteins: Western blotting was used to detect the expression levels of mIGF-1 and SIRT1 in normal cell(CK)group,paraquat induction(PQ)group,and paraquat + testosterone intervention(Tes)group.Cellular immunofluorescence staining was used to detect the localization of SIRT1 in the nucleus.(3)To explore the role of mIGF-1 in testosterone in delaying the senescence of cardiomyocytes and its correlation with SIRT1 expression: pre-construct mIGF-1 overexpression vector and silencing vector and transfect cardiomyocytes.After treatment with paraquat + testosterone,they were divided into the following 5 groups: mIGF-1 overexpression transfection(OE-mIGF-1)group,empty-load transfection(NC)group,mIGF-1 interference transfection(sh-mIGF-1)group,negative control transfection(sh-NC)group,and blank control(Tes)group,to observe the effect of mIGF-1 on the change of senescence effect of testosterone-treated cardiomyocytes and interference of mIGF-1 on SIRT1 expression.(4)To further determine the role of SIRT1 in testosterone in delaying the senescence of cardiomyocytes:SIRT1 overexpression vector and silencing vector were pre-constructed and transfected into cardiomyocytes.After treatment with paraquat + testosterone,they were divided into the following 5 groups: SIRT1 overexpression transfection(OE-SIRT1)group,and empty-load transfection(NC)group,SIRT1 interference transfection(sh-SIRT1)group,negative control transfection(sh-NC)group,and blank control(Tes)group.The effects of SIRT1 on the senescence effect of testosterone-treated cardiomyocytes were observed.(5)The qPCR method was used to detect the changes of contractile / diastolic related enzymes and proteins in each group of cells,and to further observe the effects of testosterone and mIGF-1 / SIRT1 pathway activation on the accompanying functions of senile cardiomyocytes.Results(1)Compared with the CK group,100μmol/L paraquat increased the senescence rate of β-galactosidase-stained cells in mouse HL-1 cardiomyocytes,which was statistically significant at a time of 72 hours(P<0.01).Compared with the PQ group,the testosterone group could reduce the senescence rate of β-galactosidase-stained cells and the level of intracellular reactive oxygen species in myocardial cells induced by paraquat in a dose-dependent manner.The differences were most significant at 1.0 μM(P <0.001).(2)Compared with the PQ group,the Tes group can increase the expression levels of mIGF-1 and SIRT1 protein(P<0.01)and the localization level of SIRT1 in the nucleus(P<0.05).(3)Compared to Tes group,mIGF-1 and SIRT1 proteins levels and the localization of SIRT1 in the nucleus of the OE-mIGF-1 group were significantly increased(P<0.05,P<0.01,P<0.05,respectively).The senescence rate of β-galactosidase-positive cells and ROS were significantly reduced(P< 0.05).Compared with the Tes group,sh-mIGF-1 group could down-regulate the expression of mIGF-1,SIRT1 protein and the localization level of SIRT1 in the nucleus,increase the senescence rate of β-galactosidase-positive cells and ROS level(P<0.05).Compared to Tes group,there was no statistical difference in the expression levels of mIGF-1 and SIRT1 proteins in the sh-NC group and the NC group,as well as the localization level of SIRT1 in the nucleus(P > 0.05).(4)Compared with the Tes group,the expression level of SIRT1 protein and the localization level of SIRT1 in the nucleus in the OE-SIRT1 group were significantly increased(P<0.05,P<0.01,respectively).The senescence rate of β-galactosidase-positive cells and ROS level were significantly reduced(P<0.05).Compared with the Tes group,sh-SIRT1 group could down-regulate SIRT1 protein expression and SIRT1 localization in the nucleus,increase the senescence rate of β-galactosidase-positive cells and ROS level(P<0.01).Compared with the Tes group,there was no statistical difference in the above indicators in sh-NC group and NC group(P> 0.05).(5)Compared with the CK group,the expression levels of MYH-7 and ACTA-1 mRNAs in cardiomyocytes of the PQ group increased,while the expression levels of MYH-6 and SERCA-2 mRNAs decreased(P<0.01).Compared with the PQ group,the expression levels of MYH-7 and ACTA-1 mRNAs in cardiomyocytes of the Tes group decreased,while the expression levels of MYH-6 and SERCA-2 mRNAs increased(P<0.01,P<0.001,P<0.01,P<0.01,respectively).Compared with the Tes group,the expression levels of MYH-7 and ACTA-1 mRNAs increased,while the expression of MYH-6 and SERCA-2 mRNAs decreased in the sh-mIGF-1 and sh-SIRT1 groups(P<0.05).The sh-double group,namely sh-"mIGF-1 and SIRT1",showed the same trend,and the expression levels of MYH-7 and ACTA-1 mRNAs increased(P<0.01,P<0.05,respectively),MYH-6 and SERCA-2 mRNAs expression decreased(P<0.01,P<0.05,respectively).Compared with the Tes group,the levels of MYH-6 mRNA in the OE-mIGF-1 group were increased(P<0.05),and the levels of MYH-6 mRNA and SERCA-2 mRNA in the OE-SIRT1 group were increased(P<0.05,P<0.01,respectively).There was no significant difference in the expression levels of MYH-7 and ACTA-1 mRNAs between the two groups(P> 0.05).ConclusionsTestosterone can delay paraquat-induced HL-1 cardiomyocytes senescence,and its anti-aging mechanism is related to the activation of the mIGF-1 / SIRT1 pathway.