Study On The Effect Of Klotho On Myocardial Cells Senescence Based On Oxidative Stress-mediated Ferroptosis

Objective: 1.To study the effect of ferroptosis on myocardial cells aging and its mechanism;2.To study the effect of Klotho on myocardial cells aging and ferroptosis.Methods:(1)Male BALB/C mice aged 2 months,8 months and 14 months were selected to weigh the weight of the heart and body weight of the mice and calculate the ratio of heart to body weight;The morphological changes of heart tissue were observed by HE staining,the content of malondialdehyde(MDA)in serum was detected by microplate method,the iron ion in heart tissue was detected by Prussian blue+DAB staining,the expression of Klotho protein,and the expression levels of ferroptosis-associated protein solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPx4)and aging related protein P53,P21 in mice heart tissue were detected by Western Blot.(2)The myocardial cells aging model was prepared by using different concentrations of D-galactose(D-gal)to induce H9C2 cells injury.The morphology and quantity of cells were observed under light microscope;MTT method was used to detect cell viability and determine the concentration of D-gal used in subsequent experiments.(3)After treating myocardial cells with D-gal and ferroptosis inhibitor,the cell viability was detected by MTT method,the intracellular reactive oxygen species(ROS)level was detected by 2’,7’-dichlorofluorescein diacetate(DCFH-DA)fluorescence probe method,the intracellular MDA content was detected by thiobarbituric acid method,and the expression level of SLC7A11,GPx4,P53 protein was detected by Western Blot;The intracellular aging marker β-Galactosidase(β-GAL)activity was detected by microassay,and observe the effect of ferroptosis on myocardial cells aging.(4)Targeted inhibition of P53 study the effect of P53/SLC7A11/GPx4 signal axis on cell aging.The intracellular glutathione(GSH)content was detected by microplate method;The expression level of SLC7A11,GPx4 and P53 protein was detected by Western Blot;The intracellular β-GAL activity was detected by microassay.(5)Myocardial cells were treated with D-gal and Klotho,the cell viability was detected by MTT method,the changes of mitochondrial structure in the cells were observed by transmission electron microscope,the level of ROS in the cells was detected by DCFH-DA fluorescence probe method,the content of GSH in the cells was detected by microplate method,the content of MDA in the cells was detected by thiobarbituric acid method,the content of superoxide dismutase(SOD)in the cells was detected by microplate method,and the expression levels of SLC7A11,GPx4,P53 and P21 protein were detected by Western Blot,The intracellular β-GAL activity was detected by microassay,to study the effect of Klotho on cell senescence and ferroptosis.(6)Myocardial cells were treated with ferroptosis inducers Erastin and Klotho.Cell viability was measured by MTT assay to observe the effect of Klotho on Erastin induced cell viability.Results:(1)Compared with young mice,the heart volume of aged mice increased,the ratio of heart weight to body weight increased(P<0.05);HE staining results showed that myocardial cells in aged mice were hypertrophic and disorderly arranged,and the cell boundary was not clear;The level of MDA in serum increased(P<0.05);Prussian blue+DAB staining showed that iron ions in heart tissue of aged mice increased(P<0.05);Western Blot results showed that the expression level of ferroptosis related proteins SLC7A11 and GPx4 in the heart of aged mice decreased,the expression of aging related proteins P53 and P21 increased,and the expression of Klotho protein decreased(P<0.05).(2)Compared with control group,with the increase of D-gal concentration,the morphology of myocardial cells showed flat and fat changes under light microscope;MTT results showed that cell viability decreased with the increase of D-gal concentration(P<0.05).Myocardial cells were treated with 20mg/m L D-gal,compared with the control group,the content of ROS and MDA in D-gal group increased(P<0.05);Western Blot showed that the expression of SLC7A11 and GPx4 protein was down regulated and the expression of P53 protein was up regulated(P<0.05);β-GAL activity increased in D-gal group(P<0.05).(3)Compared with D-gal group,the cell viability of the group treated with D-gal and ferroptosis inhibitor(Fer-1)increased(P<0.05);The content of ROS and MDA decreased(P<0.05);The expression levels of SLC7A11 and GPx4 protein increased,and the expression of P53 protein decreased(P<0.05);β-GAL activity decreased(P<0.05).(4)Compared with D-gal group,Western Blot results showed that expression of P53 protein decreased and the expression of SLC7A11 and GPx4 protein increased in the group of Dgal and P53 inhibitor Pifithrin-α(P<0.05);The intracellular GSH content increased(P<0.05);Intracellular β-GAL activity decreased(P<0.05).(5)Compared with the D-gal group,the MTT method showed that the cell viability of the D-gal and Klotho protein treatment group increased(P<0.05);The contents of ROS and MDA in cells decreased,while the contents of GSH and SOD increased(P<0.05);Western Blot showed that after treatment with Klotho,the expression of SLC7A11 and GPx4 protein increased,and the expression of P53 and P21 protein decreased(P<0.05);β-GAL activity decreased(P<0.05).Transmission electron microscopy showed that the mitochondrial cristae decreased and mitochondria swelled of D-gal group,while the mitochondrial structure damage of myocardial cells was reduced after treatment with Klotho.(6)Compared with the control group,the myocardial cells viability of the group treated with Erastin decreased,while that of the group treated with Erastin and Klotho protein increased(P<0.05).Conclusion:Ferroptosis is involved in the process of myocardial cells aging,Klotho can inhibit ferroptosis of myocardial cells and delay cell aging.Its mechanism may be mediated by P53/SLC7A11/GPx4 targeting.