Protective Effects Of Aspirin On Growth Differentiation Factor 15 On Cardiomyocyte Hypoxia/Reoxygenation Injury And Its Mechanism

Myocardial ischemia-reperfusion(I/R)injury is a phenomenon that the heart injury is aggravated after the blood perfusion is restored.The damage nature of the injury is related to the apoptosis.In recent years,it has been found that H/R occurs in patients with angina pectoris coronary antispasmodic,cardiopulmonary resuscitation,etc,which is one of the important causes of postoperative disease progression.GDF15,as a stress-responsive protein,is highly expressed in the prostate and placenta,and is expressed in most other tissues little,but in physiological,pathological and environmental stress situations,such as I/R injury,atherosclerosis,cardiac hypertrophy and heart failure,GDF15 in myocardial cells are a large number of expression of myocardial cells and apoptosis play a regulatory role.Therefore,GDF15 can be seen as a marker of cardiovascular disease.Objective:First,the GDF15 lentiviral expression vector was constructed and the virus was packaged to establish a stable cell line of GDF15.To investigate the protective effect of GDF15 on H9c2 cardiomyocyte hypoxia / reoxygenation(H/R)injury and its effect on autophagy.After the Pretreatment of aspirin,the expression of GDF15 in H9c2 cardiomyocytes was observed,and asp protects cells by increasing the protective effect of GDF15 protein on apoptosis and the effect on autophagy.The mechanism of GDF15 protein and Asp myocardium protection was clarified,which provided the theoretical basis for its clinical application.Methods: Part Ⅰ1.The establishment of GDF15 stable overexpressing cell line H9c2/GDF15Construction of lentiviral expression vector pCDH-CMV-MCS-GDF15-EF1-Puro,and blank vector pCDH-CMV-MCS-EF1-Puro,respectively with pMDLg / pRRE,pRSV-Rev,pMD2.G packaging lentivirus,infected H9c2 cells,Puromycin screening,and finally get GDF15 stable overexpressing cell line H9c2/GDF15 and control cell line H9c2/Sham.2.The establishment of H/R injury model of H9c2 cellWhen the H9c2 cells were grown to cover 80% ~ 90%,the simulated hypoxia solution was placed in a sealed hypoxic device for 30 min.The ischemia was simulated in the incubator.4h later replaced with serum-free medium simulate normal reperfusion.MTT was used to detect the cell activity at 2h,3h and 4h,and the most suitable reoxygenation time was selected The time of expression of GDF15 protein was screened as the experimental H/R condition to establish the H/R model of H9c2.3.The effect of GDF15 on H9c2 was examinedTo examine the cell viability and the activity of lactate dehydrogenase(LDH)expression.The expression of Caspase-3,Bcl-2,Bax and autophagy-related proteins Beclin-1 and LC3-Ⅱ were measured.Part Ⅱ1.Effects of Asp on H9c2 cell injuryH9c2 cells were pretreated with different concentrations of Asp.MTT was used to detect the survival rate of cells treated and untreated with Asp.Select the most appropriate reoxygenation time point2.The expression of GDF15 in Asp pretreatmentH9c2 cells were treated with different concentrations of Asp,and the expression of GDF15 protein was detected by Western Bolt.3.Apoptosis and autophagy-related protein expressionThe expression levels of apoptosis-related proteins Caspase-3,Bcl-2,Bax and autophagy-related proteins Beclin-1 and LC3-B were detected.Results:Part Ⅰ1.To construct the GDF15 lentiviral expression vector and to express the virus,and to establish the rat GDF15 overexpressing cell line H9c2 / GDF15Construction of the lentiviral vector pCDH-CMV-MCS-GDF15-EF1-Puro,the establishment of GDF15 stable overexpressing cell lines.Western Blot showed that the expression of GDF15 protein in H9c2/GDF15 group was significantly higher than that in H9c2/Sham group.Real-time PCR results showed that the expression of GDF15 in H9c2/GDF15 group was 6.3 times higher than that in H9c2/Sham group.It was proved that GDF15 overexpression of H9c2 cell line was successfully constructed.2.Effect of overexpression of GDF15 on H9c2MTT results showed that the cell viability of H9c2/GDF15 group was higher than that of H9c2/Sham group and positive control group after H/R(P<0.01).The LDH activity value of the supernatant of cell culture was significantly lower than that of H9c2 / Sham and H9c2 / GDF15 after H/R,and there was significant difference(P<0.01).3.Effects of overexpression of GDF15 on H/R-induced apoptosisThe effect of GDF15 overexpression on H/R-induced apoptosis: Western Blot showed that the expression of Caspase-3 in H9c2/GDF15 group was significantly lower than that in H9c2 / Sham group at H/R 2h(P<0.01).Under the condition of H/R,the Bcl-2/Bax value of H9c2/GDF15 group was significantly higher than that of H9c2/Sham group(P<0.01).4.Effects of overexpression of GDF15 on H/R-induced cell autophagyThe effect of GDF15 overexpression on H/R-induced autophagy: Western Blot showed that the expression of Beclin-1 in H9c2/GDF15 group was significantly higher than that in H9c2/Sham group at ischemia 4h(P<0.01).Compared with H9c2/Sham group,the expression of Beclin-1 in H9c2/GDF15 group was significantly higher than that in control group at H/R 2h(P<0.01).The expression of LC3Ⅱ in H9c2/GDF15 group was significantly higher than that in H9c2/Sham group at H/R 2h(P<0.01).Part Ⅱ1.Effects of Asp on H9c2 cell H/R injuryMTT results showed that the survival rate of H9c2 cells was decreased with the increase of Asp concentration,but at the same concentration,the survival rate of H9c2-Asp-H/R group was significantly higher than that of H9c2-H/R group(P< 0.01).2.Effects of Asp pretreatment on GDF15 in H9c2 Cells before and after H/RWestern Blot showed that the expression of GDF15 in H9c2-Asp-H/R group was significantly higher than that in H9c2-H/R group after 48 h of treatment with different concentrations of Asp(P< 0.01).3.Effects of Asp on H/R apoptosis and autophagy in H9c2 CellsWestern Blot showed that the expression of Caspase-3 protein in H9c2-Asp-H/R group was significantly lower than that in H9c2-H/R group(P<0.01),and the ratio of Bcl-2/Bax was significantly higher than that of H9c2-H/R group(P<0.01).The expression of Beclin-1 was significantly increased(P<0.01),and the ratio of LC3Ⅱ/LC3Ⅰ was significantly increased(P<0.01).Conclusion:1.Successfully construct GDF15 stable overexpressing cell line H9c2 / GDF15 and its negative empty vector H9c2 / Sham.2.Increased levels of GDF15 in H9c2 cells can protect H9c2 rat cardiomyocytes and reduce H/R injury.The protective mechanism may be achieved by promoting autophagy of cells.3.For H/R H9c2 cells,Asp intervention can increase the expression of GDF15,reduce H/R induced cell damage.