| ObjectiveBy establishing a hypoxia/reoxygenation(H/R)cell model,to explore the protective effect of tropomyosin receptor kinase A(TRKA)on H9c2 cardiomyocytes after hypoxia/reoxygenation(H/R)and its relationship with PI3K/Akt/m TOR signaling pathwayMethods1.H9c2 rat cardiomyocytes were cultured in vitro and different concentrations of GW441756(0,0.5,1,2,4,8,16 and 32 μM)and wortmannin(0,3.125,6.25,12.5,25,50,100,200 and 400 n M)were used to act on H9c2 cells for 24 h,and the CCK-8 assay to detect cell proliferation activity and screen the appropriate concentrations of GW441756 and wortmannin for subsequent experiments.2.In vitro hypoxia/reoxygenation cell model was established,and experimental groups were established: control group(control),hypoxia/reoxygenation model group(H/R),TRKA inhibitor group(H/R+GW441756),PI3 K inhibitor group(H/R+ wortmannin)and combined drug group(H/R+GW441756+ wortmannin).The CCK-8 method was applied to detect the effect of TRKA inhibitor and PI3 K inhibitor on the proliferation viability of H9c2 cardiomyocytes after H/R.3.Detection of lactate dehydrogenase(LDH)and malondialdehyde(MDA)content in 5 groups of cells.After DCFH-DA staining,the fluorescence intensity was observed under fluorescence microscope,and the intracellular reactive oxygen species(ROS)content of the 5 groups was analyzed using Image J software to investigate the effects of TRKA inhibitor and PI3 K inhibitor on oxidative stress in cardiomyocytes after H/R.4.JC-1 mitochondrial membrane potential(MMP)kit,Annexin V-FITC/PI apoptosis kit and Western blot were used to explore the effects of TRKA inhibitors and PI3 K inhibitors on apoptosis in cardiomyocytes after H/R.5.To explore the protein expression of PI3K/Akt/m TOR signaling pathway after using TRKA inhibitor: The protein expression of p-TRKA(Tyr490),TRKA,p-p85 PI3 K,PI3K,p-Akt(Ser473),Akt,p-m TOR(Ser2448)and m TOR in five groups of cells after GW441756 and Wolman penicillin treatment were detected by Western blot method.Results1.Under normal oxygen,after the treatment of GW441756 in the 0-4μM concentration range and wortmannin in the 0-50 n M concentration range for 24 h,the proliferative activity of cells did not change significantly,and the difference was not statistically significant.GW441756 and 100-400 n M of wortmannin in the concentration range of 8-32μM reduce cell proliferative activity(P<0.05).Therefore,4μM GW441756 and 50 n M wortmannin H9c2 cells were selected for 24 h as subsequent experimental conditions.2.Inhibition of TRKA and PI3 K pathway can inhibit the proliferative activity of H9c2 cardiomyocytes after H/R: compared with the H/R group,the proliferative activity of cells in the GW441756 and Wolman penicillin groups decreased(P<0.05);and the inhibitory effect on cell proliferation activity was stronger after the combination of the two inhibitors compared with the group using the two inhibitors alone.3.Inhibition of TRKA and PI3 K pathways increased the level of oxidative stress in cardiac myocytes after H/R: LDH,MDA and ROS levels were increased in the GW441756 and Wolman penicillin groups compared with the H/R group(P<0.05);and the intracellular LDH,MDA and ROS levels were increased in the combined use of both inhibitors group compared with the two inhibitors alone(P< 0.05).4.Inhibition of TRKA and PI3 K pathways increased the level of apoptosis in cardiomyocytes after H/R: MMP was significantly decreased in the GW441756 and Wolman penicillin groups compared with the H/R group(P<0.05);apoptosis rate was increased by flow cytometry(P<0.05);and both upregulated the pro-apoptotic proteins Bax and caspase-3 and downregulated the anti-apoptotic protein Bcl-2 expression(P<0.05).And the apoptosis rate increased in the combined group compared with the two inhibitors alone(P<0.05).5.Inhibition of TRKA pathway reduced the expression of PI3K/Akt/m TOR signaling pathway-related proteins in H9c2 cardiomyocytes after H/R: p-PI3 K,p-Akt,p-TRKA and p-m TOR expression were reduced after GW441756 treatment compared with the H/R group(P<0.05).Compared with the H/R group,p-PI3 K,p-Akt and p-m TOR expression was reduced after Wolman penicillin treatment(P<0.05).The expression of p-TRKA,p-PI3 K,p-Akt and pm TOR was increased in the combination group compared to the two inhibitors alone(P<0.05).It is suggested that the activation of PI3K/Akt/m TOR signaling pathway may be one of the mechanisms by which TRKA exerts its protective effect on post-H/R cardiomyocytes.Conclusions1.TRKA and PI3K/Akt signaling pathways can exert myocardial protective effects by reducing oxidative stress and apoptosis in cardiomyocytes after hypoxia/reoxygenation.2.The protective effect of TRKA on cardiomyocytes after hypoxia/reoxygenation is partly related to the activation of PI3K/Akt/m TOR signaling pathway. |
