Follistatin-like 1 Protects Cardiomyoblasts From Injury Induced By Excessive Nitric Oxide

Objective: Studies have shown that cardiac dysfunction and myocardial cell apoptosis are inseparable, the apoptosis of myocardial cells has a great relationship with excessive NO, sodium nitroprusside(SNP) is widely used as a clinical vascular expansion agent, also it is widely used as the NO donor, on myocardial cell injury. Therefore, the aim of this study was to study the protective effect of FSTL1 on SNP induced apoptosis in cardiomyocytes.Methods: In vitro, we used rat fetal ventricular myocytes to culture and subculture.1.Via SNP(2mM) stimulated H9C2 cell 24 h to establish SNP induced H9C2 cell apoptosis model.2. The expression of FSTL1 was detected by q-PCR and Western Blot before and after SNP stimulation in H9C2.3 By introducing exogenous FSTL1(100ng/ml) pretreatment H9C2 cell 30 min to compare the difference of H9C2 cell apoptosis before and after SNP stimulation.4 Comparison of apoptosis of H9C2 cells by transfection of endogenous FSTL1 before and after SNP stimulation.5 The total nitric oxide detection kit using nitrate reductase reduced nitrate to nitrite, and then through the Griess reagent detection of nitrite classic, which measured a total nitric oxide.6 By introducing the PI3K/Akt Smad2/3 inhibitor LY294002 and SB5253334, Smad1/5/8 signaling pathway agonist BMP-4 study on the molecular mechanism of FSTL1 protects myocytes.Results: Using the results of DAPI staining can display H9C2 cells in SNP(2mM) after 24 h stimulation compared with that without SNP stimulation, the number of apoptosis of H9C2 cells increased significantly. The apoptosis of H9C2 cells was significantly decreased after SNP stimulation with FSTL1(100ng/ml) pretreatment 30 min. Silencing of endogenous FSTL1 by transfection and then SNP stimulated H9C2 cells, then with SNP stimulation, we found that the number of apoptosis was significantly higher than that of the none silent group. Q-PCR test results showed that the expression of FSTL1 in SNP stimulated H9C2 decreased at the level of gene, Western Blot showed that the expression of FSTL1 in the protein level decreased after SNP stimulated H9C2. Total nitric oxide detection kit test results showed that the content of NO in the culture medium after SNP stimulation increased significantly than the control group.However, with FSTL1 pretreatment 30 min the content of NO was significantly lower than none pretreatment group. By introducing the PI3K/Akt signaling pathway inhibitor LY294002 interference Akt signaling pathway, the role of FSTL1 in protecting H9C2 cells was obviously decreased. Introduction of BMP-4 excited Smad1/5/8 signaling pathway showed a significant decrease in the role of FSTL1 in the protection of H9C2 cells. The introduction of SB523334 inhibited Smad2/3 signaling pathway after FSTL1 protection of H9C2 cells showed no significant changes before and after.Conclusion: FSTL1 has a protective effect on SNP induced apoptosis in H9C2 cells. The role of FSTL1 in the protection of myocardial cell apoptosis is at least by activating the Akt signaling pathway and inhibiting the Smad1/5/8 signaling pathway.