Vascular Endothelial Growth Factor Ameliorated Palmitate Induced Cardiomyocyte Injury via JNK Pathway
Authors: Wang Shiya , Zou Cao , Liu Xiaofeng , Yan Yonjin , Gu Shunzhon , Wang Qinghua , Li Xun 
Published: December 2020

Abstract
Objective To investigate the effect of palmitate (PAL) on apoptosis and the timing and activity of VEGF expression in HHHM2 myocardial cells (a human embryonic cardiomyocyte cell- line). Methods 1. Cardiomyocytes were divided into the following five groups: the control group and the 0.2 mM, 0.5 mM, 0.8 mM, and 1.2 mM PAL groups. We examined the changes in cell viability by MTT assay after PAL incubation for 24 h and the cardiomyocyte apoptosis rate by FACS examination, and thus determined the effective concentration of PAL. The transcription levels of CASP3, Bcl-2, Bax, and VEGF were detected by quantitative fluorescence PCR and the protein expression of caspase 3 and VEGF by western blot. 2. To observe the time-dependent effects on cell injury induced by 0.5 mM PAL, cardiomyocytes were divided into 0, 4, 8, 16, 24, and 48 h groups. The variation in cell viability was examined by MTT assay. The transcription levels of CASP3, Bcl-2, Bax, and VEGF were detected by quantitative fluorescence PCR and the protein expression of caspase 3 and VEGF by western blot. 3. To observe the effects of VEGF on the PAL induced apoptosis of cardiomyocytes, the cells were divided into the control group and the VEGF overexpression group. At 24 h after transfection, cells were incubated with 0.5 mM PAL for 6, 12, 24, and 48 h. Cell viability was examined by MTT assay. The apoptosis rate was measured by FACS using the Annexin V-FITC kit. The transcription levels of CASP3, Bcl-2, Bax, NF-kB p65, and VEGF were measured by quantitative fluorescence PCR, the protein expression of VEGF, caspase 3, Bcl-2, Bax, NF-κB p65, p-JNK/JNK, and p-ERK/ERK were measured by western blot, as well as caspase 3 activity. Results 1. A dose-dependent relation between the concentration of PAL and H9c2 cardiomyocyte injury was observed. In the 0.5 mM group, the apoptosis rate was increased significantly, while cell viability was decreased, indicating that 0.5 mM PAL was the ideal concentration to induce cardiomyocyte apoptosis. The expression of caspase 3 and Bax was significantly increased, and the expression of VEGF was enhanced, while the levels of Bcl-2 remained unchanged during the process. 2. A significant time-dependent relation between PAL and cardiomyocyte injury was observed. The apoptosis rate was increased greatly after 16 h treatment with 0.5 mM PAL. 3. Cell viability was restored by VEGF overexpression during treatment with 0.5 mM PAL. The apoptosis rate was also reduced by VEGF overexpression, as detected by FACS. The expression of caspase 3, Bax, and NF-κB p65 was significantly decreased, Bcl-2 and VEGF expression was dramatically increased, p-JNK/JNK expression was significantly enhanced, p-ERK/ERK levels did not exhibit a significant change, and the activity of caspase 3 was significantly decreased. Conclusions 1. PAL can induce injury and apoptosis in HHHM2 myocardial cells, and these effects are time-dependent. A PAL concentration of 0.5 mM was ideal to establish the cardiac cell injury model. 2. PAL at a concentration of 0.5 mM can effectively induce cardiomyocyte injury and enhance the expression of caspase 3, Bax, and VEGF, especially after 24 h and 48 h of PAL treatment. 3. VEGF overexpression can reverse the effects of PAL on apoptosis and cell viability. In addition, VEGF overexpression inhibited the expression of proapoptotic and inflammatory factors, caspase 3 activity, and transduction of the MAPK signaling pathway.