| Part Ⅰ The protective effects of 6-gingerol against hydrogen peroxide induced apoptosis in HUVEC through autophagyObjective:The occurrence and development of atherosclerosis is a process of multiple factors involved in the complex pathophysiology. Apoptosis is one of the important reasons of inducing cardiovascular cell injury, and many pathogenic factors of atherosclerosis can activate the apoptosis of vascular endothelial cells. In the recent research, much more attention has been paid on the relationship between autophagy and apoptosis, and the role of autophagy regulation in heart diseases. Autophagy plays a very important role in maintaining the function and morphology of cardiovascular. To study the role of autophagy in endothelial cell injury, can provide a new idea for the treatment of atherosclerosis. Many phytochemicals can induce autophagy.6-Gingero1 as a kind of phytochemical has the effect of anti-atherosclerosis, but the specific mechanism is still not completely clear. This research focuses on whether autophagy plays an important role in the process of anti-atherosclerosis by 6- gingerol.Methods:Human umbilical vein endothelial cells (HUVEC) were used as the research objects. The cells were pretreated with 6-gingerol (10-40 μM) for 24 h, and then treated with H2O2 (800μM) for another 1 h. MTT method was used to test cell viability of the cells after treated with 6-gingerol and H2O2. Moreover, Autophagy inhibitor 3-Methyladenine (3-MA) and the autophagy inducer rapamycin were added to test the cell viability of the cells after treated with 6-gingerol and H2O2.Hoechst 33342 staining and annexin V-FITC/PI double staining flow cytometry methods were used to test the apoptosis. Autophagosome staining by Cyto-ID Green fluorescence was detected by fluorescence microscopy. LC3, Bcl-12, Beclin-1, mTOR and p-mTOR releases of cytosolic protein samples were detected by Western blot. The lysosomal stability was observed by confocal laser scanning microscopy. The levels of intracellular reactive oxygen species (ROS), glutathione (GSH), and mitochondrial membrane potential were detected by fluorescence spectrophotometer. Moreover,3-MA and the rapamycin were added to test the change of ROS and GSH after treated with 6-gingerol and H2O2Result:(1) result of apoptosis:apoptotic cells were observed under fluorescence microscope after treated with H2O2 (800 μM) for 1 h. The nuclei were deeply stained, condensation or fragmentation. After pretreated with 6-gingerol (10-40μM), the numbers of apoptotic cells were significantly decreased. There was obvious dose-respones relationship between different groups. Apoptosis was also detected by annexin V-FITC/PI double staining flow cytometry method. And the apoptosis can also be protected by 6-gingerol. (2) Result of autophagy:After Cyto-ID Green staining, less green punctate fluorescence was detected in control group and H2O2 treatment group, while bright green fluorescent was detected in 6-gingerol protection group. Moreover, Western blot results showed that autophagy protein marker LC3-II expression increased with the increased concentration of 6-gingerol. There was obvious dose-respones relationship (P<0.01). (3) Result of autophagy related protein:Bcl-2, Beclin-1, mTOR, and p-mTOR release of cytosolic protein samples were detected by western blot in 6-gingerol protection group. Bcl-2 and Beclin-1 protein expression gradually increased, while the expression of p-mTOR decreased gradually (P<0.01). (4) Result of oxidative stress:the level of ROS was significantly increased in the H2O2 group compared with the control (P<0.01), but the level of ROS was significantly decreased in 6-gingerol group (P<0.05 or P<0.01). The level of GSH was significantly decreased in H2O2 group (P<0.01), but the level of ROS was significantly increased in 6-gingerol group (P <0.05 or P<0.01). Pretreated with 3- MA, the level of ROS was increased and GSH was decreased compared with the groups without 3-MA; Pretreated with rapamycin, the level of ROS was decreased and GSH was increased. (5) Result of Lysosome and mitochondria:the level of mitochondrial membrane potential decreased significantly in H2O2 group, while it was raised by 6-gingerol; Laser scanning confocal microscope showed that, the fluorescence intensity of AO was weak in H2O2 group, while the fluorescence intensity of AO is stronger in 6-gingerol groups, which suggested that 6-gingerol can improve the stability of cell lysosomes (P<0.05 or P<0.01).Conclusion:1. H2O2 (800 μM) could induce apoptosis in HUVEC, and the apoptosis could be prevented by 6-gingerol.2.6-Gingerol could induce autophagy in HUVEC.3. The expression of Bcl-2 and Beclin-1 was increased, and the expression of p-mTOR was decreased, suggesting the occurrence of autophagy may be related with mTOR signaling pathway and Beclinl complexes.4. Oxidative stress results showed that 6-gingerol could reduce the ROS level, increase the GSH level, which suggested that autophagy has the potential function against oxidative stress.5.6-Gingerol can improve the stability of cell lysosomes, and increased the level of mitochondrial membrane potential, which could prevent apoptosis.Part Ⅱ The protective effects of 6-gingerol against hydrogen peroxide induced DNA damage in HUVECObjective:a variety of factors were involved in the pathogenesis of coronary heart diseases, and the injury of vascular endothelial cells was the initial factor of cardiovascular diseases. It has been confirmed that the DNA damage of endothelial cell was related to the atherosclerosis, and the reactive oxygen is one of the direct cause leading to DNA damage. Oxidative stress could happen when the imbalance of the generation of reactive oxygen species and antioxidant ability of cells, which could induce the DNA damage. The purpose of the study was to detect whether 6- gingerol as an antioxidant, can protect the DNA damage induced by hydrogen peroxide in HUVEC, and to explore its possible mechanism.Methods:HUVEC were used as the research objects. The cells were pretreated with 6-gingerol (10-40μM) for 24 h, and then treated with H2O2 (50 μM) for another 1 h. Comet assay was used to test the DNA damage; the levels of intracellular ROS, GSH, mitochondrial membrane potential and the stability of cell lysosorties were detected by fluorescence spectrophotometer; p53 releases of cytosolic protein samples was detected by western blot.Results:(1) Results of DNA damage:DNA strand breaks were detected after treatment of H2O2 (50 μM) in HUVEC, and tail length, tail DNA/head DNA (%) and tail moment were significantly more than the control group (P<0.01), which could be prevented by 6-gingerol (10,20,40 μM) (P<0.05 or P<0.01). (2)Result of lysosome and mitochondria:the level of mitochondrial membrane potential decreased significantly in H2O2 group (P<0.01), while it was raised by 6-gingerol (P<0.05 or P <0.01); the stability of lysosomal membrane was decreased significantly in H2O2 group (P<0.01), while it was raised by 6-gingerol (P<0.0 or P<0.01). (3) Results of p53 protein expression:p53 release of cytosolic protein samples was detected by Western blot in H2O2 (50 μM) group compared with control (P<0.01). However, p53 protein expression decreased in 6-gingerol (40 μM) group (P<0.01), and p53 protein expression also decreased in NAC group (P<0.01), the positive group.Conclusion:1. The DNA damage was detected in HUVEC after treatment with H2O2(50μM) for 1 h, and the damage could be prevented by 6-gingerol.2. The ROS level was increased and the GSH level was decreased after treated by H2O2 (50 μM), while 6-gingerol could reduce the ROS level, increase the GSH level.3. It was suggested that lysosome and mitochondria may be the main damage targets caused by H2O2 in HUVEC.4. H2O2 may cause DNA damage in HUVEC through p53 pathway, and 6-gingerol can decrease the expression of p53 protein to protect the DNA damage. |
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