Study Of Mechanism On Apoptosis Of Human Pulmonary Microvascular Endothelial Cells By Simulated Microgravity

ObjectiveMicrogravity or simulated microgravity can induce the change of pulmonary histomorphology, pulmonary hypertension and pulmonary microvascular change. Meanwhile, Microgravity or simulated microgravity can induce the apoptosis of endothelial cell. However, the apoptosis of human pulmonary microvascular endothelial cells (HPMVECs) which was caused by microgravity or simulated microgravity is not reported until now. The time effect and mechanism of the apoptosis are nor clear. So, the purpose of this study is following:1. Find the optimal time of the apoptosis of human pulmonary microvascular endothelial cells which was caused by simulated microgravity by clinostat.2. Study the effect of mitochondrial signal transduction pathway on the apoptosis of human pulmonary microvascular endothelial cells which was caused by simulated microgravity.3. Observe the change of PI3K/Akt and NF-κB in the human pulmonary microvascular endothelial cells of apoptosis caused by simulated microgravity.4. Observe the change of cytoskeleton in human pulmonary microvascular endothelial cells by simulated microgravity, and find the relationship between cytoskeleton and cell apoptosis.MethodsTo obtain the aim of study above, the experimental methods were applied as following:1. The human pulmonary microvascular endothelial cells were cultured and passaged. When the adherence of human pulmonary microvascular endothelial cells and fraction of coverage was 80%, they were gathered and were applied in the experiment of simulated microgravity by clinostat.4 time point selected were 12h,24h,48h and 72h, respectively, meanwhile,1g control was set at every time point. Cell apoptosis was detected by the flow cytometry, and transmission electron microscope was used to observe the ultrastrcture of human pulmonary microvascular endothelial cells after simulated microgravity. According to the results, the optimal time of the apoptosis of human pulmonary microvascular endothelial cells.2. After the optimal time of the apoptosis of human pulmonary microvascular endothelial cells was found, some important genes and proteins, such as Bcl-2, Bax, Cytochrome C, Caspase-9 and Caspase-3, correlated with the mitochondrial signal transduction pathway were selected. Caspase-3, Bcl-2 and Bax were detected by real time PCR, and ctytochrome C, Caspase-9 and Caspase-3 were measured by Western blot.3. PI3K, Akt, P-Akt and NF-κB in the human pulmonary microvascular endothelial cells after simulated microgravity were measured by Western blot.4. By inmunfluorescence technique, phalloidin was used to incubate with human pulmonary microvascular endothelial cells to detect the change of microfilament cytoskeleton at the different time of cell apoptosis.Results1. By the flow cytometry, when 12h and 24h of simulated microgravity by clinostat, cell apoptosis was not obvious; Compared 12h with 24h, there was no significantly difference (C12:2.29±0.78, Clinostat 12:3.89±1.24, p=0.13; C24: 2.35±1.48, Clinostat 24:4.08±0.72, p=0.14). When 48h and 72h of simulated microgravity by clinostat, cell apoptosis can by observed; however, Compared 48h with 72h, the apoptosis of human pulmonary microvascular endothelial cells was most obvious at 72h (C48:2.07±0.49, Clinostat 48:3.47±0.32, p=0.0386; C72:2.55±0.78, Clinostat72:8.18±0.48, p=0.0132). The results from transmission electron microscopy showed that the cell body of endothelial cells decreased and became round, the cell process decreased, the density of cytoplasmic matrix increased, nuclear membrane shrinkaged, intranuclear chromatin concentrated and aggregated below the nuclear membrane and mitochondrion became hyperplasia and swelling. Meanwhile, the apoptotic body can be observed when 72h of simulated microgravity.2. The results by real time PCR showed that the expression of Bcl-2 was decreased, and the expression of Bax and Caspase-3 were increased in the human pulmonary microvascular endothelial cells when 72h of simulated microgravity. The results by Western blot showed the concentration of Cytochrome C in the cytoplasm was higher than that in the mitochondrion and the expression of Caspase-9 and Caspase-3 were increased when 72h of simulated microgravity.3. The results by Western blot showed that simulated microgravity significantly decreased the expression of PI3K and the phosphorylation level of Akt, meanwhile, increased the expression of NF-κB.4. The results by phallodidin showed that the expression of F-actin was decreased and appeared depolymerization at 48h, and the change of that was more obvious at 72h.Conclusion1. Simulated microgravity by clinostat can induce human pulmonary microvascular endothelial cells prophase apoptosis and the optimal time was 72h.2. The mitochondial signal transduction pathway played an important role in the apoptosis of human pulmonary microvascular endothelial cells by simulated microgravity.3. PI3K-Akt and NF-κB took part in the apoptosis of human pulmonary microvascular endothelial cells by simulated microgravity.4. The express of F-actin was decreased and appeared depolymerization in the human pulmonary microvascular endothelial cells by simulated microgravity, and this suggested that the change of F-actin can take part in the cell apoptosis.