The Apoptotic Pathway Of Human Pulmonary Microvascular Endothelial Cells Induced By Simulated Microgravity And Medicine Intervention

Microvascular endothelial cells not only constitute the main barriers between blood and tissues, but also work as powerful endocrine and metabolic organ, which have the function of synthesis, secretion and degradation biologically active substances. The abnormal responses of microvascular endothelial cells associate with hyperemia, hemorrhage, edema, inflammation and other pathological processes. Under mormal circumstances, the proliferation rate and apoptosis rate of endothelial cell are very low. Vascular endothelial cells maintain stability and normal vascular function depending on the normal balance between proliferation and apoptosis. The previous results of our research team showed that simulated microgravity can induce apoptosis of pulmonary microvascular endothelial cells. A large number of endothelial cells apoptosis for a short period of time can lead to imbalance between proliferation and apoptosis, which further caused endothelial cell dysfunction and circulatory system function loss. Therefore it is especially necessary for us to explore the apoptotic signal pathway and intervention medicine which can inhibit apoptosis.Microgravity effect was simulated by clinorotation in our study. At first, we confirmed that simulated microgravity for 72h had a remarkable effect on the occurrence of apoptosis of human pulmonary microvascular endothelial cell (HPMEC). Secondly, We observed protein and gene expression correlated with the mitochondrial signal transduction pathway and endoplasmic reticulum (ER) stress-induced apoptosis in following exposure to microgravity. At last, we used salidroside (Sal) and NG-Nitro-L-arginine Methyl Ester (L-NAME) to intervene apoptosis induced by simulated microgravity, and explore the relevant mechanisms. Main methods and results are as follows:Firstly, human pulmonary microvascular endothelial cells (HPMEC) apoptosis was detected by TUNEL method and DAPI staining after clinorotation for 72h. The rate of apoptotic cells after 72h-clinostat was much higher than that in control group. We determined mitochondrial membrane potential changes using JC-1 as a fluorescent probe to further verify the apoptosis. Quantitative real-time PCR and western blot analysis were used to detect protein and gene expression correlated with the mitochondrial signal transduction pathway and endoplasmic reticulum stress-induced apoptosis (CytC, Caspase9, Caspase3, CHOP, ATF-6, GRP78, Caspase12 and JNK). Finally we confirmed both mitochondrial signal transduction pathway and endoplasmic reticulum stress were involved in the apoptosis induced by simulated microgravity.Secondly, salidroside (Sal) is a glucoside compound isolated from traditional Chinese medicine Rhodiola rosea, which can inhibit a variety of cells apoptosis in other reports. In this study, we added salidroside and salidroside+PI3K inhibitor (LY294002) before clinorotation and determined apoptotic rate of HPMECs after 72h-clinostat by TUNEL method. We found that salidroside significantly reduced apoptotic rate. Western blot results showed that clinorotation inhibited the PI3K protein expression and phosphorylation level of AKT. Salidroside attenuated the decrease of PI3K protein expression and phosphorylation level of AKT, accompying with increased Bal-2/BAX ratio. PI3K inhibitor intervention can lower the expression of PI3K and phosphorylation level of AKT. When salidroside and PI3K inhibitor was used as intervention in comparison with salidroside intervention, apoptotic rate was increased and Bal-2/BAX ratio was decreased. Then we deduced that salidroside can reversed apoptosis induced by simulated microgravity because of the effect on PI3K/AKT pathway.Thirdly, simulated microgravity stimulates Nitric oxide synthesis and releasing for pulmonary microvascular endothelial cells in the previous results of our research team, which may be one of mechanism that simulated microgravity induced apoptosis. L-NAME can block Nitric oxide synthesis as a NOS inhibitor. Apoptotic rate was decreased by TUNEL method after L-NAME intervened. Besides that, we found that protein and gene expression correlated with endoplasmic reticulum (ER) stress declined. We concluded that Nitric oxide induced apoptosis through endoplasmic reticulum stress. Through this study, we drew the following conclusions:Microgravity simulated by clinorotation can induce human pulmonary microvascular endothelial cell apoptosis. Both mitochondrial signal transduction pathway and endoplasmic reticulum stress are involved in apoptosis. Salidroside and L-NAME intervention can inhibit apoptosis induced by simulated microgravity. Salidroside inhibit apoptosis through the PI3K/AKT pathway. NO induced apoptosis possibly through endoplasmic reticulum stress.