ABSTRACT
Objective To characterize exosomes released from human umbilical vein endothelial cells (HUVECs) after heatstroke, and explore their effects on biological behaviors of cells. Methods HUVECs were cultured and divided into the control group and the heat shock group. The heat shock group cells were exposed at 41 ℃ for 2 hours; the control group cells were incubated at 37 ℃ for 2 hours. The characteristics of exosomes secreted by the two groups were analyzed by nanoparticle tracing. The expression of exosomes marker proteins, CD63 and TSG101, were detected by Western blotting. The process of exosomes entering normal cells was observed by fluorescence labeling. To further explore the potential function of exosomes secreted from the heat-shocked cells, cells were co-cultured with exosomes collected from cells cultured at 37 ℃ or cells cultured at 41 ℃. Cells under normal culture conditions were served as a negative control (blank control). FACS was used to detect cell cycle and apoptosis; MTS was used to evaluate cell proliferation; ELISA was used to quantify coagulation related factor expression; high-throughput sequencing was used to profile microRNAs (miRNAs) in the exosomes. Results Different from the control group, the cells in 41 ℃ heat shock group secreted abnormal exosomes, which resulted in abnormal behaviors and functions of normal HUVECs. The expression of tissue factor, von Willebrand factor, plasminogen activator inhibitor-1 increased significantly (P<0.05), and the expression of tissue plasminogen activator decreased (P<0.05). Compared with the incubation of HUVECs at 37 ℃, there were 2590 miRNA regulatory changes in exosomes secreted by HUVECs after heat shock at 41 ℃, which had obvious effects on apoptosis. Conclusions Heat shock at 41 ℃ could induce the release of abnormal exosomes from HUVEC, leading to abnormal behavior and potential coagulation of normal cells.
ABSTRACT
Objective To observe the enzymatic changes of myocardial sarcoplasmic reticulum in rats after injecting endotoxin (LPS), and provide basic research results for the future study of myocardial sarcoplasmic reticulum dysfunction caused by LPS in rats. Methods Ten SD rats were randomly divided into blank control group and LPS injection group with 5 rats in each group. In the LPS injection group, endotoxin was injected into the tail vessels of the rats. Results The heart rate (HR) of the LPS injection group increased and was faster than that in the blank control group [(204±18) beat per min vs. (139±10) beat per min on the first day, and (199±22) beat per min vs. (143±17) beat per min on the next day, both P values were less than 0.05]. The mean arterial pressure (MAP) was lower than that of the blank control group on the first day [(87±12) mmHg vs. (102±7) mmHg, P<0.05]. Under light and electron microscope, the myocardial cells of rats with LPS injection were loosely arranged, with intercellular infiltration with inflammatory cells, muscle fibers broken, and difficult to identify the morphology of mitochondria and sarcoplasmic reticulum. Quantitative PCR results showed that after endotoxin injection, troponin (CASQ1), sodium-calcium exchanger (NCX), calmodulin phosphatase 1 (ppplCa), phospholipid protein (PLN), sarcoplasmic reticulum Ca2+-ATPase (SERCA2) increased significantly (P<0.05). Conclusion Endotoxin can inhibit cardiomyocyte function by affecting the activity of sarcoplasmic reticulum calcium regulatory protein-related enzymes through various mechanisms.