Cigarette Smoke Extract and Lipopolysaccharides Induce Pyroptosis in Rats Pulmonary Microvascular Endothelial Cells

Objective: To investigate the effects of cigarette smoke extract (CSE) and lipopolysaccharide (LPS) on the activity and pyroptosis of pulmonary microvascular endothelial cells (PMVECs). Methods: PMVECs were cultured without treatment or with CSE (1%-25%), LPS, or CSE+LPS. Cell viability was detected using the CCK8 method. Apoptosis was evaluated by flow cytometry. Cell morphology was evaluated using optical microscopy. The content of IL-1β and IL-18 was measured by ELISA. Results: CSE decreased cell viability in a dose-dependent manner. The cells in the CSE+LPS group showed the most obvious cytomorphological changes and the highest pyroptosis rate under the microscope. Flow cytometry showed that the CSE and LPS groups showed higher apoptosis rates than the blank group; the apoptotic rate in the CSE+LPS group was even higher (P<0.01). Compared with the bkank group, the levels of IL-18 and IL-1β in the cell supernatant of the CSE, LPS, and CSE+LPS groups increased significantly, with significant differences (P<0.01). There were no differences between the CSE and LPS groups (P>0.05). Compared with the CSE and LPS groups, the CSE+LPS group had higher IL-18 and IL-1β (P<0.01). Conclusion: The effect of CSE on cell viability is dose-dependent. CSE+LPS can induce cell pyroptosis and increase the levels of inflammatory cytokines in PMVECs. These observations demonstrated that pyroptosis caused by CSE and LPS might play an important role in pulmonary vascular remodeling.

Expression of Zinc-Finger Antiviral Protein in hCMEC/D3 Human Cerebral Microvascular Endothelial Cells: Effect of a Toll-Like Receptor 3 Agonist

<b><i>Introduction:</i></b> Invasion of viruses into the brain causes viral encephalitis, which can be fatal and causes permanent brain damage. The blood-brain barrier (BBB) protects the brain by excluding harmful substances and microbes. Brain microvascular endothelial cells are important components of the BBB; however, the mechanisms of antiviral reactions in these cells have not been fully elucidated. Zinc-finger antiviral protein (ZAP) is a molecule that restricts the infection of various viruses, and there are 2 major isoforms: ZAPL and ZAPS. Toll-like receptor 3 (TLR3), a pattern-recognition receptor against viral double-stranded RNA, is implicated in antiviral innate immune reactions. The aim of this study was to investigate the expression of ZAP in cultured hCMEC/D3 human brain microvascular endothelial cells treated with an authentic TLR3 agonist polyinosinic-polycytidylic acid (poly IC). <b><i>Methods:</i></b> hCMEC/D3 cells were cultured and treated with poly IC. Expression of ZAPL and ZAPS mRNA was investigated using quantitative reverse transcription-polymerase chain reaction, and protein expression of these molecules was examined using western blotting. The role of nuclear factor-κB (NF-κB) was examined using the NF-κB inhibitor, SN50. The roles of interferon (IFN)-β, IFN regulatory factor 3 (IRF3), tripartite motif protein 25 (TRIM25), and retinoic acid-inducible gene-I (RIG-I) in poly IC-induced ZAPS expression were examined using RNA interference. Propagation of Japanese encephalitis virus (JEV) was examined using a focus-forming assay. <b><i>Results:</i></b> ZAPS mRNA and protein expression was upregulated by poly IC, whereas the change of ZAPL mRNA and protein levels was minimal. Knockdown of IRF3 or TRIM25 decreased the poly IC-induced upregulation of ZAPS, whereas knockdown of IFN-β or RIG-I did not affect ZAPS upregulation. SN50 did not affect ZAPS expression. Knockdown of ZAP enhanced JEV propagation. <b><i>Conclusion:</i></b> ZAPL and ZAPS were expressed in hCMEC/D3 cells, and ZAPS expression was upregulated by poly IC. IRF3 and TRIM25 are involved in poly IC-induced upregulation of ZAPS. ZAP may contribute to antiviral reactions in brain microvascular endothelial cells and protect the brain from invading viruses such as JEV.