Autophagy Attenuates MnCl2-induced Apoptosis in Human Bronchial Epithelial Cells

Objective To investigate the role of autophagy in MnC l2-induced apoptosis in human bronchial epithelial 16 HBE cells.Methods Cell proliferation was measured by MTT assay.Mitochondrial membrane potential（MMP） and apoptosis were measured by flow cytometry.Autophagic vacuoles were detected by fluorescence microscopy.Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting.Results 16 HBE cell proliferation was inhibited by Mn Cl2 in a dose-and time-dependent manner.Mn Cl2-induced 16 HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis.Our data revealed that Mn Cl2-induced apoptosis in 16 HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3.It was observed that when we exposed 16 HBE cells to MnCl2 in a dose-dependent manner,the formation of autophagic vacuoles and the levels of LC-3B-II were elevated.RNA interference of LC3 B in these Mn Cl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced.Additionally,the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis,but did not affect the cellular levels of LC3 B in Mn Cl2-treated 16 HBE cells.Conclusion Mn Cl2 dose-and time-dependently inhibits 16 HBE cell proliferation and induces MMP loss and apoptosis.Autophagy acts in a protective role against Mn Cl2-induced apoptosis in 16 HBE cells.

Connexin43 Modulates X-Ray-Induced Pyroptosis in Human Umbilical Vein Endothelial Cells

Objective Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43(Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells(HUVECs). Methods HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay(FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively. Results Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with si RNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1(Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with si RNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown. Conclusion Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1.

S Phase Cell Percentage Normalized BrdU Incorporation Rate, a New Parameter for Determining S Arrest

In this study, a new parameter, S phase cell percentage （S fraction） normalized BrdU （SFN-BrdU） incorporation rate, was introduced to detect $ arrest. The results showed a positive linear correlation between the BrdU incorporation rate and the S fraction in unperturbed 16HBE cells. Theoretical analysis indicated that only S arrest could result in a decrease in the SFN-BrdU incorporation rate. Additionally, the decrease in SFN-BrdU incorporation rate and the activation of DNA damage checkpoints further demonstrated that S arrest was induced by diethyl sulfate treatment of 16HBE cells. In conclusion, $FN-BrdU incorporation rate can be used to detecting S arrest.