Vasoactive Intestinal Peptide Protects Alveolar Epithelial Cells Against Hyperoxia Via Promoting The Activtion Of STAT3

Partâ… Hyperoxia on Typeâ…¡Alveolar Epithelial Cells SurvivalObjective To observe the death and proliferation in typeâ…¡alveolar epithelial cells (AECâ…¡) exposed to hyperoxia and understand the effect of hyperoxia on AECâ…¡survival.Methods MLE-12 cells were exposed to hyperoxia (>90% oxygen) at different lengths of time. The early apoptosis or late apoptosis and necrosis were measured by flow cytometry. Activity of caspase-3 was analyzed by microplate spectrophotometer. The morphological changes of cell nucleus and chromatin were studied after DAPI stain by Fluorescence microscopy. The ultrastructural transformations of mitochondria were observed by transmission electron microscope (TEM) and mitochondrial membrane potential was evaluated by fluorescent dye JC-1 to understand mitochondrial function and cell damage. The proliferation of AECâ…¡was detected by MTT cell proliferation assay,Results Compared with air control, there is no difference in early apoptosis or late apoptosis and necrosis after 0.5 h, 1h, 2 h, 4 h hyperoxia exposure, but early apoptosis or late apoptosis and necrosis increased notably after hyperoxia exposure for 6 h (p< 0.01) and more cells exposed to hyperoxia were in apoptosis or necrosis at 12 h and 24 h (p< 0.01), dead cells mostly concentrated in the right upper quadrant on behalf of late apoptosis and necrosis. Caspase-3 activity indicated no significant difference between the air control cells and cells exposed to hyperoxia for 24 h. As seen in air group, the morphology of cell nucleus and chromatin had no obvious changes after 0.5 h,1 h,2 h, 4 h hyperoxia exposure. When the exposure lasted 6 h, 12 h and 24 h, we observed the swelled nuclear but no nuclear condensation and chromatin aggregation. MTT showed that, with the same point of the air control, OD_490nm didn't change significantly at 0.5 h, 1 h, 2 h, 4 h exposed to hyperoxia, the downtrend in OD_490nm was obvious gradually with time after 6 h exposure (p<0.01). The cell ratio of G₀/G₁,S,G2 / M phases showed no significant difference between 0.5 h, 1 h, 2 h, 4 h hyperoxia exposure and the air control and most of the cells distributed in S or G₂/M of DNA synthesis and mitosis. The cells in G₀/G₁ were markedly increased while the cells in S,G2/M were observably decreased after 6 h exposure and most of the cells distributed in the G₀/G₁ phase of prophase and quiescence.Conclusion Hyperoxia could induce injury and death in AECâ…¡and inhibit cell proliferation in time-dependent manner, leading to the ruin in survival. Exposed to 90% concentration oxygen, AECâ…¡manifested the characteristics of oncosis such as destruction of the cell membrane, nucleus and mitochondrial swelling and there was no phosphatidylserine translation, nuclear condensation, chromatin aggregation and caspase protease activating of apoptosis.Partâ…¡Vasoactive intestinal peptide on repair and regeneration of hyperoxic typeâ…¡alveolar epithelial cellsObjective To observe the effect of VIP on cell injury, death and proliferation in AECâ…¡exposed to hyperoxia and explore the consequence of VIP on repair and regeneration of AECâ…¡after hyperoxia-induced injury.Methods MLE-12 cells were exposed to hyperoxia (>90% oxygen) and VIP. The cell proliferation was detected by MTT cell proliferation assay. The early apoptosis or late apoptosis and necrosis were measured by flow cytometry, the morphological changes of cell nucleus and chromatin were studied after DAPI stain by Fluorescence microscopy and mitochondrial membrane potential was evaluated by fluorescent dye JC-1 to learn the effect of VIP on hyperoxia-induced cell death.Results OD_490nm of MLE-12 after 24h hyperoxia treatment was significantly reduced compared with the air control group (p<0.05). OD_490nm was increased with the concentration-increase of VIP, and reached to the maximum activity at the concentration of VIP 10^-7M. 24 h after hyperoxia exposure, the number of MLE-12 labeled as Annexin V+/PI- and Annexin V+/PI+ was significantly decreased in the presence of VIP 10^-7M compared with those in the hyperoxia alone group(p<0.01). Dead cells with hyperoxia and additional VIP mostly concentrated in the right upper quadrant on behalf of late apoptosis and necrosis. 10^-7M VIP administration could repress the decline of mitochondrial membrane potential induced by hyperoxia (p<0.01). DAPI stained and observed under fluorescence microscope, the nucleus still swelled and there was no nuclear condensation and chromatin aggregation after hyperoxia and 10^-7 M VIP interference. VIP10^-7M hyperoxia group compared with hyperoxia alone, cells in G₀/G₁ phase decreased significantly (p<0.01) and cells in S phase, G₂/M phase was markedly increased (p<0.01), but both less than the air control group.Conclusion VIP could promote cell proliferation of AECâ…¡exposed to hyperoxia in dose-dependent manner and reduce hyperoxic cell injury and death by stabilizing mitochondrial function to improve AECâ…¡survival. However, hyperoxia-exposed cells still showed membrane damage, and nuclear swelling of oncosis after VIP intervention, suggesting that VIP could affect oncosis induced by hyperoxia and oncosis may be regulated as apoptosis.Partâ…¢Vasoactive intestinal peptide promote repair and regeneration of typeâ…¡alveolar epithelial cells under hyperoxia via STAT3 signaling mechanismObjective By analyzing the activation of JAK2/STAT3 after hyperoxia and VIP intervention and the effect of VIP on cell proliferation and death in STAT3-knockdown cells exposed to hyperoxia, the possible signaling mechanism of VIP regulation was to be discussed.Methods MLE-12 cells were random to hyperoxia (>90% oxygen) and VIP treatment with or without STAT3 siRNA transfection. The activation of JAK2/STAT3 was assessed by Western Blot detection of phosphorylated JAK2/STAT3 and Electrophoretic Mobility Shift Assay (EMSA) of STAT3 DNA banding activity. The cell proliferation was detected by MTT cell proliferation assay. The cell death was measured by flow cytometry.Results The JAK2 phosphorylation (p-JAK2) and STAT3 phosphorylation (P-STAT3) induced by hyperoxia stimulation occurred at 2 h and persisted 6 h and the proteins were expressed best at 4 h, and the increase in p-JAK2 or p-STAT3 was not due to enhanced protein expression. There was an increase in p-STAT3 as exemplified by change on the p-STAT3/STAT3 ratio after hyperoxia exposure with VIP 10^-7M in comparison with hyperoxia alone (p<0.05), but the time points of STAT3 phosphorylation couldn't make a difference, while p-JAK2 expression was no alteration even in the presence of VIP, and similar results were obtained by EMSA. Compared with hyperoxia exposure alone, additional VIP treatment promoted cell proliferation and reduced the cell death (p<0.01), but the protective effects aforesaid were weakened after STAT3 expression was down regulated by siRNA. Cells with STAT3 siRNA transfection had a lower proliferation compared with wild-type cells after hyperoxia exposure with VIP administration (p<0.01).Conclusion VIP would be a regulator in AECâ…¡regeneration after hyperoxia-induced injury, which might be associated with the promotion of STAT3 activation. However, the activation of STAT3 by VIP was distinct from the activation of the classical JAK2/STAT3 pathways without affecting the activity of JAK2.