Effects of Ghrelin on iNOS-Derived NO Promoted LPS-Induced Pulmonary Alveolar Epithelial A549 Cells Apoptosis

Abstract

Background/Aims: In the process of abnormal apoptosis of pulmonary alveolar type II epithelial A549 cells in acute respiratory distress syndrome (ARDS), inducible nitric oxide synthase (iNOS) activity in the lung, nitric oxide (NO) production, and the level of protein S-nitrosylation were increased. However, the role of excessive NO production in sepsis-induced ARDS is controversial. Additionally, ghrelin is a growth hormone that exerts an inhibitory role in cell apoptosis. We examined the effect of NO and S-nitrosylation on apoptosis of A549 cells induced by Lipopolysaccharide (LPS) and molecular mechanism underlying the anti-apoptotic effect of ghrelin in this process. Methods: Flow cytometry and qPCR were used to detect lentiviral infection efficiency and iNOS gene level, respectively. Extracellular and intracellular NO levels were observed by Griess assay kit and DAF-FM DA. Mitochondrial transmembrane potential, apoptosis rate and SNO levels were determined by flow cytometry, Biotin-Switch method and immunofluoresence staining. The expression of iNOS, apoptotic proteins and JNK were assessed by immunoblot analysis. Results: The results showed about two times increase in iNOS expression and intracellular NO levels response to LPS exposure at 24 hours (P< 0.05), while not in extracellular NO levels. NO donors, S-nitroso-N-acetylpenicillamine (SNAP) significantly raised (36.7%, P< 0.05; 38.4%, P< 0.05; 41.8%, P< 0.05) extracellular NO levels without influencing the intracellular NO levels. LPS increased the apoptosis rate (42.4%±2.6% vs 2.8%±1%, P< 0.05) of A549 accompanied by increased Bax levels and decreased Bcl-2 levels through activating JNK signaling, which was reversed when we diminished the iNOS expression in A549 cells using lentiviral vectors encoding iNOS shRNA in the presence of LPS (24.8%±3.8% vs 42.4%±2.6%, P< 0.05). However, the apoptosis rate was increased when SNAP was added (38.8%±1.3% vs 24.8%±3.8%, P< 0.05). Furthermore, we investigated whether ghrelin exert a protective role against LPS-induced apoptosis and the potential mechanism involved in. Ghrelin alone appeared to decrease iNOS expression (32.3%, P< 0.05; 42.3%, P< 0.05), which showed no signifiant difference between LPS+ghrelin group and LPS group. However, this study showed that ghrelin decreased the intracellular NO production (38.9%, P< 0.05), protein S-nitrosylation levels (33.5%, P< 0.05), Bax protein expression (70.2%, P< 0.05), whereas increasing Bcl-2 protein expression (14.1%, P< 0.05) and mitochondrial transmembrane potential (∆ΨM) (20.7%, P< 0.05) in the presence of LPS. Conclusion: The data suggested that NO derived from iNOS induced by LPS stimulation exerts an important role in promoting apoptosis of A549 cells, and ghrelin abolished intracellular NO production and protein S-nitrosylation levels, abrogating the apoptosis of A549 cells partly through inhibiting mitochondrial-dependent pathways.

S-nitrosylation of the Peroxiredoxin-2 promotes S-nitrosoglutathione-mediated lung cancer cells apoptosis via AMPK-SIRT1 pathway

“…2b–e). Lipopolysaccharides (LPS), which could induce inducible NO synthase 19 and contribute NO production (Supplementary Fig. 2f), caused similar Caspase-3-mediated apoptosis as GSNO (Supplementary Fig.…”

Two-Step In Vitro Model to Evaluate the Cellular Immune Response to SARS-CoV-2

“…In the pathway to investigate the output of cellular responses on the alveolar epithelial lung cells (A549) after in vitro exposure to SARS-CoV-2 antigens, the nitric oxide synthase (iNOS) and caspase-3 expression by A549 were assessed aiming at evaluating if their expression can be regulated by cytokines among other mediators produced by infected cells [ 66 , 67 , 68 , 69 , 70 ]. The antiviral activity in supernatants provided alterations in the alveolar epithelial lung cells (A549) during our in vitro assay.…”

Long non-coding RNA MALAT1 exacerbates acute respiratory distress syndrome by upregulating ICAM-1 expression via microRNA-150-5p downregulation

“…The primer sequences (Table 1) for MALAT1, miR-150-5p, ICAM-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). The relative expression of the target gene was calculated using the 2 -ΔΔCt method with GAPDH serving as the internal reference [42][43][44][45].…”