(P30-034-23) Melatonin Attenuates Cigarette Smoke Extract-Induced Inflammation and Autophagy

Objectives: Cigarette smoking and other nicotine-based products cause chronic lung inflammation. Despite improved therapeutic options, the prognosis of cigarette smoke (CS) induced inflammation and associated pathologies have not changed significantly. Diet supplementation strategies are alluring because the implementation is relatively easy even in population groups with limited income and resources. Though N-acetyl-5-methoxy-tryptamine (melatonin) is produced by the pineal gland and other human tissues, its role as a dietary supplement is being studied in various experimental models of inflammatory diseases. Based on earlier studies, we hypothesize that melatonin supplementation can rescue CS-extract (CSE)-induced inflammation and autophagy process in lung epithelial cells.

Methods: We used human type II alveolar epithelial cells (A549) and pre-treated them with melatonin (0.25 µM) for 1 hour prior to CSE (250 ng/ml; 24h)-challenge. We performed ELISA for cytokine/chemokine production, qPCR, and immunoblotting for expression profile. Autophagy Flux Assay to detect autophagosomes and machine learning approach using Software FIJI- Image J plugin to count autophagosomes.

Results: Our results showed increased production of cytokine/chemokine (IL-6, IL-8, CCL2, and CCL5); expression of DAMPs (HMGB1 and Hsp70); inflammatory mediators (NF-kB, STAT3, and p38 MAPK); autophagy-related proteins (Beclin1 and LC3B) and autophagy flux; expression of NADPH oxidase subunits (p22^phox, p47^phox, p67^phox, and gp91^phox); and antioxidants (MnSOD, Catalase and GPx)-in CSE-challenged A549 cells. Interestingly, pretreatment with melatonin was able to rescue CSE-induced inflammatory responses and autophagy flux in A549 cells. It was critical to observe that the rescue effect of melatonin on CSE-induced inflammation and autophagy was almost of similar magnitude when compared with ROS inhibitor [N-Acetyl cysteine (NAC)] or autophagy inhibitor [3-Methyladenine (3-MA)].

Conclusions: Overall, our findings demonstrate the important role of Melatonin in CSE-induced inflammatory responses. Further studies are in progress to delineate the molecular mechanisms in detail.

Funding Sources: USDA-NIFA-CBG Award to SB, Young Clinical Scientist Award (FAMRI) to SB, Institutional Development Award (IDeA) from National Institutes of Health to SB.