Cigarette smoking is the major cause of chronic inflammatory diseases such as Chronic Obstructive Pulmonary Disease (COPD). It is paramount to develop pharmacological interventions and delivery strategies against the cigarette smoke (CS) associated oxidative stress in COPD. This study in Wistar rats examined cysteamine in nanoemulsions to counteract the cigarette smoke distressed microenvironment. In vivo, 28 days of cigarette smoke and 15 days of cysteamine nanoemulsions treatment starting on 29th day consisting of oral and inhalation routes were established in Wistar rats. Additionally, we conducted inflammatory and epithelial-to-mesenchymal transition (EMT) studies in vitro in human bronchial epithelial cell lines (BEAS2B) using 5% cigarette smoke extract. Inflammatory and anti-inflammatory markers such as TNF-α, IL-6, IL-1ß, IL-8, IL-10, IL-13, have been quantified in bronchoalveolar lavage fluid (BALF) to evaluate the effects of the cysteamine nanoemulsions in normalizing the diseased condition. Histopathological analysis of the alveoli and the trachea showed the distorted, lung parenchyma and ciliated epithelial barrier, respectively. To obtain mechanistic insights into the cigarette smoke COPD rat model, “shotgun” proteomics of the lung tissues have been carried out using high-resolution mass spectrometry wherein genes such as ABI1, PPP3CA, PSMA2, FBLN5, ACTG1, CSNK2A1, and ECM1 exhibited significant differences across all the groups. Pathway analysis showed autophagy, signaling by receptor tyrosine kinase, cytokine signaling in immune system, extracellular matrix organization, and hemostasis, as the major contributing pathways across all the studied groups. This work offers new preclinical findings on how cysteamine taken orally or inhaled can combat cigarette smoke-induced oxidative stress.

In this study, cysteamine nanoemulsions were prepared using the high speed homogenization (HSH) technique and characterized for size, charge and encapsulation efficiency using cryo-HRTEM, Zetasizer Nano ZS and LC-MS/MS, respectively. Prepared nanoemulsions were tested in vitro for cellular uptake, inflammatory and EMT assays in A549 and BEAS2B cell lines, respectively. In vivo, cigarette smoke model was established in Wistar rats for 28 days. A total of 28 Wistar rats were divided into six groups, normal control, diseased control and four treatment groups were evaluated namely plain cysteamine given orally, cysteamine nanoemulsions given orally, cysteamine nanoemulsions given via inhalation, and finally, eugenol nanoemulsions given via inhalation. Treatment was started on 29th day and continued for 15 days. On 44th day, all the rats were sacrificed for the biological examination of the BALF, serum, and lung tissues. Further, proteomics analysis was carried out to further study the proteins altered under the exposure of CS and different treatment strategies. Finally, bioinformatics approaches were adopted to screen the significant proteins and the pathways playing significant role in CS exposed rats and different treatment modalities. Significant proteins were further validated using the multiple reaction monitoring (MRM) assay.

A total of 84 runs were performed corresponding to 28 samples against three transition lists in TSQ Altis for MRM assay belonging to normal control (NC; n=3), COPD control (DC; n=5), plain cysteamine given orally (CPO; n=5), cysteamine nanoemulsion given orally (CFO; n=5), cysteamine nanoemulsion given via inhalation (CFI; n=5), and eugenol nanoemulsion given via inhalation (EFI; n=5). After data acquisition, all samples were analysed in skyline. msms file from discovery proteomics (performed in thermo orbitap fusion) and mqpar file from maxquant, were used to build the library in skyline.