Extraction and Biochemical Characterization of Alligator mississippiensis glycosaminoglycans and an Ex-vivo Murine Pilot Study to Test their Potential Effect on a Selected Panel of Genes Associated with Cystic Fibrosis

Cystic fibrosis (CF) is a genetic disorder with a median survival age of 40.7 years. Chronic airway inflammation and dehydration are critical features of CF. The size and structure-dependent hydration and anti-inflammatory properties of glycosaminoglycans (GAGs) such as hyaluronic acid (HA) may help ameliorate these symptoms. The GAGs contained in farmed Alligator mississippiensis by-products offer a potential to improve waste management practices and increase revenue in the alligator industry, through their development for use in biomedical applications. This study aimed to efficiently extract and characterize GAGs from alligator carcasses (ACS), backstraps (ABS), feet (AFT) and eyeballs (AEB), to evaluate their effects on CF-like airways ex-vivo. Samples were collected from local alligator processing facilities and extractions were conducted according to a randomized complete block design. The contents of sulfated and non-sulfated GAGs were determined using dimethylmethylene blue and HA ELISA assays, respectively. Total GAG content was confirmed by the carbazole reaction assay. Protein content, mineral content and molecular weight (MW) were determined by the bicinchoninic acid assay, inductively coupled plasma and 0.75% agarose electrophoresis, respectively. FTIR spectroscopy was conducted to fingerprint the structures of the extracted GAGs. Multi-step processes for the extraction of GAGs from alligator by-products were developed and ABS had the highest (p≤0.05) total GAG content (15.53±0.27 mg/g), followed by AFT (4.72±0.05 mg/g), AEB (0.79±0.01 mg/g) and ACS (0.60±0.00 mg/g). These results are equivalent to ~2.13 g GAGs per harvest-size farmed alligator or an estimated GAG production of ~0.73 tons/year in Louisiana. The GAGs in all samples were predominantly (\u3e97%) HA with a poly-disperse MW of up to 1600 kDa. FTIR revealed spectra showing characteristic GAG features such as -OH, -NH, -CH and amide signals from residual protein, comparable to previous studies on animal-sourced GAGs. Ex-vivo gene expression analysis of Scnn1b-Tg mice tracheal epithelial cells indicated that AEB GAGs (0.5 mg/12 mm insert) have the potential to regulate the expression of genes which may aid in restoring the protease/anti-protease balance, reducing inflammation, and regulating ASL osmotic homeostasis. Alligator GAGs may also aid in the treatment of other inflammatory conditions such as wound healing and arthritis in humans and animals