Porcine elastase

The amidolytic activity was determined using 50 μg of venom protein, to measure trypsin-like, chymotrypsin-like, and elastase-like activities using BApNA, SAAPFpNA, and 10 mM N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) in dimethylsulfoxide as substrates, respectively. The mixture was brought to a final volume of 120 μL with 0.1 M Tris (pH 8) buffer and 10 μL of substrate was added to start the reaction. Activity was determined by the release of p-nitroanilide that absorbs at 405 nm, reporting as activity units (AU)/min/µg venom protein. One AU corresponded to an increase of absorbance of 0.01 at 405 nm [68 (link)]. Analyses were done in triplicate. In each case, bovine chymotrypsin, bovine trypsin and porcine elastase (Sigma-Aldrich) were used as positive controls.

C57BL/6 J mice were purchased from Orientbio (Seongnam, Korea). The mice were bred in specific pathogen-free facilities at Asan Medical Center. Female C57BL/6 J mice (7 weeks of age) were exposed to commercial cigarette smoke based on the protocol used in our previous report^{22 (link)}.
Briefly, mice were exposed to smoke for 1 day, 4 days, and 6 months (5 days/week) in an inhalation box (50 × 40 × 30 cm) connected to a pump to assess changes in FGF-2 levels. To evaluate the effect of short-term smoke exposure on the mice, mice were exposed to smoke for 4 days, and 0.1, 10, or 1,000 pg of rFGF-2 was intranasally administered to each mouse immediately after smoke exposure on each day to define the effects of rFGF-2 on smoke exposure. After completing the 4-day smoke exposure with or without rFGF-2, the mice were killed to collect their lung tissues on day 5.
For the elastase-induced mouse model, 0.4 U of porcine elastase (Sigma-Aldrich, St. Louis, MO, USA) was intratracheally injected into C57BL/6 mice on day 0. Then, rFGF-2 (10 pg) was administered intranasally once daily from days 7–13, and PD173074 was intraperitoneally injected once per day during the same period. The mice were killed to collect their lung tissues on day 14.
All mouse experiments were approved by the Institutional Animal Care and Use Committee of Asan Medical Center (South Korea).

Prolastin^® powder for infusion, containing 1000 mg of human plasma-purified AAT (Grifols, Barcelona, Spain, Batch G45BE00351) was divided into aliquots and dissolved in ultrapure water produced by an Arium^® purification system (Sartorius, Goettingen, Germany). A 25 mM potassium phosphate buffer solution was prepared by dissolving 2.336 g of K₂HPO₄ and 1.577 g of KH₂PO₄, both from A.C.E.F. (Fiorenzuola, Italy), in 1 L of ultrapure water. The Bradford assay kit was from Bio-Rad Laboratories (Milan, Italy). Porcine elastase was from Sigma-Aldrich (St. Louis, Missouri, USA). All other materials were from Sigma-Aldrich, unless otherwise stated.