3.9. In Vivo Anti-Inflammatory Experiments

All animal experiments and processes were fulfilled with the approval of the Institutional Animal Care and Use Committee (IACUC) of Sunchon National University, Republic of Korea (SCNU IACUC-2019-10). Seven-week-old female Balb/c mice (Koatech Co., Pyongtaek, Gyeonggi-do, Republic of Korea) were anesthetized by inhalation of 5% (induction) and 2% (maintenance) isoflurane in pure oxygen and LPS (0.02 µg) was intradermally injected into the mice ears. After 1 h, the indicated peptides (32 µM) were injected, followed by further incubation for 3 days. The ears were digitally examined and collected for staining using hematoxylin and eosin (H&E). Histological observations using H&E staining were performed according to the following processes.

P. aeruginosa LPS (500 ng) in 50 µL PBS was intratracheally instilled into seven-week-old female Balb/c mice. One hour after exposure, each peptide was intratracheally administered at the indicated dose in 50 µL PBS. Control mice were injected with 50 µL PBS without the peptides. Mice were monitored for signs of morbidity for 3 days after LPS exposure. Mice were euthanized using CO₂ inhalation, and the lung tissues were excised and fixed in 4% paraformaldehyde. The fixed tissues were dehydrated using a series of ethanol solutions (50−100%) and embedded in paraffin. The tissues embedded with paraffin were sectioned with a thickness of 5 µm (Leica microtome, Deerfield, MA, USA). Histopathology of the lung tissues was performed using alcian blue and eosin and H&E staining. An immunohistology was performed using monoclonal antibodies of PE-conjugated anti-IL-6 and anti-TNF-α (BioLegend, San Diego, CA, USA). The stained tissue sections were observed under a fluorescence microscope (OPTINIT KCS3-160S; Korea Lab Tech, Seongnam, Gyeonggi-do, Republic of Korea).