Reagents and antibodies
BPF (purity > 98%) was purchased from Selleck Chemicals (Houston, TX, USA). LPS derived from bacteria (serotype: 0111:B4, L5293) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-iNOS and anti‐β‐actin were obtained from Cell Signaling Technology (Beverly, MA, USA).
Primary human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex Bio Science (Charles City, IA, USA) and maintained as previously described . Cells were cultured in EBM-2 basal media (Cambrex Bio Science) at 37℃ under 5% CO2. THP-1 cells, derived from a human monocyte line, were purchased from the ATCC (Manassas, VA, USA) and cultured as previously described . Differentiation of THP-1 cells into macrophages was induced by incubating them in complete medium containing PMA (50 μM) for 48 h followed by incubation in complete medium without PMA for 48 h.
Male C57BL/6 mice (8-week-old, 27 ± 0.5 g) were purchased from Orient Bio Co. (Sungnam, Republic of Korea). All animal experiment protocols were approved by the Animal Care Committee at Kyungpook National University prior to conducting the study (IRB No. KNU 2020-107). Each animal sepsis model was established as below-
LPS-induced septic shock model: LPS (2.5 mg/kg) was administered to mice by intraperitoneal injection. Simultaneously, BPF was administered intravenously. For cytokine analysis, the mice were euthanized 4 h later. The blood and ascitic fluid were collected and centrifuged at 9500 g at 4 ° C for 10 min. The supernatant was used for cytokine analysis using ELISA. For lung histological analysis, the septic shock was extended to 24 h.
LPS-induced lethal septic shock model: Lethal dose of LPS (25 mg/kg) was administered to mice by intraperitoneal injection. Simultaneously, BPF was administered intravenously. Mouse survival was observed every 4 h for 36 h.
Cecal Ligation and Puncture (CLP) sepsis model: Mice were prepared as described previously . Sham operated animals underwent laparotomy without ligation and puncture of the cecum. BPF was injected intravenously and the mice were observed every 12 h for 96 h.
Murine peritoneal macrophage isolation
Mice were administered 3 mL of 3% thioglycolate by intraperitoneal injection. Three days later mice were euthanized. Peritoneal macrophages were collected and washed with PBS. Subsequently, they were seeded in 48-well plates at a density of 5 x 105 cells/mL.
Methyl-thiazolyl-tetrazolium (MTT) cell viability assay
The cultured HUVECs and THP-1-macrophages were incubated with BPF at the indicated concentrations in serum-free medium. After 48 h, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to estimate cell viability [14, 15].
Enzyme-linked immunosorbent assay (ELISA)
The activities of pp65, pp38, pJNK, and pERK cellular proteins were determined by using commercially available ELISA kits (Cell Signaling Technology). The levels of IL-6, TNF-α, CXCL1, and CXCL2 in the cell culture supernatants were determined by using ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer's instructions.
Quantitative real-time polymerase chain reaction (qPCR)
RNA was purified using TRIzol Reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) extraction. The purified RNA was reverse transcribed with a PX2 Thermal Cycler (Thermo Fisher Scientific) using 0.5 mg/ml of the oligo (dT)-adapter primer (Thermo Fisher Scientific) and M-MLV reverse transcriptase (Thermo Fisher Scientific) in a 20 μL reaction mixture. The primer sequences were as follows: IL-6; forward, 5′-GCA CTG GCA GAA AAC AAC CT-3′ and reverse, 5′-TCA AAC TCC AAA AGA CCA GTG A-3′. TNF-α; forward, 5′-CCC AGG GAC CTC TCT CTA ATC-3′ and reverse, 5′-ATG GGC TAC AGG CTT GTC ACT-3′. iNOS; 5′-TCC AGG AGG ACA TGC AGC AC3′ and reverse, 5′-CGC CCT TCC GCA GTT CT-3′. β-actin; forward, 5′-CCT GGC ACC CAG CAC AAT-3′ and reverse, 5′-GCC GAT CCA CAC GGA GTA CT-3′. Quantitative gene expression levels are normalized to the expression levels of β-actin.
Lysis buffer containing Nonidet P‐40, complete protease inhibitor cocktail (Roche, Mannheim, Germany), 2 mM dithiothreitol, and phosphatase inhibitor cocktail 2 (Sigma-Aldrich) was used to lyse the cells. The proteins in the treated cell lysate were separated using SDS-PAGE and transferred to nitrocellulose membranes. Transferred proteins were incubated with primary antibodies corresponding to each target protein and then with the relevant secondary antibodies.
Measurement of nitrite levels in cell culture supernatants
Conditioned cell culture supernatants were harvested and nitric oxide concentrations were determined using the Griess reaction assay.
Hematoxylin and eosin (H&E) staining
Five mice were used for histopathological analysis. Mice from LPS-induced septic shock model were euthanized and the left lobes of lungs were fixed in 10% neutral formalin for 24 h, followed by tissue processing and paraffin embedding. The paraffin blocks were sectioned into 2-μm thick sections and stained with H&E. Histopathological scoring was done by three experimental veterinary medicine experts using a random score index based on the degree of inflammatory cell infiltration and the extent of the injury area (0, normal; 1, mild; 3, moderate; 5, severe) with slight modifications from previous studies [16, 17].
Results are expressed as mean ± standard deviation (SD) of three independent experiments. Results were analyzed using ANOVA (one-way) followed by Tukey’s post-hoc tests and differences at p < 0.05 were considered significant. The Kaplan–Meier method was used to compare differences in survival outcomes following LPS- or CLP-induced sepsis experiments.