Figure 1.
Effect of PAF on the survival rate of LPS-induced endotoxemic mice.
(A) Survival of mice i.p. with varying doses of PAF (0.1 to 5 µg/mouse) following LPS challenge (20 mg/kg,) was monitored for 6 d. Shown is one of six experiments with similar results. (B) Treatment with PAF-R antagonist, BN-52021 (80 µg/mouse, i.p), blocked the protective effect of PAF (2.5 µg/mouse) against lethality. (C) Delayed treatment of PAF (5 µg/mouse) had no protective effect against LPS-induced lethality. (D) Effect of cPAF (1 µg/mouse) on survival. N = 8–10 mice per group. *P<0.05; **P<0.001 compared with LPS challenge.
Figure 2.
Effects of PAF on LPS-induced inflammatory cytokine production.
(A) Mice were injected with vehicle alone (PBS containing 0.25% BSA), or LPS (20 mg/kg, i.p.), or PAF (0.1–5 µg/mouse) immediately following LPS challenge. Blood was collected at 2 h or 6 h (for INF-γ measurement) after injection. PAF treatment altered the serum level of different cytokines in response to LPS in a dose-dependent manner. The mean±SEM values shown represent three independent experiments. *P<0.05; **P<0.001 compared with LPS challenge. (B) Peritoneal macrophages were isolated 3 d following i.p. injection with 2 ml of 4% thioglycollate. Macrophages were then incubated with LPS alone (100 ng/ml) or a combination of LPS and PAF (0.4 µM) for 3 h or 6 h. (C) BMDCs were generated from murine bone marrow cells as described in the Materials and Methods section. BMDCs were stimulated with LPS(100 ng/ml) in the presence of PAF (0.4 µM) for the indicated times. Cytokines concentrations in culture supernatants were measured in triplicate by ELISA. The mean±SEM values shown represent three independent experiments. *P<0.05; **P<0.001.
Figure 3.
Effect of PAF treatment on organ injury induced by LPS.
(A) Lung sections, as well as sera, were obtained from mice 20 h after treatment with vehicle alone, PAF (5 µg/mouse), LPS (10 mg/kg) or LPS plus PAF. Lungs were perfused and fixed with formalin, and sections of lung were stained with hematoxylin and eosin. Shown are representative images of lung sections from each group of mice. (B) The MPO level in the liver, lung, and kidney from each group of mice was assessed 20 h post-injection. (C) The amounts of AST, ALT, and BUN in the sera were measured. The mean±SEM values shown represent three independent experiments. *P<0.05; **P<0.001. (D) Changes in MABP were measured in animals injected with PAF (5 µg/mouse, i.p.), LPS (10 mg/kg, i.p.), or PAF plus LPS. The data points indicate the mean MABP of mice for the 10 min. Results are expressed as a percentage of baseline±SEM. The baseline was set to the mean value over a 10 min period before injection at time 0. The basal MABP of mice were 76.2±5.7 mmHg. Numbers in parentheses are the numbers of animals alive at the indicated times. MABP of dead mice was taken to be zero. (E) PAF inhibits nitric oxide production in LPS-challenged mice. Blood samples were collected 20 h after treatment with vehicle alone, PAF (5 µg/mouse), LPS (10 mg/kg) or LPS plus PAF, and serum nitrite levels were measured by chemiluminescence assay. Data are presented as the mean±SEM of five independent experiments. *P<0.001 compared with LPS-challenged mice group.
Figure 4.
Exogenous PAF prevents lymphocyte apoptosis in LPS-induced endoxemia.
BALB/c mice (7 wk old, n = 3) were injected with either vehicle alone, PAF (5 µg/mouse, i.p.), LPS (10 mg/kg, i.p.), or both and sacrificed after 20 h. (A) Apoptotic cells fluorescently labeled using the TUNEL assay. Fixed paraffin-embedded spleens were labeled using the TUNEL assay to detect apoptotic cells and then examined by laser scanning confocal microscopy at magnifications of×100. (B) Hematoxylin and eosin staining, which illustrated the morphological changes (compacted and fragmented nuclei) in cells undergoing apoptosis, revealed splenocytes from endotoxic mice underwent extensive apoptosis while spleen from endotoxic mice treated with PAF did not. (C) Flow cytometric analysis of apoptosis. Splenocytes from each group of mice were examined for apoptosis by flow cytometry using Annexin V staining. T and B cells were distinguished by staining with PE-conjugated anti-CD3 and anti-CD19 Abs, respectively. LPS-induced endotoxemia increased apoptosis in both cell types, while PAF protected them against endotoxemia-induced cell death. The mean±SEM values shown represent three or four independent experiments. *P<0.05 compared with LPS-challenged mice group.