Figure 1.
SIN attenuates lung damage at 24 hour after acute lung injury in a dose-dependent manner.
One hour before LPS injection, 0, 30, 60 or 120 mg/kg SIN was administrated to mice. At 24 hour after ALI, lung damage was assessed. The mice intratracheally treated with 40 µl PBS were served as the control. (A) Lung water content. (B) PaO2/FIO2 (P/F) ratio. (C) HE staining for histopathological changes in lung tissues. * p<0.01 compared to the injured group without SIN treatment (0 mg/kg SIN treatment); # p<0.01 compared to the two indicated groups; NS: no significant difference between the two groups. (n = 8∼10 mice per group).
Figure 2.
SIN inhibits neutrophil infiltration at 24 hour after acute lung injury in a dose-dependent manner.
Neutrophil infiltration into lung tissue was evaluated at 24 hour after acute lung injury by immunofluorescence using a CD177 primary antibody and a FITC-conjugated secondary antibody. Mice treated intratracheally with 40 µl PBS served as controls. (A) CD177-positive cells in the lung of control injured mice. (B) Cell counting and statistical analysis. * p<0.01 compared to the injured group without SIN treatment (0 mg/kg SIN treatment); # p<0.01 compared to the two indicated groups; NS: no significant difference between the two groups. (n = 8∼10 mice per group).
Figure 3.
SIN suppresses acute lung injury-induced TNF-α and IL-1β expression levels in a dose-dependent manner.
The protein expression levels of TNF-α and IL-1β in injured murine lung tissue were assayed at 24 hour post-acute lung injury by ELISA. Mice treated intratracheally with 40 µl PBS served as controls. (A) TNF-α protein levels. (B) IL-1β protein levels. * p<0.01 compared to the injured group without SIN treatment (0 mg/kg SIN treatment); # p<0.01 compared to the two indicated groups; NS: no significant difference between the two groups. (n = 8∼10 mice per group).
Figure 4.
A2AR expression in lung tissues is upregulated by SIN.
Expression profiles of murine lung tissues with or without SIN treatment were assayed by microarray and validated by real-time PCR. (A) Expression profiles in the murine lung tissue. The selected genes showed different expression patterns for the mice either with or without SIN treatment. LPS: LPS-induced ALI mice without SIN treatment; LPS+SIN: LPS-induced ALI mice pre-treated with 120 mg/kg SIN (n = 4 mice per group for microarray). (B) Relative A1R, A2AR, A2BR and A3R mRNA expression levels. Mice treated intratracheally with 40 µl PBS served as controls. * p<0.01 compared to LPS-induced ALI group without SIN treatment; # p<0.01 compared to the two indicated groups; NS: no significant difference between the two groups. (n = 8∼10 mice per group).
Figure 5.
Protection of SIN against lung damages is not observed in A2AR KO mice.
One hour before LPS injection to induce acute lung injury, 120 mg/kg SIN was administered to A2AR KO mice. At 24 hour after ALI, lung damage of both the injured control and SIN-treated A2AR KO mice were assayed. Normal A2AR KO mice treated intratracheally with 40 µl PBS served as controls. (A) Lung water content. (B) PaO2/FIO2 (P/F) ratio. (C) HE staining for the histopathological changes in the lung tissue. NS: no significant difference between the two groups. (n = 8∼10 mice per group).
Figure 6.
The anti-inflammatory effect of SIN is abolished in A2AR KO mice.
Thirty minutes before LPS injection to induce acute lung injury, 120 mg/kg SIN was administered to A2AR KO mice. At 24 hour after ALI, neutrophil infiltration was detected by immunofluorescence using a CD177 primary antibody and a FITC-conjugated secondary antibody, and the protein expression levels of the inflammatory cytokines TNF-α and IL-1β were assayed by ELISA. The normal A2AR KO mice intratracheally treated with 40 µl PBS were served as the control. (A) TNF-α protein levels. (B) IL-1β protein levels. (C) CD177-positive cells in the murine lung tissue. (D) Cell counting and statistical analysis. NS: no significant difference between the two groups. (n = 8∼10 mice per group).
Figure 7.
SIN inhibits inflammatory cytokine expression levels in LPS-stimulated murine neutrophils in an A2AR-dependent manner.
In isolated WT and A2AR KO murine neutrophils, SIN (1 mol/L) was applied 30 minutes before LPS stimulation. The neutrophils treated with PBS served as control. At 4 h after LPS administration, the A2AR-associated effects of SIN were detected. (A) Relative A2AR mRNA levels. (B) Relative TNF-α mRNA levels. (C) Relative IL-1β mRNA levels. * p<0.01 compared to the two groups; # p<0.01 compared to the LPS-treated WT neutrophil group; NS: no significant difference between the two groups. (n = 8∼10 mice per group).
Figure 8.
A2AR-cAMP-PKA signaling is involved in the anti-inflammatory effect of SIN.
In isolated murine neutrophils, SIN (1 mol/L) was applied 30 minutes before LPS stimulation, and the A2AR-associated mechanism for the anti-inflammatory effect of SIN was further investigated by assaying for cAMP and using the PKA inhibitor H-89. The neutrophils treated with PBS were served as control. (A) cAMP levels. (B) TNF-α protein levels. (C) IL-1β protein levels. * p<0.01 compared to the LPS treatment groups; NS: no significant difference between the two groups. (n = 8∼10 mice per group).