Fig 1.
The severity of lung injury in LPS-induced ARDS is concentration-dependent.
(A) H&E staining of lung tissues under different doses of LPS administration (200×). Atelectasis was marked with black arrow and alveolar fusion was marked with *. (B) Lung injury score under different doses of LPS administration. The mice were instilled with different concentrations of LPS at doses of 10 mg/kg~40 mg/kg, respectively, while the sham group was instilled with nothing, and the control group was instilled with PBS. The mice were sacrificed 24 h after LPS instillation, and the left lungs were obtained for H&E. The data were presented as mean±s.e.m; (n = 5). **P<0.01 versus the control group; #P<0.05, ##P<0.01 versus the LPS 10 mg/kg group.
Fig 2.
Apoptosis was increased in low dose LPS-induced mild ARDS but was decreased in high dose LPS-induced server ARDS.
(A) The expression of total and cleaved caspase-3 and XIAP as detected by western blotting. (B) The expression of total and cleaved caspase-8 as detected by western blotting. (C) The number of apoptotic cells by TUNEL staining (400×). (D) TUNEL-positive cell counts per area. The mice were instilled with different concentrations of LPS at doses of 10 mg/kg~40 mg/kg, respectively, while the control group was instilled with PBS. The mice were sacrificed 24 h after LPS instillation. The data are presented as mean±s.e.m; (n = 5). *P<0.05, **P<0.01 versus the control group; ##P<0.01 versus the LPS 10 mg/kg group.
Fig 3.
Necroptosis was increased in the lung in high dose but not in low dose LPS-induced ARDS.
(A) The expression of RIP3 as detected by western blotting. (B) p-RIP3 and MLKL expression as detected by western blotting. (C) Immunohistochemistry of lung section for detecting of RIP3. RIP3 was increased in the cytoplasm of alveolar wall epithelial cells (as directed by black arrow with bifurcated tail) and macrophage-like cells (as directed by black arrow without bifurcated tail). The mice were instilled with different concentrations of LPS at doses of 10 mg/kg~40 mg/kg, respectively, while the control group was instilled with PBS. The mice were sacrificed 24 h after LPS instillation. The data are presented as mean±s.e.m; (n = 5). **P<0.01 versus the control group; ##P<0.01 versus the LPS 10 mg/kg group.
Fig 4.
RIP3 depletion ameliorated lung injury and improved survival rate in LPS-induced ARDS in mice.
(A-B) Pathological changes of lung tissues as determined by H&E staining (200×, n = 5). (C) Body temperature of LPS treated RIP3-WT and RIP3-KO mice (n = 10~16). (D) Survival rate of LPS treated RIP3-WT and RIP3-KO mice (n = 20). RIP3-WT and RIP3-KO mice were both divided into control and LPS groups, respectively. The LPS group mice were instilled with 30 mg/kg LPS, while the control group mice were instilled with PBS. The mice were sacrificed 24 h after LPS instillation. The data are presented as mean±s.e.m; **P<0.01 versus the respective control group; &P<0.05 versus the LPS treated RIP3-WT group.
Fig 5.
RIP3 depletion decreased the levels of IL-1α, IL-1β, IL-6 and HMGB1 in BALF in LPS-induced ARDS.
(A) The concentration of IL-1β in BALF. (B) The concentration of IL-6 in BALF. (C) The concentration of TNF-α in BALF. (D) The concentration of IL-1α in BALF. (E) The concentration of IL-10 in BALF. (F) The concentration of HMGB1 in BALF. RIP3-WT and RIP3-KO mice were divided into two groups. The LPS group mice were instilled with 30 mg/kg LPS, while the control group mice were instilled with PBS. The mice were sacrificed at 12 h or 24 h after LPS instillation. The data are presented as mean±s.e.m; (n = 10). **P<0.01 versus the respective control group; &P<0.05 versus the LPS treated RIP3-WT group.
Fig 6.
RIP3 depletion reduced the tissue MPO activity, neutrophil influx and total protein concentration in BALF in LPS-induced ARDS.
(A) The MPO activity in lung tissue. (B) The neutrophil counts in BALF. (C) The total protein concentration in BALF. RIP3-WT and RIP3-KO mice were divided into two groups. The LPS group mice were instilled with 30 mg/kg LPS, while the control group mice were instilled with PBS. The mice were sacrificed 12 h or 24 h after LPS instillation. The data were presented as mean±s.e.m. (n = 10). **P<0.01 versus the respective control group; &P<0.05 versus the LPS treated RIP3-WT group.
Fig 7.
RIP3 depletion reduced the necrotic cells in the lung and decreased the expression of MLKL, but had no impact on cleaved caspase-3 in LPS-induced ARDS.
(A) The number of necrotic cells as determined by PI staining (100×). Frozen sections were stained with propidium iodide (PI, red) and nuclear stain (DAPI, blue). The cells were counted by the Cell^P imaging software. (B) MLKL expression as detected by western blotting. (C) Cleaved caspase-3 expression as detected by western blotting. RIP3-WT and RIP3-KO mice were divided into control and LPS groups, respectively. The LPS group mice were instilled with 30 mg/kg LPS, while the control group mice were instilled with PBS. The mice were sacrificed 24 h after LPS instillation. The data are presented as mean±s.e.m; (n = 10). **P<0.01 versus the respective control group; &P<0.05 versus the LPS treated RIP3-WT group.