Accelerated inflammation and oxidative stress induced by LPS in acute lung injury: Ιnhibition by ST1926

Abstract

Bioavailable and less toxic synthetic retinoids, such as the atypical adamantyl retinoid ST1926, have been well developed and investigated in clinical trials for many diseases. The aim of our study was to explore the role of ST1926 in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to reveal the possible molecular mechanism. Mice were treated with LPS to induce acute lung injury followed by ST1926 administration. After LPS induction, mice administered with ST1926 showed lower inflammation infiltration in bronchoalveolar lavage (BAL) fluid, and pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-18, IL-6 and tumor necrosis factor-α (TNF-α) in serum and lung tissue samples obtained from mice. In addition, western blot assays suggested that ST1926 suppressed nuclear factor-κB (NF-κB), inhibitor-κB kinase-α (IκBα) and IκB kinase (IKKα), as well as Toll-like receptor 4 (TLR4) induced by LPS. In addition, reactive oxygen species (ROS) stimulated by LPS was also suppressed for ST1926 through inhibiting p38 and extracellular receptor kinase (ERK) signaling pathway. Taken together, the data here indicated that ST1926 may be of potential value in treating acute lung injury through inflammation and ROS suppression via inactivating TLR4/NF-κB and p38/ERK1/2 signaling pathways.

Figure 1

ST1926 reduces inflammatory cell infiltrate in lipopolysaccharide (LPS)-induced mice with acute lung injury. The number of cells in inflammation infiltration was determined. (A) Total cells, (B) total neutrophils, (C) total lymphocytes, (D) total macrophages, and (E) total eosinophils. (F) Eotaxin levels in bronchoalveolar lavage (BAL) were assessed. The data are represented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 2

Lipopolysaccharide (LPS)-induced acute lung injury in mice is ameliorated by ST1926 administration. (A) The representative images of lung injury are shown by hematoxylin and eosin (H&E) staining. (B) The quantification of inflammatory response following H&E staining. (C) PAS staining was used to observe goblet cells in LPS-induced mice with acute lung injury. (D) The quantification of PAS-positive cells. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 3

ST1926 improves pro-inflammatory cytokine releases caused by lipopolysaccharide (LPS). Serum pro-inflammatory cytokines were measured, including (A) tumor necrosis factor-α (TNF-α), (B) interleukin-6 (IL-6), (C) IL-5, (D) IL-1β, (E) IL-18 and (F) IL-17. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 4

ST1926 suppresses inflammation response in lipopolysaccharide (LPS)-induced lung injury. Western blotting and RT-qPCR assays were used to calculate (A) TGF-β1, (B) Foxp3, (C) IFNγ, (D) Granzyme B and (E) Tbx-21 protein and gene levels in LPS-treated lung tissue samples from mice in the presence or absence of ST1926. Serum levels of (F) interleukin-12 (IL-12) and (G) IL-10 were assessed. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 5

ST1926-ameliorates inflammation response in mice induced by lipopolysaccharide (LPS) is dependent on nuclear factor-κB (NF-κB) signaling pathway. Immnohistochemical analysis of pro-inflammatory cytokines of (A) tumor necrosis factor-α (TNF-α) and (B) interleukin-1β (IL-1β) were assessed. (C) RT-qPCR assays were conducted to explore mRNA levels of TNF-α, IL-1β, IL-18 and IL-6. (D) Western blot assay was performed to investigate NF-κB signaling pathway, including signals of inhibitor-κB kinase (IKKα), inhibitor-κB kinase-α (IκBα) and NF-κB phosphorylation, and the quantified levels are displayed. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 6

ST1926 suppresses Toll-like receptor 4 (TLR4)/MyD88 signal pathway to inactivate nuclear factor-κB (NF-κB) activity. (A) TLR4 and MyD88 protein levels were evaluated through western blot analysis. (B) The quantification of TLR4 and MyD88 is shown following immunoblotting analysis. (C) RT-qPCR was used to determine TLR4 and MyD88 gene levels. (D) Immunofluorescence was included to further test TLR4 expression levels. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. lipopolysaccharide (LPS)-induced mice (LPS).

Figure 7

ST1926 impedes oxidative stress in mice with acute lung injury induced by lipopolysaccharide (LPS). (A) SOD activity, CAT activity and MDA levels were measured in the serum of mice after LPS induction in the absence or presence of ST1926 at different doses. (B) SOD activity, CAT activity and MDA levels were measured in the lung tissue samples of mice after LPS induction in the absence or presence of ST1926 at different doses. (C) O₂⁻ and H₂O₂, as well as nitrate levels in the LPS-induced lung tissue samples were measured. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 8

ST1926 reduced reactive oxygen species (ROS) generation from iNOS/MPO inhibition. (A) NOx levels in the lung tissue samples were examined. (B) MPO activity was measured in the lung tissue specimens obtained from mice pre-treated with lipopolysaccharide (LPS) and/or ST1926 under different conditions. (C) The representative images of iNOS and MPO assessed by western blot analysis and the quantification was shown. The data are represented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 9

AMPK/p38/extracellular receptor kinase 1/2 (ERK1/2) signaling pathway was inhibited by ST1926 administration in lipopolysaccharide (LPS)-induced mice. (A) Images of reactive oxygen species (ROS) production are shown, and the quantified levels of ROS generation are displayed. (B) SOD1 and SOD2 protein levels were calculated through western blot assay. The quantification of SOD1 and SOD2 is shown. (C) p38/ERK1/2 signaling pathway was investigated by immunoblotting, and the phosphorylated p38 and ERK1/2 protein levels were quantified. (D) Western blot analysis was used to calculate CAT, haeme oxygenase-1 (HO-1) and Nrf2 protein levels, and the quantification of these molecules are shown. (E) Phosphorylated AMPK at T172 was analyzed by western blot assays. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 10

ST1926 shows no toxicity in vitro and in vivo. (A) MTT analysis was included to explore the lung epithelia cell viability with different concentrations of ST1926 for 24 h. (B) ST1926 (8 µM) was administered to lung epithelial cells for different times to determine cell viability. (C) Hematoxylin and eosin (H&E) staining analysis of mouse liver without lipopolysaccharide (LPS) and ST1926 administration. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 11

ST1926 suppresses inflammation response and reactive oxygen species (ROS) production in lung epithelia cells in vitro. (A) Immunofluorescence of phosphorylated nuclear factor-κB (NF-κB) was evaluated. (B) Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-18 and IL-6 mRNA levels were calculated via RT-qPCR analysis. (C) Western blot analysis was used determine Toll-like receptor 4 (TLR4) and MyD88 expression levels in lipopolysaccharide (LPS)-induced cells with or without ST1926. (D) Inhibitor-κB kinase (IKKα) and inhibitor-κB kinase-α (IκBα) protein expression levels were calculated through western blot analysis. (E) ROS levels were calculated in different groups of cells. (F) SOD1 and SOD2 mRNA levels were determined via RT-qPCR analysis. (G) Phosphorylated p38, and (H) extracellular receptor kinase 1/2 (ERK1/2) was determined by the use of western blot analysis. (I) MLE-12 cells were treated with 100 ng/ml LPS for different times as indicated. Then, western blot analysis was conducted to investigate p38 and ERK1/2 phosphorylation. (J) ST1926 reduced p38 and ERK1/2 phosphorylation in MLE-12 cells after LPS treatment in the presence or absence of ST1926 for different times from 0 to 48 h. Western blot assays were used to explore p38 and ERK1/2 activation. (K) Western blotting was carried out to investigate nuclear NF-κB levels. (L) Cytoplasm p-IκBα and IκBα levels were determined by western blot analysis. The ratio of p-IκBα to IκBα was exhibited. The data are represented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 11

ST1926 suppresses inflammation response and reactive oxygen species (ROS) production in lung epithelia cells in vitro. (A) Immunofluorescence of phosphorylated nuclear factor-κB (NF-κB) was evaluated. (B) Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-18 and IL-6 mRNA levels were calculated via RT-qPCR analysis. (C) Western blot analysis was used determine Toll-like receptor 4 (TLR4) and MyD88 expression levels in lipopolysaccharide (LPS)-induced cells with or without ST1926. (D) Inhibitor-κB kinase (IKKα) and inhibitor-κB kinase-α (IκBα) protein expression levels were calculated through western blot analysis. (E) ROS levels were calculated in different groups of cells. (F) SOD1 and SOD2 mRNA levels were determined via RT-qPCR analysis. (G) Phosphorylated p38, and (H) extracellular receptor kinase 1/2 (ERK1/2) was determined by the use of western blot analysis. (I) MLE-12 cells were treated with 100 ng/ml LPS for different times as indicated. Then, western blot analysis was conducted to investigate p38 and ERK1/2 phosphorylation. (J) ST1926 reduced p38 and ERK1/2 phosphorylation in MLE-12 cells after LPS treatment in the presence or absence of ST1926 for different times from 0 to 48 h. Western blot assays were used to explore p38 and ERK1/2 activation. (K) Western blotting was carried out to investigate nuclear NF-κB levels. (L) Cytoplasm p-IκBα and IκBα levels were determined by western blot analysis. The ratio of p-IκBα to IκBα was exhibited. The data are represented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 12

ST1926 inhibits inflammation response and reactive oxygen species (ROS) generation in human bronchial epithelial cells. (A) Human bronchial epithelial cells were treated with different concentrations of ST1926 at different concentrations for 24 h. Then, the cell viability was calculated through MTT analysis. (B) Human bronchial epithelial cells were treated with 8 µM ST1926 for different time, followed by MTT assays. (C) The phosphorylated nuclear factor-κB (NF-κB) levels were measured by immunofluorescent analysis. (D) ROS generation was evaluated and the quantification was displayed. (E) RT-qPCR assays were performed to determine tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-18 and IL-6 gene levels in cells under different treatment. (F) SOD1, SOD2, CAT, haeme oxygenase-1 (HO-1) and Nrf2 mRNA levels were calculated through RT-qPCR assays. (G) Toll-like receptor 4 (TLR4), MyD88 and inhibitor-κB kinase (IKKα) protein expression levels were measured by western blot analysis. (H) Western blotting was carried out to investigate nuclear NF-κB levels. The cytoplasm p-inhibitor-κB kinase-α (IκBα) and IκBα levels were assessed through western blot analysis. (I) Phosphorylated p38, and (H) extracellular receptor kinase 1/2 (ERK1/2) was determined by western blot analysis. (J) NHBE cells were exposed to 100 ng/ml lipopolysaccharide (LPS) for different times as indicated, followed by western blot analysis. (K) ST1926 reduced p38 and ERK1/2 phosphorylation in human bronchial epithelial cells after LPS treatment with or without ST1926 administration from 0 to 48 h. Then, western blot assays were used to explore p38 and ERK1/2 activation. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).

Figure 12

ST1926 inhibits inflammation response and reactive oxygen species (ROS) generation in human bronchial epithelial cells. (A) Human bronchial epithelial cells were treated with different concentrations of ST1926 at different concentrations for 24 h. Then, the cell viability was calculated through MTT analysis. (B) Human bronchial epithelial cells were treated with 8 µM ST1926 for different time, followed by MTT assays. (C) The phosphorylated nuclear factor-κB (NF-κB) levels were measured by immunofluorescent analysis. (D) ROS generation was evaluated and the quantification was displayed. (E) RT-qPCR assays were performed to determine tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-18 and IL-6 gene levels in cells under different treatment. (F) SOD1, SOD2, CAT, haeme oxygenase-1 (HO-1) and Nrf2 mRNA levels were calculated through RT-qPCR assays. (G) Toll-like receptor 4 (TLR4), MyD88 and inhibitor-κB kinase (IKKα) protein expression levels were measured by western blot analysis. (H) Western blotting was carried out to investigate nuclear NF-κB levels. The cytoplasm p-inhibitor-κB kinase-α (IκBα) and IκBα levels were assessed through western blot analysis. (I) Phosphorylated p38, and (H) extracellular receptor kinase 1/2 (ERK1/2) was determined by western blot analysis. (J) NHBE cells were exposed to 100 ng/ml lipopolysaccharide (LPS) for different times as indicated, followed by western blot analysis. (K) ST1926 reduced p38 and ERK1/2 phosphorylation in human bronchial epithelial cells after LPS treatment with or without ST1926 administration from 0 to 48 h. Then, western blot assays were used to explore p38 and ERK1/2 activation. The data are presented as mean ± SD (n=8). ^*p<0.05 and ^**p<0.001 vs. the control (Con); ⁺p<0.05, ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 vs. LPS-induced mice (LPS).