Protective effects of HY1702 on lipopolysaccharide-induced mild acute respiratory distress syndrome in mice

Abstract

Acute respiratory distress syndrome is an inflammatory disease with no effective pharmacological treatment. We investigated the therapeutic effect of HY1702, a new small molecule diterpene obtained from the processing and modification of Glaucocalyxin A and may exhibit anti-inflammatory activity. Specifically, we studied the anti-inflammatory effects of HY1702 on lipopolysaccharide-induced inflammatory responses in RAW264.7 and THP-1 cells in vitro and its protective efficacy on lipopolysaccharide-induced mild acute respiratory distress syndrome in mice. Our results showed that HY1702 significantly decreased lipopolysaccharide-induced inflammatory cytokine expression in RAW264.7 and THP-1 cells and attenuated the secretion of nitric oxide and prostaglandin E2 by down-regulating the expression of inducible nitric oxide synthase and cyclooxygenase 2 in RAW264.7 cells. In mice with lipopolysaccharide-induced mild acute respiratory distress syndrome, HY1702 alleviated histological alterations in the lungs and reduced the alveolar cavity protein leakage and inflammatory cytokine expression in murine bronchial alveolar lavage fluid. HY1702 decreased the myeloperoxidase activity and lung wet to dry weight ratio. In our mechanism studies in lipopolysaccharide-exposed RAW264.7 cells, HY1702 suppressed the inflammation stimulated by lipopolysaccharide through inhibiting phosphorylation of inhibitor of nuclear factor κB kinase subunit α/β (IKKα/β) and inhibitor of nuclear factor κB subunit α (IκBα), further affecting the nuclear transfer of phosphorylated p65. Meanwhile, phosphorylation of p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) was inhibited. These data suggest that HY1702 can reduce inflammation on lipopolysaccharide-stimulated macrophages and attenuate the symptoms of mild acute respiratory distress syndrome in a murine model by regulating the nuclear factor κB and MAP kinase signalling pathways.

Keywords: HY1702; Inflammation; MAP kinase; Mild acute respiratory distress syndrome; Nuclear factor κB.

Fig. 1

Effect of HY1702 on RAW264.7 and THP-1 cell viabilitys. RAW264.7 and THP-1 cells were treated with different concentrations of HY1702 for 24 h, then 10 μl of Cell Counting Kit-8 solution was added to each well to incubate with the cells for another 1 h. The absorbance was measured at 450 nm. A: The chemical structure of HY1702. B: Effects of HY1702 on the viability of RAW264.7 cells. C: Effects of HY1702 on the viability of THP-1 cells. The values presented are mean ± S.E.M (n = 6) of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with control group.

Fig. 2

Effect of HY1702 on lipopolysaccharide-induced inflammatory response in RAW264.7 and THP-1 cells. RAW264.7 and THP-1 cells were pretreated with HY1702 for 1 h and further treated with lipopolysaccharide for 24 h, then cellular supernatant and protein were collected. Tumor necrosis factor-α, interleukin-1, and interleukin-6 secretion from RAW264.7 (A) and THP-1 (B) cells was detected by using an enzyme-linked immunosorbent assay. Nitric oxide (C) and prostaglandin E2 (D) secretion in RAW264.7 were detected by Griess reagent and enzyme-linked immunosorbent assay. E: Cyclooxygenase 2 and inducible nitric oxide synthase protein level in the lysates of RAW264.7 cells were detected by Western blot. The values presented are mean ± S.E.M (n = 3) of three independent experiments. ^###P < 0.001, ^##P < 0.01 compared with control; *P < 0.05, **P < 0.01, ***P < 0.001 compared with LPS mode.

Fig. 3

Effect of HY1702 on histopathology of lung tissues of lipopolysaccharide-induced mild acute respiratory distress syndrome. Mice were treated with HY1702, dexamethasone or vehicle 1 h before lipopolysaccharide challenge. Lung tissues (n = 6) from each experimental group were processed for histological evaluation at 6 h after the lipopolysaccharide challenge. A: Control group. B: Lipopolysaccharide-only group. C: Lipopolysaccharide + HY1702 (5 mg/kg) group. D: Lipopolysaccharide + HY1702 (10 mg/kg) group. E: LPS + HY1702 (30 mg/kg) group. F: Lipopolysaccharide + dexamethasone (5 mg/kg) group. Representative histological lung sections were stained with hematoxylin and eosin at 400 × magnification.

Fig. 4

HY1702 attenuates mild acute respiratory distress syndrome in mice. Mice were intragastrically administratered with HY1702 (5, 10 or 30 mg/kg), dexamethasone (5 mg/kg) or vehicle 1 h before lipopolysaccharide challenge. 6 h after lipopolysaccharide administration, bronchial alveolar lavage fluid, and lung tissue were collected to quantify the tumor necrosis factor-α, interleukin-1β and interleukin-6 expression by enzyme-linked immunosorbent assay (A) and verse transcription-polymerase chain reaction (B). C: Effects of HY1702 on the lung wet to dry weight ratio in lipopolysaccharide-induced mild acute respiratory distress syndrome. D: Effects of HY1702 on protein content of bronchial alveolar lavage fluid in lipopolysaccharide-induced mild acute respiratory distress syndrome. The values presented are mean ± S.E.M (n = 6) of three independent experiments. ^##P < 0.01, ^###P < 0.001 compared with control; * P < 0.05, ** P < 0.01, ***P < 0.001 compared with LPS mode.

Fig. 5

Effect of HY1702 on myeloperoxidase activity in lung tissues of lipopolysaccharide-induced mild acute respiratory distress syndrome. Mice were intragastrically administratered with HY1702 (5, 10 or 30 mg/kg), dexamethasone (5 mg/kg) or vehicle 1 h before LPS challenge. 6 h after lipopolysaccharide administration, the myeloperoxidase activity in the lung tissue of mice was detected in lung tissue. The values presented are mean ± S.E.M (n = 6) of three independent experiments. ^###P < 0.001 compared with control; * P < 0.05, ** P < 0.01, ***P < 0.001 compared with LPS mode.

Fig. 6

Application of Proteomics mass spectrometry to detect the alteration of protein expressions in RAW264.7 cells following the treatment with LPS or LPS and HY1702. Pathway analysis involved in inflammation (nuclear factor κB and MAP kinase signaling pathways) following the treatment with LPS or LPS and HY1702 (2 μM) (From http://www.kegg.jp/).

Fig. 7

HY1702 extenuates the lipopolysaccharide-induced activation of nuclear factor κB and mitogen-activated protein kinase in RAW264.7 macrophages. RAW264.7 cell were pretreated with HY1702 for 1 h and further treated with lipopolysaccharide for 24 h, at 6 h, protein was extracted for experiments. A: Expression of phosphorylated-IKBα/β, phosphorylated-IkBα/β in cytoplasm and nuclear phosphorylated-p65 level. B: The phosphorylated and total extracellular signal–regulated kinase (ERK) and p38 levels were determined by Western blot analysis. Total ERK (ERK), total p38 (p38), or total p65 (p65) protein was used as the loading control. C: The nuclear translocation of nuclear factor κB p65 was visualized by immunofluorescence. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; (blue)). Scale bar = 200 μm. The values presented are mean ± S.E.M of three independent experiments. ^###P < 0.001, ^##P < 0.01 compared with control; *P < 0.05, **P < 0.01, ***P < 0.001 compared with LPS mode. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)