Luteolin alleviated neutrophilic asthma by inhibiting IL-36γ secretion-mediated MAPK pathways.

CONTEXT: Luteolin can affect multiple biological functions, such as anti-inflammatory, antioxidant and immune enhancement processes. Luteolin can inhibit inflammation of T2-high asthma, but its role in neutrophilic asthma has been insufficently studied. OBJECTIVE: This study determines the effect of luteolin on IL-36γ secretion-mediated MAPK pathway signalling in neutrophilic asthma. MATERIALS AND METHODS: The asthma model was established by using ovalbumin/lipopolysaccharide (OVA/LPS). Female 6-8-week-old C57BL/6 mice were divided into control, asthma, luteolin (20 mg/kg) and asthma + luteolin (20 mg/kg) groups. To explore the mechanism of anti-inflammatory effects of luteolin in neutrophilic asthma, Beas-2B cells were treated with luteolin (20 µmol/L), LPS (100 ng/mL), recombinant human IL-36γ protein (rhIL-36γ; 100 ng/mL) or IL-36γ siRNA. RESULTS: IL-36γ secretion and MAPK/IL-1ß signalling were significantly increased in the asthma mouse model compared with the control (p < 0.05). However, the levels of IL-36γ secretion and MAPK/IL-1ß signalling were reduced by luteolin (p < 0.05). In addition, luteolin inhibited IL-36γ and MAPK/IL-1ß levels after LPS (100 ng/mL) stimulation of Beas-2B cells (p < 0.05). We found that in Beas-2B cells, luteolin inhibited activation of the MAPK pathway and IL-1ß secretion following stimulation with rhIL-36γ (100 ng/mL; p < 0.05). Finally, IL-1ß and phosphorylated MAPK levels were found to be lower in the IL-36γ siRNA + LPS (100 ng/mL) group than in the nonspecific control (NC) siRNA + LPS group (p < 0.05). DISCUSSION AND CONCLUSIONS: Luteolin alleviated neutrophilic asthma by inhibiting IL-36γ secretion-mediated MAPK pathways. These findings provided a theoretical basis for the application of luteolin in the treatment of neutrophilic asthma.

Syndecan-1 Shedding by Matrix Metalloproteinase-9 Signaling Regulates Alveolar Epithelial Tight Junction in Lipopolysaccharide-Induced Early Acute Lung Injury.

INTRODUCTION: Alveolar epithelial tight junction damage and glycocalyx syndecan-1 (SDC-1) degrading are key factors to pulmonary edema of acute lung injury (ALI). Matrix metalloproteinase-9 (MMP-9) was involved in glycocalyx shedding, which was vital in SDC-1 degrading. This study aimed to investigate the effects of MMP-9-mediated SDC-1 shedding on tight junction in LPS-induced ALI. METHODS: Mice were intratracheally atomized with 5 mg/kg LPS to stimulate different periods and LPS stimulation for 6 hours for further studies. A549 cells was stimulated for 6 hours by active MMP-9 protein to assess the effects of active MMP-9 protein on SDC-1 and tight junction. Afterward, the mice treated with MMP-9 shRNA or A549 cells were treated with MMP-9 siRNA before LPS stimulation for 6 hours to explore the effects on glycocalyx SDC-1 and tight junction. Moreover, the mice were treated with recombinant SDC-1 protein or A549 cells were over-expressed by pc-SDC-1 before LPS stimulation for 6 hours to explore the effects of SDC-1 on tight junction. RESULTS: The mice persistent exposure to LPS showed that MMP-9 expression, glycocalyx SDC-1 shedding (SDC-1 decreased in alveolar epithelium and increased in the BALF), tight junction impairment, FITC-albumin infiltration, and other phenomena began to appear after 6 hours of LPS treatment in this study. The levels of SDC-1 and tight junction significantly decreased by active MMP-9 protein stimulation for 6 hours in the A549 cells. Therefore, LPS stimulation for six hours was selected for investigating the underlying effects of MMP-9-mediated SDC-1 shedding on the alveolar epithelial tight junction and pulmonary edema. Further vivo analysis showed that down regulation MMP-9 expression by MMP-9 shRNA significantly alleviated glycocalyx SDC-1 shedding (SDC-1 increased in alveolar epithelium and decreased in the BALF), tight junction (occludin and ZO-1) damage, and FITC-albumin infiltration in LPS-induced early ALI mice. The vitro results also showed that MMP-9 siRNA alleviated glycocalyx SDC-1 shedding (SDC-1 increased in cell culture medium and decreased in cell surface) and tight junction damage by downregulating MMP-9 expression in LPS-stimulated A549 cells. In addition, pretreatment with recombinant mouse SDC-1 protein significantly alleviated glycocalyx (SDC-1 increased in alveolar epithelium) and tight junction damage, and FITC-albumin infiltration in LPS-induced early ALI mice. Overexpression SDC-1 by pc-SDC-1 also significantly decreased tight junction damage in LPS-stimulated A549 cells. CONCLUSION: Glycocalyx SDC-1 shedding mediated by MMP-9 significantly aggravated tight junction damage, which further increased the pulmonary edema.

Syndecan-1 Amplifies Ovalbumin-Induced Airway Remodeling by Strengthening TGFß1/Smad3 Action.

Syndecan-1 (SDC-1) is a transmembrane proteoglycan of heparin sulfate that can regulate various cell signal transduction pathways in the airway epithelial cells and fibroblasts. Airway epithelial cells and human bronchial fibroblasts are crucial in airway remodeling. However, the importance of SDC-1 in the remodeling of asthmatic airways has not been confirmed yet. The present study was the first to uncover SDC-1 overexpression in the airways of humans and mice with chronic asthma. This study also validated that an increase in SDC-1 expression was correlated with TGFß1/Smad3-mediated airway remodeling in vivo and in vitro. A small interfering RNA targeting SDC-1 (SDC-1 siRNA) and homo-SDC-1 in pcDNA3.1 (pc-SDC-1) was designed to assess the effects of SDC-1 on TGFß1/Smad3-mediated collagen I expression in Beas-2B (airway epithelial cells) and HLF-1 (fibroblasts) cells. Downregulation of the SDC-1 expression by SDC-1 siRNA remarkably attenuated TGFß1-induced p-Smad3 levels and collagen I expression in Beas-2B and HLF-1 cells. In addition, SDC-1 overexpression with pc-SDC-1 enhanced TGFß1-induced p-Smad3 level and collagen I expression in Beas-2B and HLF-1 cells. Furthermore, the levels of p-Smad3 and collagen I induced by TGFß1 were slightly increased after the addition of the recombinant human SDC-1 protein to Beas-2B and HLF-1 cells. These findings in vitro were also confirmed in a mouse model. A short hairpin RNA targeting SDC-1 (SDC-1 shRNA) to interfere with SDC-1 expression considerably reduced the levels of p-Smad3 and remodeling protein (α-SMA, collagen I) in the airways induced by ovalbumin (OVA). Similarly, OVA-induced p-Smad3 and remodeling protein levels in airways increased after mice inhalation with the recombinant mouse SDC-1 protein. These results suggested that SDC-1 of airway epithelial cells and fibroblasts plays a key role in the development of airway remodeling in OVA-induced chronic asthma.