Fig 1.
Chemical structure of kaempferol (A), and epithelial mucus hypersecretion in trachea and lung tissues (B).
BALB/c mice were OVA-sensitized and orally supplemented with 10–20 mg/kg kaempferol. Lung tissue extracts were prepared for Western blot analysis with a primary antibody against MUC5AC. Representative blot data were obtained from 3 experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the right panel represent quantitative results of blots. Values in bar graphs not sharing a letter indicate significant different at P<0.05. Tissue sections of mouse trachea and lung were stained by using PAS reagents and counterstained with hematoxylin (C and D). A strong PAS positive staining (red arrows) in many areas of mucous cells of airway epithelium was observed. Each photograph is representative of four mice.
Fig 2.
Time course responses of MUC5AC induction to TGF-β and tunicamycin (A and C), and inhibition of MUC5AC induction by kaempferol (B and D).
BEAS-2B cells were treated with 10 ng/ml TGF-β or 1 μM tunicamycin up to 72 h in the absence and presence of 1–20 μM kaempferol. For the measurement of MUC5AC induction, total cell lysates were subject to Western blot analysis with a primary antibody against MUC5AC. β-Actin was used as an internal control. The bar graphs (mean ± SEM) in the bottom panels represent quantitative results of blots. Values not sharing a common letter are significantly different at P<0.05.
Fig 3.
Inhibitory effects of kaempferol on tunicamycin-induced expression and activation of ATF6 and IRE1α (A), and GRP78 and HSP70 (B) in BEAS-2B cells.
Cells were treated with 1–20 μM kaempferol and simulated with 1 μM tunicamycin. Cell lysates were prepared for Western blot analysis with a primary antibody against ATF6, phospho-IRE1α, GRP78 and HSP70. Representative blot data were obtained from 3 experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panels represent quantitative results of upper blots. Values in bar graphs not sharing a letter indicate significant different at P<0.05.
Fig 4.
RT-PCR data showing XBP-1 mRNA splicing (A and B) and Western blot data showing XBP-1 induction (C) in tunicamycin-stimulated and kaempferol-supplemented BEAS-2B cells.
Cells were treated with 1–20 μM kaempferol and simulated with 1 μM tunicamycin. GAPDH was used as a housekeeping gene for the co-amplification with XBP-1 (A and B). Cell lysates were prepared for Western blot analysis with a primary antibody against XBP-1 (C). Representative blot data were obtained from 3 experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panel represent quantitative results of upper blots. Values in bar graphs not sharing a letter indicate significant different at P<0.05.
Fig 5.
Suppressive effects of kaempferol on induction and activation of SMAD4, IRE1α and GRP78 (A), and XBP-1 protein induction (B) and XBP-1 mRNA transcription and splicing (C and D) in TGF-β-stimulated BEAS-2B cells.
Cells were treated with 1–20 μM kaempferol and simulated with 10 ng/ml TGF-β. Cell lysates were prepared for Western blot analysis with a primary antibody against SMAD4, phospho-IRE1α, GRP78 and XBP-1. Representative blot data were obtained from 3 experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) represent quantitative results of blots. Values in bar graphs not sharing a letter indicate significant different at P<0.05. RT-PCR was conducted to determine XBP-1 mRNA splicing (C and D). GAPDH was used as a housekeeping gene for the co-amplification with XBP-1.
Fig 6.
Immunofluorescent data showing inhibition of XBP-1 induction in OVA-challenged mouse lung tissues by kaempferol.
Epithelial XBP-1 protein was identified as reddish and/or pinkish staining. XBP-1 was visualized with a Cy3-conjugated secondary antibody and nuclear staining was done with DAPI. Each photograph is representative of four mice. Magnification: 200-fold. The bar graphs (mean ± SEM) represent quantitative results of Cy3 staining. Values in bar graphs not sharing a letter indicate significant different at P<0.05.
Fig 7.
Immunohistochemical staining showing inhibition of IRE1α activation in OVA-challenged mouse lung tissues by kaempferol.
Epithelial IRE1α was identified as brown staining and was visualized with DAB and the counter-staining was done with hematoxylin. Each photograph is representative of four mice. Magnification: 200-fold. The bar graphs (mean ± SEM) represent quantitative results of DAB staining. Values in bar graphs not sharing a letter indicate significant different at P<0.05.
Fig 8.
Inhibition of JNK activation and TRAF2 induction in BEAS-2B cells treated with kaempferol and exposed to 1 μM tunicamycin (A) or 10 ng/ml TGF-β (B).
Cell extracts were subject to 12% SDS-PAGE and western blot analysis with a primary antibody against total JNK, phospho-JNK and TRAF2. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panels represent quantitative results of blots obtained from a densitometer. Values not sharing a common letter are significantly different at P<0.05. BALB/c mice were OVA-sensitized and orally supplemented with 10–20 mg/kg kaempferol (C). Epithelial TRAF2 was identified as reddish staining and was visualized with ACE and the counter-staining was done with hematoxylin. Each photograph is representative of four mice. Magnification: 200-fold.
Fig 9.
Inhibition of XBP-1 mRNA splicing (A and B) and MUC5AC induction (C and D) by JNK inhibitor.
BEAS-2B cells were treated with kaempferol and exposed to 1 μM tunicamycin or 10 ng/ml TGF-β. RT-PCR was conducted to determine XBP-1 mRNA splicing (A and B). GAPDH was used as a housekeeping gene for the co-amplification with XBP-1. Cell extracts were subject to 12% SDS-PAGE and Western blot analysis with a primary antibody against MUC5AC. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panels represent quantitative results of blots obtained from a densitometer. Values not sharing a common letter are significantly different at P<0.05.