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, 7 (9), e45734

An Extract of Crataegus Pinnatifida Fruit Attenuates Airway Inflammation by Modulation of Matrix metalloproteinase-9 in Ovalbumin Induced Asthma

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An Extract of Crataegus Pinnatifida Fruit Attenuates Airway Inflammation by Modulation of Matrix metalloproteinase-9 in Ovalbumin Induced Asthma

In Sik Shin et al. PLoS One.

Abstract

Background: Crataegus pinnatifida (Chinese hawthorn) has long been used as a herbal medicine in Asia and Europe. It has been used for the treatment of various cardiovascular diseases such as myocardial weakness, tachycardia, hypertension and arteriosclerosis. In this study, we investigated the anti-inflammatory effects of Crataegus pinnatifida ethanolic extracts (CPEE) on Th2-type cytokines, eosinophil infiltration, expression of matrix metalloproteinase (MMP)-9, and other factors, using an ovalbumin (OVA)-induced murine asthma model.

Methods/principal finding: Airways of OVA-sensitized mice exposed to OVA challenge developed eosinophilia, mucus hypersecretion and increased cytokine levels. CPEE was applied 1 h prior to OVA challenge. Mice were administered CPEE orally at doses of 100 and 200 mg/kg once daily on days 18-23. Bronchoalveolar lavage fluid (BALF) was collected 48 h after the final OVA challenge. Levels of interleukin (IL)-4 and IL-5 in BALF were measured using enzyme-linked immunosorbent (ELISA) assays. Lung tissue sections 4 µm in thickness were stained with Mayer's hematoxylin and eosin for assessment of cell infiltration and mucus production with PAS staining, in conjunction with ELISA, and Western blot analyses for the expression of MMP-9, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 protein expression. CPEE significantly decreased the Th2 cytokines including IL-4 and IL-5 levels, reduced the number of inflammatory cells in BALF and airway hyperresponsiveness, suppressed the infiltration of eosinophil-rich inflammatory cells and mucus hypersecretion and reduced the expression of ICAM-1, VCAM-1 and MMP-9 and the activity of MMP-9 in lung tissue of OVA-challenged mice.

Conclusions: These results showed that CPEE can protect against allergic airway inflammation and can act as an MMP-9 modulator to induce a reduction in ICAM-1 and VCAM-1 expression. In conclusion, we strongly suggest the feasibility of CPEE as a therapeutic drug for allergic asthma.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CPEE inhibits the recruitment of inflammatory cells in bronchoalveolar lavage fluid (BALF) of mice.
Cells were isolated by centrifugation and stained with Diff-Quik® stain reagent. Cell numbers were determined using a light microscope to count cells in at least five squares of a hemocytometer after excluding dead cells using Trypan blue. NC; normal control mice treated with PBS only; OVA; OVA-sensitized/challenged mice; Mon; Montelukast (30 mg/kg) + OVA-sensitized/challenged mice; CPEE-1; CPEE (100 mg/kg) + OVA-sensitized/challenged mice; CPEE-2; CPEE (200 mg/kg) + OVA-sensitized/challenged mice. Values are expressed as mean ± SD (n = 6/group). *Significantly different from NC, P<0.05; †significantly different from OVA, P<0.05.
Figure 2
Figure 2. CPEE inhibits the recruitment of inflammatory cells to lung tissue of mice.
(A) Histological examination of lung tissue was performed 48 h after the last OVA challenge. Lung tissues were fixed, sectioned at 4 µm thickness, and stained with H&E solution (magnification x200). (B) Scoring the extent of inflammation by quantitative analysis of inflammatory cell infiltration in lung sections were performed using an image analyzer (Molecular Devices Inc., CA, USA). Quantitative analysis was assessed in at least four squares of a sample slide stained with H&E. NC; normal control mice treated PBS only; OVA; OVA-sensitized/challenged mice; Mon; Montelukast (30 mg/kg) + OVA-sensitized/challenged mice; CPEE-1; CPEE (100 mg/kg) + OVA-sensitized/challenged mice; CPEE-2; CPEE (200 mg/kg) + OVA-sensitized/challenged mice. Values are expressed as mean ± SD (n = 6/group). *Significantly different from NC, P<0.05; †significantly different from OVA, P<0.05.
Figure 3
Figure 3. CPEE reduces mucus production in lung tissues of mice.
(A) Histological examination of mucus secretion in lung tissue 48 h after the last OVA challenge. Lung tissue was fixed, sectioned at 4 µm thickness, and stained with periodic acid Schiff (PAS) for mucus production (magnification ×200). (B) Scoring of mucus production in lung sections were performed an image analyzer (Molecular Devices Inc., CA, USA). Quantitative analysis was assessed in at least four squares of a sample slide stained with PAS. NC; normal control mice treated with PBS only; OVA; OVA-sensitized/challenged mice; Mon; Montelukast (30 mg/kg) + OVA-sensitized/challenged mice; CPEE-1; CPEE (100 mg/kg) + OVA-sensitized/challenged mice; CPEE-2; CPEE (200 mg/kg) + OVA-sensitized/challenged mice. Values are expressed as mean ± SD (n = 6/group). *Significantly different from NC, P<0.05; †significantly different from OVA, P<0.05.
Figure 4
Figure 4. CPEE reduces total IgE, OVA-specific IgE, and OVA-specific IgG1 levels in serum of mice.
(A) Total IgE level (B) OVA-specific IgE level (C) OVA-specific IgG1 level. NC; normal control mice treated with PBS only; OVA; OVA-sensitized/challenged mice; Mon; Montelukast (30 mg/kg) + OVA-sensitized/challenged mice; CPEE-1; CPEE (100 mg/kg) + OVA-sensitized/challenged mice; CPEE-2; CPEE (200 mg/kg) + OVA-sensitized/challenged mice. Values are expressed as mean ± SD (n = 6/group). *Significantly different from NC, P<0.05; †significantly different from OVA, P<0.05.
Figure 5
Figure 5. CPEE reduces the levels of IL-4 and IL-5 in BALF of mice. (A) IL-4 level (B) IL-5 level.
NC; normal control mice treated with PBS only; OVA; OVA-sensitized/challenged mice; Mon; Montelukast (30 mg/kg) + OVA-sensitized/challenged mice; CPEE-1; CPEE (100 mg/kg) + OVA-sensitized/challenged mice; CPEE-2; CPEE (200 mg/kg) + OVA-sensitized/challenged mice. Values are expressed as mean ± SD (n = 6/group). *Significantly different from NC, P<0.05; †significantly different from OVA, P<0.05.
Figure 6
Figure 6. CPEE reduces airway hyperresponsiveness (AHR) by OVA-challenge.
AHR was measured using plethysmography 24 h after the final OVA challenge, in mice given various doses of methacholine (6.25–25 mg/mL). NC; normal control mice treated with PBS only; OVA; OVA-sensitized/challenged mice; Mon; Montelukast (30 mg/kg) + OVA-sensitized/challenged mice; CPEE-2; CPEE (200 mg/kg) + OVA-sensitized/challenged mice. Values are expressed as mean ± SD (n = 6/group). *Significantly different from NC, P<0.05; †significantly different from OVA, P<0.05.
Figure 7
Figure 7. CPEE decreases the MMP-9 activities and protein expression in lung tissues of mice.
The protein of supernatants was loaded for gelatin zymography (60 µg/lane). SDS−PAGE zymography was performed according to the method of Heussen and Dowdle (1980). (A) MMP-9 activities (B) MMP-9 protein expression. NC; normal control mice treated with PBS only; OVA; OVA-sensitized/challenged mice; Mon; Montelukast (30 mg/kg) + OVA-sensitized/challenged mice; CPEE-1; CPEE (100 mg/kg) + OVA-sensitized/challenged mice; CPEE-2; CPEE (200 mg/kg) + OVA-sensitized/challenged mice.
Figure 8
Figure 8. CPEE reduces the expression of ICAM-1 and VCAM-1 proteins in lung tissues of mice.
NC; normal control mice treated with PBS only; OVA; OVA-sensitized/challenged mice; Mon; Montelukast (30 mg/kg) + OVA-sensitized/challenged mice; CPEE-1; CPEE (100 mg/kg) + OVA-sensitized/challenged mice; CPEE-2; CPEE (200 mg/kg) + OVA-sensitized/challenged mice. Values are expressed as mean ± SD (n = 6/group). *Significantly different from NC, P<0.05; †significantly different from OVA, P<0.05.
Figure 9
Figure 9. CPEE reduces the activity of MMP-9. The protein of supernatants was loaded for gelatin zymography (60 µg/lane).
SDS−PAGE zymography was performed according to the method of Heussen and Dowdle (1980). (A) MMP-9 activities, (B) Quantitative analysis of MMP-9. NC; normal control mice treated with PBS only; OVA; OVA-sensitized/challenged mice; Mon; Montelukast (30 mg/kg) + OVA-sensitized/challenged mice; CPEE-2; CPEE (200 mg/kg) + OVA-sensitized/challenged mice; CPEE-2+ MMPI-I; CPEE (200 mg/kg) + MMPI-I (20 mg/kg) + OVA-sensitized/challenged mice; MMPI-I; MMPI-I (20 mg/kg) + OVA-sensitized/challenged mice. Values are expressed as mean ± SD (n = 6/group). *Significantly different from NC, P<0.05; †significantly different from OVA, P<0.05.
Figure 10
Figure 10. CPEE has no effect on AST and ALT levels in serum of mice.
Each sample was analyzed using commercial kits (Beckman Coulter, Inc., Fullerton, CA).

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This research was supported by grants from the Korea Research Council of Fundamental Science and Technology of the Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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