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. 2008 Dec;12(6A):2381-94.
doi: 10.1111/j.1582-4934.2008.00258.x. Epub 2008 Feb 4.

Activation of Cannabinoid Receptors Prevents Antigen-Induced Asthma-Like Reaction in Guinea Pigs

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Free PMC article

Activation of Cannabinoid Receptors Prevents Antigen-Induced Asthma-Like Reaction in Guinea Pigs

L Giannini et al. J Cell Mol Med. .
Free PMC article

Abstract

In this study we evaluated the effects of the CB1/CB2 cannabinoid receptor agonist CP55, 940 (CP) on antigen-induced asthma-like reaction in sensitized guinea pigs and we tested the ability of the specific CB2 receptor antagonist SR144528 (SR) and CB1 receptor antagonist AM251 (AM) to interfere with the effects of CP. Ovalbumin-sensitized guinea pigs placed in a respiratory chamber were challenged with the antigen given by aerosol. CP (0.4 mg/kg b.wt.) was given i.p. 3 hrs before ovalbumin challenge. Sixty minutes before CP administration, some animals were treated i.p. with either AM, or SR, or both (0.1 mg/kg b.wt.). Respiratory parameters were recorded and quantified. Lung tissue specimens were then taken for histopathological and morphometric analyses and for eosinophilic major basic protein immunohistochemistry. Moreover, myeloperoxidase activity, 8-hydroxy-2-deoxyguanosine, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) levels, and CB1 and CB2 receptor protein expression by Western blotting were evaluated in lung tissue extracts. In the bronchoalveolar lavage fluid, the levels of prostaglandin D2 and tumour necrosis factor-alpha TNF-alpha were measured. Ovalbumin challenge caused marked abnormalities in the respiratory, morphological and biochemical parameters assayed. Treatment with CP significantly reduced these abnormalities. Pre-treatment with SR, AM or both reverted the protective effects of CP, indicating that both CB1 and CB2 receptors are involved in lung protection. The noted treatments did not change the expression of cannabinoid receptor proteins, as shown by Western blotting. These findings suggest that targeting cannabinoid receptors could be a novel preventative therapeutic strategy in asthmatic patients.

Figures

Figure 1
Figure 1
Occurrence of respiratory abnormalities in guinea pigs of the different experimental groups. (A) latency time (sec.) for the onset of cough; (B) cough severity; (C) latency time (sec.) for the appearance of dyspnea. Compared with the sensitized, ovalbumin (OV)-challenged animals (group 2), a 3-hrs treatment with the CB1/CB2 receptor agonist CP of the sensitized guinea pigs before OV challenge (group 3) resulted in a statistically significant reduction of the respiratory abnormalities. Pretreatment of the sensitized guinea pigs with the CB1 receptor antagonist SR (SR+CP, group 4) or the CB2 receptor antagonist AM (AM+CP, group 5) or both (SR+AM+CP, group 6) before CP administration markedly reverted the protection afforded by CP. Significance of differences (one-way ANOVA; each group: n= 8): #P < 0.001 versus naive; ***P < 0.001 versus OV; +P < 0.05 and +++P < 0.001 versus CP.
Figure 2
Figure 2
Representative light micrographs of lung tissue from naive guinea pigs given OV aerosol (A; group 1), sensitized guinea pigs challenged with OV aerosol (B; group 2), sensitized guinea pigs treated with the CB1/CB2 receptor agonist CP before OV challenge (C; group 3), sensitized guinea pigs pre-treated with CP in combination with the CB1 receptor antagonist SR (D; group 4), the CB2 receptor antagonist AM (E; group 5) or both (F; group 6). Compared with the animals of group 1, those of group 2 show reduction of bronchiolar lumen and dilation of the respiratory air spaces. These alterations are not evident in the CP-treated guinea pigs of group 3, but persist in the animals given SR, AM or both 3 hours before CP (groups 4, 5 and 6, respectively). Haematoxylin and eosin. Bars = 100 μm.
Figure 3
Figure 3
Surface area of alveolar air spaces (A) and small-sized bronchial lumina (B) in the lungs of guinea pigs from the different experimental groups. Compared to the OV-challenged, untreated animals (group 2) in the guinea pigs treated with the CB1/CB2 receptor agonist CP (group 3) the mean surface area of alveolar air spaces nearly returned to the control values and the mean surface area of bronchiolar lumina was significantly increased. With respect to the CP-treated group, pre-treatment of the sensitized guinea pigs with the CB1 receptor antagonist SR (SR+CP, group 4) or the CB2 receptor antagonist AM (AM+CP, group 5) or both (SR+AM+CP, group 6) before CP administration significantly prevented the reduction of the mean surface area of alveolar air spaces but not the increase of the mean surface area of bronchiolar lumina. Significance of differences (one-way ANOVA): #P < 0.001 versus naive; ***P < 0.001 and *P < 0.05 versus OV; +++P < 0.001 versus CP.
Figure 4
Figure 4
Representative images of Astra Blue-stained mast cells from naive guinea pigs given OV aerosol (A, group 1), sensitized guinea pigs challenged with OV aerosol (B, group 2), sensitized guinea pigs treated with the CB1/CB2 receptor agonist CP before OV challenge (C, group 3), sensitized guinea pigs pretreated with CP in combination with the CB1 receptor antagonist SR (D, group 4), the CB2 receptor antagonist AM (E, group 5) or both (F, group 6). Compared with the animals of group 1, those of group 2 show a clear-cut reduction of mast cell staining intensity. These alterations are not evident in the CP-treated guinea pigs of group 3, but persist in the animals given SR, AM or both 3 hrs before CP (groups 4, 5 and 6, respectively). Astra blue staining. Bars = 10μm. The visual observations are confirmed and objectified by computer-aided densitometry on selected mast cell profiles (G). Significance of differences (one-way ANOVA): #P < 0.001 versus naive; **P < 0.01 versus OV; +P < 0.05 and +++P < 0.001 versus CP.
Figure 5
Figure 5
Lung tissue myeloperoxidase (MPO) activity in the guinea pigs from the different experimental groups. In the guinea pigs treated with the CB1/CB2 receptor agonist CP (group 3) MPO activity is significantly decreased compared with the OV-challenged untreated guinea pigs (group 2). Pre-treatment with the CB1 receptor antagonist SR (SR+CP, group 4) or SR and the CB2 receptor antagonist AM (SR+AM+CP, group 6) before CP administration prevented the reduction of MPO activity, while pre-treatment with AM alone (AM+CP, group 5) had no effects. Significance of differences (one-way ANOVA): #P < 0.001 versus naive; ***P < 0.001 versus OV; +++P < 0.001 versus CP.
Figure 6
Figure 6
Eosinophils positive for eMBP in the lung tissue from naive guinea pigs given OV aerosol (A, group 1), sensitized guinea pigs challenged with OV aerosol (B, group 2), sensitized guinea pigs treated with the CB1/CB2 receptor agonist CP before OV challenge (C, group 3), sensitized guinea pigs pre-treated with CP in combination with the CB1 receptor antagonist SR (D, group 4), the CB2 receptor antagonist AM (E, group 5) or both (F, group 6). Pre-treatment with CP (group 3) reduces the amount of eMBP-positive eosinophils compared with the untreated, OV-challenged guinea pigs (group 2). Pre-treatment with the CB1 receptor antagonist SR (SR+CP, group 4), the CB2 receptor antagonist (AM+CP, group 5) or both (SR+AM+CP, group 6) before CP administration prevents the reduction eMBP-positive eosinophils. Morphometrical analysis (G) confirmed the visual observations. Significance of differences (one-way ANOVA): #P < 0.001 versus naive; ***P < 0.001 versus OV; +P < 0.05 and +++P < 0.001 versus CP.
Figure 7
Figure 7
Lung tissue levels of 8-hydroxy-2´-deoxyguanosine (8-OHdG) in the guinea pigs from the different experimental groups. In the guinea pigs treated with the CB1/CB2 receptor agonist CP (group 3), the levels of 8-OHdG were significantly decreased compared with the untreated, OV-challenged animals (group 2). Pre-treatment with the CB1 receptor antagonist SR (SR+CP, group 4) or SR and the CB2 receptor antagonist AM (SR+AM+CP, group 6) before CP administration prevented the reduction of 8-OHdG. Significance of differences (one-way ANOVA): #P < 0.001 versus naive; ***P < 0.001 versus OV; +++P < 0.001 versus CP.
Figure 8
Figure 8
Levels of tumour necrosis factor-αTNF-α (A) and PGD2 (B) in BAL fluid of guinea pigs from the different experimental groups. In the guinea pigs treated with the CB1/CB2 receptor agonist CP (group 3), the values of TNFα and PGD2 were significantly lower than in the untreated, OV-challenged animals (group 2). Pre-treatment with the CB1 receptor antagonist SR (SR+CP, group 4) or the CB2 receptor antagonist AM (AM+CP, group 5) or both (SR+AM+CP, group 6) before CP administration prevented the CP-induced decrease of TNF and PGD2 levels. Significance of differences (one-way ANOVA): #P < 0.001 versus naive; ***P < 0.001 versus OV; +P < 0.05 and +++P < 0.001 versus CP.
Figure 9
Figure 9
Lung tissue levels of cAMP (A) and cGMP (B) in the guinea pigs from the different experimental groups. In the guinea pigs treated with the CB1/CB2 receptor agonist CP (group 3), the values of cAMP were significantly lower than in the untreated, OV-challenged animals (group 2) and in the naive animals (group 1). Pre-treatment with the CB1 receptor antagonist SR (SR+CP, group 4) or the CB2 receptor antagonist AM (AM+CP, group 5) or both (SR+AM+CP, group 6) before CP administration prevented this decrease. The levels of cGMP were not different among the experimental groups. Significance of differences (one-way ANOVA): ***P < 0.001 versus OV; +++P < 0.001 versus CP.
Figure 10
Figure 10
Western blot analysis of CB1 and CB2 cannabinoid receptors in lung tissue of the guinea pigs from the different experimental groups. The treatment with the CB1/CB2 receptor agonist CP (group 3), alone or in combination with the CB1 receptor antagonist SR (SR+CP, group 4), the CB2 receptor antagonist AM (AM+CP, group 5) or both (SR+AM+CP, group 6) did not cause any modification in the receptor protein expression when compared to the naive (group 1) or the untreated, OV-challenged animals (group 2).
Figure 11
Figure 11
Schematic diagram summarizing the effects of the CB1/CB2 receptor agonist CP55,940 in the current model of allergic asthma-like reaction. CB1, CB2, cannabinoid receptors 1 and 2; MPO, myeloperoxidase; ROS, reactive oxygen species.

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