| M3 muscarinic receptors are localized on inflammatory cells, airway smooth muscle, and submucosal glands, known to mediate bronchoconstriction, mucus secretion and airway remodeling. It is hypothesized bencycloquidium bromide (BCQB), a novel M3 receptor antagonist, might have potential effects on airway hyperresponsiveness, inflammation and airway remodeling in a murine model of asthma. Mice sensitized and challenged with ovalbumin developed airway inflammation. Inhalation administration of BCQB.Bronchoalveolar lavage fluid was examined to determine the total and differential cell counts, and cytokine levels. Lung tissues were evaluated for cell infiltration, mucus hypersecretion, airway remodeling, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. On 4 consecutive days mice were exposed to cigarette smoking(CS). CS exposure mice were administered inhaled BCQB. Bronchoalveolar lavage fluid was examined to determine the total and differential cell counts, and cytokine levels. Lung tissues were evaluated for cell infiltration, mRNA expression of cytokines and chemotactic factor. The results include:1. The effect of BCQB on airway inflammation in sensitized mice:Aerosol pretreatment with BCQB at 60,120 and 240μg/ml significatly inhibited the infiltration of inflammatory cells in a dose-dependent manner.The inhibitory rates were 30.8%, 45.8% and 65.5%. The ID50 (95%CI) is 133.69(106.53-167.78)μg/ml; The inhibitory rates of eosinophil were 49.1%,54.0% and 76.7%, The ID50 (95%CI) is 72.96(51.04-104.28)μg/ml; It is no significantly effect of lymphocytes and macrophages. Histologically, Ovalbumin-vehicle mice exhibited an obvious infiltration of inflammatory cells into the peribronchiolar and perivascular connective tissues. BCQB 120 or 240μg/ml and ipratropium bromide markedly attenuated ovalbumin-induced eosinophil infiltration.2. The effect of BCQB on Chemokine and cytokine in the lung tissue in sensitized mice. BCQB 240μg/ml significatly inhibited the eotaxin mRNA expression. BCQB significantly inhibited IL-4 and IL-5 mRNA expression in a dose-dependent manner, increased the IFN-y mRNA expression and up-regulation the rate of IFN-y/IL-4 IFN-y/IL-5 mRNA. Aerosol pretreatment with BCQB at 60,120 and 240μg/ml significatly increased the IFN-y level in BALF, but no effect of IL-4.3. The effect of BCQB on airway remodeling in sensitized mice:MMP-9 and TIMP-1 mRNA expression were not inhibited by BCQB and 60μg/mL Ipra but BCQB 120 and 240μg/ml markedly increase the value of TIMP-1 divided by MMP-9 mRNA expressing. On 7 consecutive days sensitized mice were exposed to OVA and goblet cell hyperplasia was markedly increase as compared to control group. BCQB 120ug/ml, BCQB 240μg/ml and Ipra 60μg/ml significantly decreased the ratio of goblet cell hyperplasia as compared to model group. The effects of BCQB 60μg/ml on goblet cell hyperplasia was not significant. The collagen deposition was markly increase in model group and BCQB 60,120 and 240μg/ml were dose-dependently decreased collagen deposition in airway. The effects of BCQB 60μg/ml on collagen deposition was not significant,4. The effect of BCQB on airway hyperresponsiveness (AHR) in sensitized mice: BCQB 60μg/mL,120μg/mL and 240μg/mL dose-dependently decrease Raw and increase Cdyn as compared with the mice of asthma model. The provocation concentration (PC) of methacholine which cumulatively produced a 100% increase in lung resistance (PC100) and which cumulatively produced a 25% decrease in lung compliance (PC25) were dose-dependently increased by BCQB 60μg/mL,120μg/mL和240μg/mL.5. The anti-inflammation effects of BCQB in cigarette-induced airway inflammation:BCQB 75,150,300μg/ml inhaleted from airway were dose-dependently decreased the total cells, neutrophil and macrophage numbers in the broncho alveolarlavage fluid (BALF). The effects of BCQB on lymphocytes numbers in the BALF were not significantly compared to model group. The neutrophil and macrophage infiltration were markly increase in lung tissues of model group. The neutrophil and macrophage infiltration were decreased by BCQB 150,300μg/ml and Tio 150μg/ml in the small airway6. The effects of BCQB on MPO and SOD in cigarette-exposed lung tissue. BCQB 300μg/ml markly decreased the MPO level and increased the SOD level in lung tissue compared to model group; Tio150μg/ml markly decreased the MPO level but didn't improve the SOD level in lung tissue.7. The effects of BCQB on cytokines in lung: BCQB 75.150 and 300μg/ml dose-dependently decreased the TNF-a mRNA expression; BCQB 300μg/ml markley attenuated the IL-1βand IL-8 mRNA expression increasing in lung tissues compared to model group. TNF-a and IL-1βprotein level in lung tissues was significantly reduced by BCQB 300μg/ml.8. The effects of BCQB on KC and MCP-1 in lung:Aerosol pretreatment with BCQB at 75,150 and 300μg/ml significantly inhibited the KC and MCP-1 mRNA expression in a dose-dependent manner. The Tio at 150μg/ml can significantly inhibited KC and MCP-1 mRNA expression.Conclusion:BCQB significantly inhibited the airway inflammation, remodeing and hyperresponsiveness in OVA-induced mice. The mechanism is related to regulation of Thl/Th2 balance and decreased the cytokine mRNA expression.BCQB significantly inhibited the airway inflammation and oxidative stress. The mechanism is related to inhibited the expressions of inflammatory cytokine mRNA and protein. |
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