Tuberc Respir Dis.  2019 Jan;82(1):71-80. 10.4046/trd.2018.0049.

Expression of Muscarinic Receptors and the Effect of Tiotropium Bromide in Aged Mouse Model of Chronic Asthma

• Kang JY
• Kim IK
• Hur J
• Kim SC
• Lee SY
• Kwon SS
• Kim YK

• KMID: 2441732
• DOI: http://doi.org/10.4046/trd.2018.0049

Abstract

BACKGROUND
Efficacy and safety of tiotropium bromide, a muscarinic receptor antagonist, in treatment of asthma have been reported. However, its effect on airway remodeling in chronic asthma of the elderly has not been clearly verified. The objective of this study was to investigate the effect of tiotropium and expression of muscarinic receptors as its related mechanism in an aged mouse model of chronic asthma with airway remodeling.
METHODS
BALB/c female mice age 6 weeks, 9 and 15 months were sensitized and challenged with ovalbumin (OVA) for three months. Tiotropium bromide was administered during the challenge period. Airway hyperresponsiveness (AHR) and pulmonary inflammation were measured. Parameters of airway remodeling, and expression levels of M2 and M3 receptors were examined.
RESULTS
Total cell with eosinophils, increased in the OVA groups by age, was decreased significantly after treatment with tiotropium bromide, particularly in the age group of 15 months. AHR and levels of interleukin (IL)-4, IL-5, and IL-13 were decreased, after tiotropium administration. In old aged group of 9- and 15-months-treated groups, hydroxyproline contents and levels of α-smooth muscle actin were attenuated. Tiotropium enhanced the expression of M2 but decreased expression of M3 in all aged groups of OVA.
CONCLUSION
Tiotropium bromide had anti-inflammatory and anti-remodeling effects in an aged mouse model of chronic asthma. Its effects seemed to be partly mediated by modulating expression M3 and M2 muscarinic receptors. Tiotropium may be a beneficial treatment option for the elderly with airway remodeling of chronic asthma.

Keyword

Receptors, Muscarinic; Ageing; Asthma; Tiotropium Bromide; Airway Remodeling

MeSH Terms

Actins
Aged
Airway Remodeling
Animals
Asthma*
Eosinophils
Female
Humans
Hydroxyproline
Interleukin-13
Interleukin-5
Interleukins
Mice*
Ovalbumin
Ovum
Pneumonia
Receptors, Muscarinic*
Tiotropium Bromide*
Actins
Hydroxyproline
Interleukin-13
Interleukin-5
Interleukins
Ovalbumin
Receptors, Muscarinic
Tiotropium Bromide

Figure

• 

  Figure 1 Effect of tiotropium bromide on airway hyperresponsiveness in a mouse model of chronic asthma with aging: 6-week-old group (A), 9-month-old group (B), and 15-month-old group (C). 6w: 6 weeks old; 9M: 9 months old; 15M: 15 months old; OVA: ovalbumin; Tio: tiotropium bromide. *p<0.05, **p<0.01, ***p<0.001 compared with the OVA group.

• 

  Figure 2 Effects of tiotropium bromide on pulmonary inflammation in a mouse model of chronic asthma with aging. (A) Inflammatory cell counts in bronchoalveolar lavage fluids. (B) Hematoxylin and eosin–stained section of lungs (×200). Values represent mean±standard error of the mean (n=5–8 per group). 6w: 6 weeks old; 9M: 9 months old; 15M: 15 months old; OVA: ovalbumin; Tio: tiotropium bromide. *p<0.05 and ***p<0.001 compared with the OVA group.

• 

  Figure 3 Effects of tiotropium bromide on levels of type 2 cytokines (A, interleukin [IL]-4; B, IL-5; and C, IL-13) in bronchoalveolar lavage fluid in a mouse model of chronic asthma with aging. Values are presented as mean±standard error of the mean (n=5–8 per group). 6w: 6 weeks old; 9M: 9 months old; 15M: 15 months old; OVA: ovalbumin; Tio: tiotropium bromide. *p<0.05 and ***p<0.001 compared with the OVA group.

• 

  Figure 4 Effect of tiotropium bromide on goblet cell hyperplasia in airway remodeling in a mouse model of chronic asthma with aging. (A) Representative photomicrographs of periodic acid Schiff (PAS)-stained lung sections (×200) and quantification of the PAS-stained area by point scoring as described in Materials and Methods. (B) Expression of mRNA of MUC5AC gene by real-time polymerase chain reaction. 6 w: 6 weeks old; 9M: 9 months old; 15M: 15 months old; OVA: ovalbumin; Tio: tiotropium bromide. ***p<0.001 compared with the OVA group.

• 

  Figure 5 Effect of tiotropium bromide on collagen deposition in airway remodeling in a mouse model of chronic asthma with aging. (A) Immunohistochemical staining for collagen V in lung tissues (×200). (B) Measurement of hydroxyproline content to quantify collagen expression in lung tissues. 6 w: 6 weeks old; 9M: 9 months old; 15M: 15 months old; OVA: ovalbumin; Tio: tiotropium bromide. **p<0.01 compared with the OVA group.

• 

  Figure 6 Effect of tiotropium bromide on smooth muscle hyperplasia in airway remodeling in a mouse model of chronic asthma with aging. (A) Representative photomicrographs of lung sections immunostained α-smooth muscle actin (α-SMA) (×200). (B) Image analysis of areas of immunostaining per micron length of basement membrane in bronchioles. 6 w: 6 weeks old; 9M: 9 months old; 15M: 15 months old; OVA: ovalbumin; Tio: tiotropium bromide. **p<0.01 and ***p<0.001 compared with the OVA group.

• 

  Figure 7 Effect of tiotropium bromide on the expression of M2 and M3 muscarinic receptors in a murine model of chronic asthma with aging. (A) Western blot of M2 and M3 subtypes relative to β-actin in lung tissues. (B) Densitometric measurements for M2 and M3 subtypes. 6w: 6 weeks old; 9M: 9 months old; 15M: 15 months old; OVA: ovalbumin; Tio: tiotropium bromide.

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