Effects of Z-VaD-Ala-Asp-Fluoromethyl Ketone (Z-VAD-FMK) and Acetyl-Asp-Glu-Val-Asp-Aldehyde(Ac-DEVD-CHO) on Inflammation and Mucus Secretion in Mice Exposed to Cigarette Smoke

Abstract

Background and Objectives

Smoking can lead to airway inflammation and mucus secretion through the nucleotide-binding domain-like receptor protein 3/caspase-1 pathway. In this study, z-VaD-Ala-Asp-fluoromethyl ketone(Z-VAD), a pan-caspase inhibitor, and acetyl-Asp-Glu-Val-Asp-aldehyde(Ac-DEVD), a caspase-3 inhibitor, were used to investigate the effect of caspase inhibitors on the expression of interleukin(IL)-1β and IL-8, airway inflammation, and mucus secretion in mice exposed to cigarette smoke(CS).

Methods

Thirty-two C57BL/6J male mice were divided into a control group, Smoke group, Z-VAD group, and Ac-DEVD group. Except for the control group, the animals were all exposed to CS for three months. After the experiment, lung function was measured and hematoxylin and eosin staining and periodic acid–Schiff staining were performed. The levels of IL-1β, IL-8, and mucin 5ac(Muc5ac) in serum and bronchoalveolar lavage fluid(BALF) were determined by enzyme-linked immunosorbent assay.

Results

Compared with the control group, the lung function of mice exposed to smoke was poorer, with a large number of inflammatory cells infiltrating around the airway, collapse of alveoli, expansion and fusion of distal alveoli, and formation of emphysema. The Z-VAD group was relieved compared with the smoke group. Airway inflammation was also reduced in the Ac-DEVD group compared with the Smoke group, but the degree of emphysema was not significantly improved. Although Z-VAD relieved airway inflammation and emphysema, Ac-DEVD only relieved inflammation. Z-VAD and Ac-DEVD decreased serum IL-1β and IL-8 levels. In BALF, IL-1β was decreased in Z-VAD group and IL-8 was highest in Smoke +Ac-DEVD group compared with control group and Ac-DEVD group. There was no significant difference in the expression of Muc5ac in serum. However, in BALF, levels of Muc5ac were higher in the smoking group and the lowest in the Ac-DEVD group.

Conclusion

Mice exposed to smoke had decreased lung function and significant cilia lodging, epithelial cell shedding, and inflammatory cell infiltration, with significant emphysema formation. The pan-caspase inhibitor, Z-VAD, improved airway inflammation and emphysema lesions in the mice exposed to smoke and reduced IL-1β and IL-8 levels in serum. The caspase-3 inhibitor, Ac-DEVD, reduced airway inflammation, serum IL-1β and IL-8 levels, and Muc5ac levels in BALF, but it did not improve emphysema.

Keywords:

• cigarette smoke
• Z-VAD-FMK
• Ac-DEVD-CHO
• inflammation
• mucin 5ac

Abbreviations

NLRP3, NOD-like receptor family, pyrin domain containing 3; IL, interleukin; HE, hematoxylin and eosin; PAS, Periodic Acid Schiff; BALF, bronchoalveolar lavage fluid; ELISA, Enzyme-linked immunosorbent assay; Muc5ac, mucin 5ac; Muc5b, mucin 5b; COPD, chronic obstructive pulmonary disease; AAALAC, American Association for Accreditation of Laboratory Animal Care; IACUC, Institutional Animal Care and Use committee; CS, cigarette smoke; RL, lung resistance; Cdyn, dynamic compliance; MVV, maximum minute ventilation; AAI, average alveolar intercept; ANOVA, analysis of variance; SD, standard deviation; ROS, reactive oxygen species; RNS, reactive nitrogen species; TNF, tumor necrosis factor.

Acknowledgments

We thank Bao Wuping for essential expertise, Yu Qiang for assistance with animal feeding, Du Wei (RuiJin Hospital) for his help with lung function assessment, and Deng Jingjing for assistance in pathological sections preparation and instruction of pathology interpretation.

Disclosure

The authors declare no conflicts of interest in this work.

Additional information

Funding

This work was supported by the Shanghai Baoshan District Science and Technology Commission Fund (Grant No. 19-E-47).