Investigating the Effects of a New Peptide, Derived from the Enterolobium contortisiliquum Proteinase Inhibitor (EcTI), on Inflammation, Remodeling, and Oxidative Stress in an Experimental Mouse Model of Asthma-Chronic Obstructive Pulmonary Disease Overlap (ACO)

Abstract

The synthesized peptide derived from Enterolobium contortisiliquum (pep3-EcTI) has been associated with potent anti-inflammatory and antioxidant effects, and it may be a potential new treatment for asthma-COPD overlap-ACO). Purpose: To investigate the primary sequence effects of pep3-EcTI in an experimental ACO. BALB/c mice were divided into eight groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep3-EcTI (treated with inhibitor), ACO-DX (treated with dexamethasone), ACO-DX-pep3-EcTI (treated with dexamethasone and inhibitor), and SAL-pep3-EcTI (saline group treated with inhibitor). We evaluated the hyperresponsiveness to methacholine, exhaled nitric oxide, bronchoalveolar lavage fluid (BALF), mean linear intercept (Lm), inflammatory markers, tumor necrosis factor (TNF-α), interferon (IFN)), matrix metalloproteinases (MMPs), growth factor (TGF-β), collagen fibers, the oxidative stress marker inducible nitric oxide synthase (iNOS), transcription factors, and the signaling pathway NF-κB in the airways (AW) and alveolar septa (AS). Statistical analysis was conducted using one-way ANOVA and t-tests, significant when p < 0.05. ACO caused alterations in the airways and alveolar septa. Compared with SAL, ACO-pep3-EcTI reversed the changes in the percentage of resistance of the respiratory system (%Rrs), the elastance of the respiratory system (%Ers), tissue resistance (%Gtis), tissue elastance (%Htis), airway resistance (%Raw), Lm, exhaled nitric oxide (ENO), lymphocytes, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, TNF-α, INF-γ, MMP-12, transforming growth factor (TGF)-β, collagen fibers, and iNOS. ACO-DX reversed the changes in %Rrs, %Ers, %Gtis, %Htis, %Raw, total cells, eosinophils, neutrophils, lymphocytes, macrophages, IL-1β, IL-6, IL-10, IL-13, IL-17, TNF-α, INF-γ, MMP-12, TGF-β, collagen fibers, and iNOS. ACO-DX-pep3-EcTI reversed the changes, as was also observed for the pep3-EcTI and the ACO-DX-pep3-EcTI. Significance: The pep3-EcTI was revealed to be a promising strategy for the treatment of ACO, asthma, and COPD.

Keywords: airway remodeling; asthma–chronic obstructive pulmonary disease overlap; inflammation; oxidative stress; protease inhibitors.

Figure 1

Mechanical evaluation of (a) %Rrs, respiratory system resistance; (b) %Ers, respiratory system elastance; (c) %Gtis, tissue resistance; (d) %Htis, lung tissue elastance; and (e) %Raw, airway resistance. * p < 0.05 compared to the SAL group; # p < 0.05 compared to OVA group; $ p < 0.05 compared to ELA group; ** p < 0.05 compared to ACO group; N = 8 for each group.

Figure 1

Mechanical evaluation of (a) %Rrs, respiratory system resistance; (b) %Ers, respiratory system elastance; (c) %Gtis, tissue resistance; (d) %Htis, lung tissue elastance; and (e) %Raw, airway resistance. * p < 0.05 compared to the SAL group; # p < 0.05 compared to OVA group; $ p < 0.05 compared to ELA group; ** p < 0.05 compared to ACO group; N = 8 for each group.

Figure 2

Evaluation of the number of cells in BALF. (a) Total numbers of cells; (b) eosinophils, (c) lymphocites, (d) macrophages, and (e) neutrophils in ×10⁴ cells/mL. * p < 0.05 compared to the SAL group; # p < 0.05 compared to the OVA group; $ p < 0.05 compared to the ELA group; ** p < 0.05 compared to the ACO group, + p < 0.05 compared to the ACO-pep3-ECTI group; +++ p < 0.05: compared to the ACO-DX-pep3-ECTI group. N = 8 for each group.

Figure 2

Evaluation of the number of cells in BALF. (a) Total numbers of cells; (b) eosinophils, (c) lymphocites, (d) macrophages, and (e) neutrophils in ×10⁴ cells/mL. * p < 0.05 compared to the SAL group; # p < 0.05 compared to the OVA group; $ p < 0.05 compared to the ELA group; ** p < 0.05 compared to the ACO group, + p < 0.05 compared to the ACO-pep3-ECTI group; +++ p < 0.05: compared to the ACO-DX-pep3-ECTI group. N = 8 for each group.

Figure 3

Qualitative analysis of the inflammatory marker (IL-5), the remodeling marker (MMP-12), oxidative stress (iNOS), and the signaling pathway (NF-κB). Photomicrographs of the results of the immunohistochemical analyses show the presence of inflammation in the alveolar septa (magnification of 400×). The experimental groups include SAL, OVA, ELA, ACO, ACO-pep3-EcTI, ACO-DX, and ACO-DX-pep3-EcTI.

Figure 4

Schematic diagram describing the study protocol. (a) The control SAL group received intraperitoneal saline (days 1 and 14) and nebulization with saline (days 21, 23, 25, and 27). The control SAL-pep3-EcTI group received intraperitoneal saline (days 1 and 14), nebulization with saline (days 21, 23, 25, and 27), and pep3-EcTI (days 22, 23, 25, and 27). (b) The OVA group was sensitized with intraperitoneal ovalbumin (days 1 and 14) and received nebulization with ovalbumin (days 21, 23, 25, and 27). The ELA group received intratracheal elastase (day 21). The ACO group received intraperitoneal ovalbumin (days 1 and 14), intratracheal elastase (day 21), and nebulization with ovalbumin (days 21, 23, 25, and 27). (c) The treatment group ACO-pep3-EcTI received intraperitoneal ovalbumin (days 1 and 14), intratracheal elastase (day 21), nebulization with ovalbumin (days 21, 23, 25, and 27), and intraperitoneal pep3-EcTI (days 22, 23, 25, and 27). The treatment group ACO-DX received intraperitoneal ovalbumin (days 1 and 14), intratracheal elastase (day 21), nebulization with ovalbumin (days 21, 23, 25, and 27), and intraperitoneal dexamethasone (days 22, 23, 25, and 27). The treatment group ACO-DX-pep3-EcTI received intraperitoneal ovalbumin (days 1 and 14), intratracheal elastase (day 21), nebulization with ovalbumin (days 21, 23, 25, and 27), and intraperitoneal dexamethasone and pep3-EcTI (days 22, 23, 25, and 27). IP, intraperitoneal injection; IT, intratracheal instillation; N, nebulization, PPE, porcine pancreatic elastase; BALF, bronchoalveolar lavage.