Effects of a Peptide Derived from the Primary Sequence of a Kallikrein Inhibitor Isolated from Bauhinia bauhinioides (pep-BbKI) in an Asthma-COPD Overlap (ACO) Model

Abstract

(1) There are several patients with asthma-COPD overlap (ACO). A peptide derived from the primary sequence of a kallikrein inhibitor isolated from Bauhinia bauhinioides (pep-BbKI) has potent anti-inflammatory and antioxidant effects. Purpose: To investigate the effects of pep-BbKI treatment in an ACO model and compare them with those of corticosteroids. (2) BALB/c mice were divided into groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep-BbKI (treated with inhibitor), ACO-DX (dexamethasone treatment), ACO-DX-pep-BbKI (both treatments), and SAL-pep-BbKI (saline group treated with inhibitor). We evaluated: hyperresponsiveness to methacholine, bronchoalveolar lavage fluid (BALF), exhaled nitric oxide (eNO), IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ, TNF-α, MMP-9, MMP-12, TGF-β, collagen fibers, iNOS, eNO, linear mean intercept (Lm), and NF-κB in airways (AW) and alveolar septa (AS). (3) ACO-pep-BbKI reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, neutrophils, IL-5, IL-10, IL-17, IFN-γ, TNF-α, MMP-12 (AW), collagen fibers, iNOS (AW), and eNO (p > 0.05). ACO-DX reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, total cells and differentials, IL-1β(AS), IL-5 (AS), IL-6 (AS), IL-10 (AS), IL-13 (AS), IFN-γ, MMP-12 (AS), TGF-β (AS), collagen fibers (AW), iNOS, and eNO (p > 0.05). SAL was similar to SAL-pep-BbKI for all comparisons (p > 0.05). (4) Pep-BbKI was similar to dexamethasone in reducing the majority of alterations of this ACO model.

Keywords: ACO; Bauhinia; airway remodeling; inflammation; oxidative stress; serine proteinase inhibitors.

Figure 1

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

Figure 2

Evaluation of the number of cells in bronchoalveolar lavage fluid (BALF). The (a) total numbers of cells, (b) macrophages, (c) neutrophils, (d) eosinophils, and (e) lymphocytes are expressed in ×10⁴ cells/mL. * p < 0.05 compared to SAL group; ** p < 0.05 compared to OVA and ELA groups; # p < 0.05 compared to ACO group; + p < 0.05 compared to SAL and OVA groups; ++ p < 0.05 compared to OVA group; $ p < 0.05 compared to ACO-DX group. N = 8 for each group.

Figure 3

Evaluation of mean linear intercepts; the results are expressed in micrometers (μm). * p < 0.05 compared to SAL group; ** p < 0.05 compared to OVA and ELA groups; # p < 0.05 compared to ACO group; $ p < 0.05 compared to ACO-DX group. N = 8 for each group.

Figure 4

Graphs of evaluation of oxidative stress and signaling pathway: (a) iNOS in airways and (b) iNOS in alveolar septa are expressed in cells/10⁴ µm²; (c) exhaled nitric oxide is expressed in ppb; (d) NF-κB in airways and (e) NF-κB in alveolar septa are expressed in cells/10⁴ µm². * p < 0.05 compared to SAL group; # p < 0.05 compared to ACO group; ++ p < 0.05 compared to OVA group; $$ p < 0.05 compared to ACO-pep-BbKI group. N = 8 to each group. iNOS, inducible nitric oxide synthase; NF, nuclear factor.

Figure 5

Qualitative analysis of inflammatory marker (IL-5), remodeling marker (MMP-12), oxidative stress (iNOS), and signaling pathway (NF-κB). Photomicrographs of the results of the immunohistochemical analyses show the presence of inflammation around the airways. The red arrows indicate positive cells for IL-5, MMP-12, iNOS and NF-κB. Magnification of 400×. The experimental groups include SAL, OVA, ELA, ACO, ACO-pep-BbKI, ACO-DX, and ACO-DX-pep-BbKI. IL, interleukin; MMP, metalloproteinase; iNOS, inducible nitric oxide synthase; NF, nuclear factor.

Figure 6

Qualitative analysis of inflammatory marker (IL-5), remodeling marker (MMP-12), oxidative stress (iNOS), and signaling pathway (NF-κB). Photomicrographs of the results of the immunohistochemical analyses show the presence of inflammation in the alveolar septa. The red arrows indicate positive cells for IL-5, MMP-12, iNOS and NF-κB. Magnification of 400×. The experimental groups include SAL, OVA, ELA, ACO, ACO-pep-BbKI, ACO-DX, and ACO-DX-pep-BbKI. IL, interleukin; MMP, metalloproteinase; iNOS, inducible nitric oxide synthase; NF, nuclear factor.

Figure 7

Schematic of the experimental protocols. (a) Control mice received saline intraperitoneal (Days 1 and 14) and nebulization with saline (Days 21, 23, 25, and 27); (b) Mice were sensitized with ovalbumin intraperitoneally (Days 1 and 14) and received nebulization with ovalbumin (Days 21, 23, 25 and 27); (c) Mice received intratracheal elastase (Day 21); (d) Mice received intraperitoneal ovalbumin (Days 1 and 14), intratracheal elastase (Day 21) and nebulization with ovalbumin (Days 21, 23, 25 and 27); (e) Mice received intraperitoneal ovalbumin (Days 1 and 14), intratracheal elastase (Day 21), nebulization with ovalbumin (Days 21, 23, 25 and 27) and were treated with intraperitoneal pep-BbKI (Days 22, 23, 25 and 27); (f) Mice received intraperitoneal ovalbumin (Days 1 and 14), intratracheal elastase (Day 21) and nebulization with ovalbumin (Days 21, 23, 25 and 27), and were treated with intraperitoneal dexamethasone (Days 22, 23, 25 and 27); (g) Mice received intraperitoneal ovalbumin (Days 1 and 14), intratracheal elastase (Day 21), nebulization with ovalbumin (Days 21, 23, 25 and 27) and were treated with intraperitoneal dexamethasone and pep-BbKI (Days 22, 23, 25 and 27); (h) Mice received intraperitoneal saline (Days 1 and 14) and nebulization with saline (Days 21, 23, 25, and 27) and were treated with pep-BbKI (Days 22, 23, 25 and 27). IP, intraperitoneal injection; IT, intratracheal instillation; N, nebulization; PPE, porcine pancreatic elastase.