Oral Angiotensin-(1-7) formulation after established elastase-induced emphysema suppresses inflammation and restores lung architecture

Abstract

Background: Chronic obstructive pulmonary disease (COPD), a prevalent age-related condition, ranks among the leading causes of global mortality. It is characterized by chronic inflammation, cellular senescence, and irreversible lung tissue damage, with no curative treatments currently available. Angiotensin-(1-7) [Ang-(1-7)] has demonstrated anti-inflammatory and regenerative potential in preclinical models. This study aimed to investigate the therapeutic effects of oral Ang-(1-7) on senescence, inflammation, and tissue regeneration in a model of elastase-induced pulmonary emphysema.

Methods: Male C57BL/6 mice were subjected to emphysema induction through three intratracheal instillations of porcine pancreatic elastase (PPE). One week after the final elastase instillation, the mice were treated with Ang-(1-7) encapsulated in hydroxypropyl-β-cyclodextrin to enhance its bioavailability. The treatment was administered daily for 4 weeks. Histological assessments, gene expression analysis, and protein quantification through Western blot were performed to evaluate lung architecture, inflammation, and senescence markers.

Results: The results showed that elastase exposure led to significant lung damage, including enlarged airspaces, increased collagen deposition and upregulated expression of collagen I/III and MMP9. Markers of inflammation and senescence were significantly elevated in the untreated emphysema group. However, treatment with Ang-(1-7) reversed these changes, reducing collagen deposition, restoring alveolar structure, and suppressing inflammation and senescence. Additionally, Ang-(1-7) modulated key signaling pathways, reactivating the Wnt/β-catenin pathway for tissue regeneration and inhibiting NF-κB activation, critical for inflammation suppression.

Conclusion: These findings suggest that Ang-(1-7), when administered after disease establishment, demonstrates potential to reverse structural lung damage and suppress chronic inflammation in experimental models, indicating a promising direction for future translational and clinical research in COPD.

Keywords: NF-κB modulation; Wnt/β-catenin pathway; alveolar regeneration; cellular senescence; chronic inflammation.

FIGURE 1

Characterization of 3 weeks evolution elastase-induced pulmonary emphysema (EIPE) Schematic timeline of the experimental design: (A) The pulmonary emphysema (PE) group received three intratracheal instillations of pancreatic porcine elastase (PPE) at weekly intervals (0.2 IU in 30 μL saline, n = 6). The control (CTRL) group received saline instillations at the same volume and intervals (n = 5–6). (B) Representative histological images of lung sections stained with picrosirius red (scale bar 100 µm) from CTRL and PE, with the graph showing quantification of collagen fibers in the lungs. As seen, elastase-challenged mice exhibited significant peribronchial and perivascular fibrosis (indicated by asterisks). (C) Representative histological images of lung sections stained with hematoxylin/eosin (scale bar 50 µm) from CTRL and PE. The elastase-challenged group showed increased airspace (asterisks) and reduced alveolar area compared to CTRL mice. Gene expression for (D,E) collagen types I and III, (F) MMP9, and (G) VEGF. Bars show mean ± SEM from four to six animals per group. *p ≤ 0.05 compared to CTRL (Student’s t-test).

FIGURE 2

Characterization of inflammatory and pro-senescence markers in EIPE (A–C) Gene expression levels for inflammatory markers (n = 5–6): NF-κB1, NLRP3, and IL-1β. (D) Protein levels of phosphorylated NF-κB (p-NF-κB). (E–G) Gene expression of cellular senescence markers: p53, p16, and p21. (H,I) Expression of anti-senescent and antioxidant factors Klotho and Nrf2. Data are presented as mean ± SEM from four to six animals per group. *p ≤ 0.05 compared to CTRL (Student’s t-test).

FIGURE 3

Effects of Oral Ang-(1-7) Treatment on Lung Remodeling in EIPE Schematic timeline of the experimental design: (A) Mice were allocated into control (CTRL) and pulmonary emphysema (PE) groups. The PE group received three weekly intratracheal elastase instillations (0.2 IU in 50 μL saline). One week after the final installation, PE mice were subdivided into PE and PE + Ang-(1-7) groups, with the latter receiving Ang-(1-7) (60 μg/kg and 92 μg/kg HPβCD) via gavage for 4 weeks. Control and PE groups received the vehicle (92 μg/kg HPβCD) via the same route (n = 5–7). (B) Seven weeks post-elastase exposure, the PE group showed increased lung collagen deposition (asterisk in picrosirius red-stained sections, scale bar: 100 μm). The graph represents the quantification of collagen fibers in the lungs. (C) Elastase exposure led to enlarged air spaces (asterisk in Hematoxylin/Eosin-stained sections, scale bar: 50 μm) and decreased alveolar area. The graph quantifies the changes in tissue area and air space percentage. (D–G) Gene expression levels of collagen I, collagen III, MMP9 and VEGF in the different groups. Bars represent mean ± SEM from four to six animals per group. *p ≤ 0.05 compared to CTRL, #p ≤ 0.05 compared to PE (one-way ANOVA followed by Tukey post hoc test).

FIGURE 4

Angiotensin-(1-7) Mitigates Inflammation and Cellular Senescence in EIPE. Gene expression of inflammatory markers (A) NF-κB1, (B) NLRP3, and (C) IL-1β, along with senescence markers (D) p53, (E) p16, and (F) p21. (G) (D) Protein levels of p53 (n = 5–7). Gene expression of (H) Klotho and (I) Nrf2. Bars represent mean ± SEM from four to six animals per group. *p ≤ 0.05 compared to CTRL, #p ≤ 0.05 compared to PE (one-way ANOVA followed by Tukey post hoc test).

FIGURE 5

Ang-(1-7) Promotes Lung Repair by Modulating Cell Signaling Pathways in EIPE. (A) Expression of non-phosphorylated β-catenin and (B) phosphorylated NF-κB (p-NF-κB) in lung tissue after elastase exposure and treatment with Ang-(1-7). Bars represent mean ± SEM from four to six animals per group (n = 5–6). *p ≤ 0.05 compared to CTRL, #p ≤ 0.05 compared to PE (one-way ANOVA followed by Tukey post hoc test).

FIGURE 6

Schematic representation of the pathological mechanisms driving emphysema and the therapeutic actions of Ang-(1-7). On the left, the cycle of alveolar destruction induced by elastase is depicted, where inflammatory mediators activate senescence pathways, which in turn amplify inflammation. This vicious cycle leads to persistent tissue damage and ineffective repair. On the right, Ang-(1-7) exerts robust therapeutic effects by markedly reducing inflammatory mediators and senescence markers, suppressing NF-κB signaling, and activating the β-catenin pathway, which may contribute to the inhibition of NF-κB and plays a critical role in promoting effective alveolar regeneration. These findings highlight the powerful pro-regenerative and anti-inflammatory potential of Ang-(1-7) in the context of chronic lung injury.