Ion therapy of pulmonary fibrosis by inhalation of ionic solution derived from silicate bioceramics

Abstract

Pulmonary fibrosis (PF) is a chronic and progressively fatal disease, but clinically available therapeutic drugs are limited due to efficacy and side effects. The possible mechanism of pulmonary fibrosis includes the damage of alveolar epithelial cells II (AEC2), and activation of immune cells such as macrophages. The ions released from bioceramics have shown the activity in stimulating soft tissue derived cells such as fibroblasts, endothelia cells and epithelia cells, and regulating macrophage polarization. Therefore, this study proposes an "ion therapy" approach based on the active ions of bioceramic materials, and investigates the therapeutic effect of bioactive ions derived from calcium silicate (CS) bioceramics on mouse models of pulmonary fibrosis. We demonstrate that silicate ions significantly reduce pulmonary fibrosis by simultaneously regulating the functions of AEC2 and macrophages. This result suggests potential clinical applications of ion therapy for lung fibrosis.

Keywords: Alveolar epithelial cells II; Lung fibrosis; Macrophages; Silicate bioceramics.

Graphical abstract

Fig. 1

The therapeutic effect of CS or 1/8CS ionic solution on BLM-induced lung fibrosis in mice. (A) Treatment of lung fibrosis with ionic solution derived from silicate bioceramics. (B) The mortality of mice within 49 days after BLM modeling. (C) ALI score results of mice in each group on D7 and D21. (D) H&E staining results of mouse lung tissue sections on D7 and D21 (Scale = 1 mm). (n = 5, *P < 0.05).

Fig. 2

The effect of CS or 1/8CS ionic solution on BLM-induced lung fibrosis in mice. (A) Masson stained images of mouse lung tissue sections on D7 and D21. (Scale = 200 μm). (B) Results of lung fibrosis score. (C) Quantitative results of hydroxyproline. (n = 5, *P < 0.05, **P < 0.01).

Fig. 3

The number of various types of inflammatory cells in BALF after the treatment of lung fibrosis mice with CS or 1/8CS ionic solution. (A) Total inflammatory cell count in BALF of mice on D7 and D21. (B) Count of macrophages, (C) CD3⁺CD8+T lymphocytes, (D) CD3⁺CD4+T lymphocytes, and (E) neutrophils in BALF of mice on D7 and D21. (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 4

The effect of different concentrations of CS ionic solution on the cell viability (A) and migration (B) of A549, Beas-2B and MRC-5. (n = 4, *P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 5

(A–C) ELISA detects the concentration of TNF-α (A), TGF-β (B) and IL-10 (C) after stimulating Raw 264.7 with different concentrations of CS ionic solution. (D–I) Analysis of inflammatory factors (TNFα(D), G-CSF(E), IL-6(F), MIP-1α(G), KC(H) and IL-1α (I)) in BALF of BLM-induced lung fibrosis mice after the treatment with 1/8 CS or CS ionic solutions on D7 and D21. (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 6

Flow cytometry analysis of cells after treatment with different concentrations of CS ionic solution directly or indirectly affect the differentiation of naïve T cells into Treg (Directly affect the differentiation (CS): Lymphocytes were cultured by using CS solution.; Indirectly affect the differentiation (CM-CS): Lymphocytes were cultured through the supernatant (CS solution) produced by culturing macrophages for 72 h). (A) The proportion of Treg cells. (B) The proportion of Treg cells in the direct stimulation group. (C) The proportion of Treg cells in the indirect stimulation group. (n = 4) (D, E) On D7 (D) and D21 (E) days after BLM modeling, cells in mouse BALF were collected and the proportion of Treg was detected by flow cytometry.

Fig. 7

The mechanism hypothesis of CS ionic solution in the treatment of lung fibrosis. SiO₃²⁻ can promote the cell viability and migration of AECⅡ to repair lung injury and reduce lung fibrosis. SiO₃²⁻ can inhibit the secretion of inflammatory factors (IL-1α, TNF-α, etc.) of macrophages and reduce lung fibrosis. The reduction of TNFα can inhibit the maturation and chemotaxis of neutrophils, and reduce the differentiation and expansion of Treg, thereby reducing lung fibrosis.