Polydatin-curcumin formulation alleviates CTD-ILD-like lung injury in mice via GABBR/PI3K/AKT/TGF-β pathway

Abstract

Connective tissue disease-associated interstitial lung disease (CTD-ILD) is a systemic autoimmune disease with high morbidity and hazard, characterized by progressive pulmonary inflammation and fibrosis. The monomer formulation of polydatin and curcumin (PD + Cur) for lung injury in CTD-ILD was optimized from Curcumae Longae Rhizoma (Curcuma Longa L.) and Polygoni Cuspidati Rhizoma Et Radix (Polygonum cuspidatum Sieb. et Zucc.). Mice with CTD-ILD-like lung injury were established by a single intratracheal drip of bleomycin. After intervening in model mice for 4 weeks, PD + Cur attenuated alveolar atrophy, fibrillar collagen formation, and thickened alveolar septa in the lung, improved serum biomarkers TOLLIP, MUC5B, KL-6, SP-D, and RCN3, and suppressed serum immunoinflammatory factors IL-6, CCL-18, and SF. The transcriptome sequencing showed that PD + Cur ameliorated CTD-ILD mainly by regulating aberrant immunoinflammation, which was further confirmed by proteomics that the PI3K/AKT/TGF-β pathway was a key pathway. Further, PD + Cur was found to affect amino acid metabolism in the serum significantly. The B-type receptor for GABA (GABBR) agonist baclofen was further found to attenuate CTD-ILD-like lung injury and modulate PI3K/AKT/TGF-β signaling. However, the inhibition of AKT, transforming growth factor beta receptor type 3 (TGFβR3), a key indicator downstream of PI3-kinase subunit p85-alpha (PI3KR1), by PD + Cur was reversed after intervention with the GABBR receptor inhibitor CGP52432. PD + Cur has an ameliorative effect on CTD-ILD-like lung injury by targeting GABBR to modulate the PI3K/AKT/TGF-β pathway.

Keywords: CTD-ILD; GABBR; PI3K/AKT/TGF-β; curcumin; polydatin.

FIGURE 1

Model evaluation of CTD-ILD-like lung injury in mice (A) H&E staining of lung tissue (×200): mild to moderate atrophy of alveoli (white arrow), mild to moderate fibrous tissue proliferation in the interstitium and thickening of alveolar septa (black arrow), and increased fibroblasts (green arrow) and inflammatory cells (red arrow) in the model group, and alveolar septal thickening and interstitial inflammatory cell infiltration scores; (B) Masson staining of lung tissue (×200): a large amount of fibrillar collagen formation (blue arrows) in the model group, and fibrous hyperplasia score; (C–D) The level of serum TOLLIP and MUC5B, biomarkers of risk and predisposition in CTD-ILD (E–G) The level of serum KL-6, SP-D, and RCN3, biomarkers of epithelial cell dysfunction and extracellular matrix remodeling in CTD-ILD; (H–J) The level of immunoinflammation-related indicators IL-6, CCL18, and SF in serum. Data are presented as mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001 for vs Control; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 for vs Model; Mann-Whitney U for figure (A–B), and the Least Significant Difference test for figure (C–J). n = 3 in figure (A–J).

FIGURE 2

Efficacy of PD + Cur on CTD-ILD-like lung injury of model mice (A) H&E staining of lung tissue (×200): mild to moderate atrophy of alveoli (white arrow), and mild to moderate fibrous tissue proliferation in the interstitium and thickening of alveolar septa (black arrow) in the model group; (B) Masson staining of lung tissue (×200): a large amount of fibrillar collagen formation (blue arrows) in the model group; (C–D) The level of serum TOLLIP and MUC5B, biomarkers of risk and predisposition in CTD-ILD; (E–G) The level of serum KL-6, SP-D, and RCN3, biomarkers of epithelial cell dysfunction and extracellular matrix remodeling in CTD-ILD; (H–J) The level of immunoinflammation-related indicators IL-6, CCL18, and SF in serum. Data are presented as mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001 for vs Control; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 for vs Model; Games-Howell for RCN3, the Least Significant Difference test for others. n = 3.

FIGURE 3

The results of RNA-seq and untargeted proteomics between PD + Cur vs Model (A–B) Differentially expressed genes (blue or red dots) and pathway enrichment between PD + Cur vs Model by RNA-seq analysis, n = 6; (C–D) Differentially expressed genes (blue or red dots) and pathway enrichment between PD + Cur vs. Model by untargeted proteomics. n = 6.

FIGURE 4

Metabolomics results of CTD-ILD-like lung injury of model mice with PD + Cur intervention (A–B) Differentially expressed genes (blue or red dots) and pathway enrichment between Model vs. Control; (C–D) Differentially expressed genes (blue or red dots) and pathway enrichment between PD + Cur vs. Model. n = 6.

FIGURE 5

Alleviating immunoinflammatory injury in lung tissue of bleomycin–treating model by activating GABBR (A) H&E staining of lung tissue (×200): mild to moderate atrophy of alveoli (white arrow), and mild to moderate fibrous tissue proliferation in the interstitium and thickening of alveolar septa (black arrow) in the model group; (B) Masson staining of lung tissue (×200): a large amount of fibrillar collagen formation (blue arrows) in the model group; (C–D) The level of serum TOLLIP and MUC5B, biomarkers of risk and predisposition in CTD-ILD; (E–G) The level of serum KL-6, SP-D, and RCN3, biomarkers of epithelial cell dysfunction and extracellular matrix remodeling in CTD-ILD; (H–J) The level of immunoinflammation-related indicators IL-6, CCL18, and SF in serum (K) Relative expression levels of PI3KR1 in lung detected by PRM-based targeted proteomics; (L–M) Differentially expressed genes (blue or red dots) and pathway enrichment between Model vs. Control by RNA-seq analysis; (N–O) Differentially expressed genes (blue or red dots) and pathway enrichment between baclofen vs Model by untargeted proteomics; (P–Q) Relative expression levels of AKT and TGFβR3 in lung detected by PRM-based targeted proteomics. Data are presented as mean ± S. D in Fig. (D–G) and (L–N), and as median in Fig. (C) *P < 0.05, **P < 0.01, ***P < 0.001 for vs Control; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 for vs Model; Kruskal–Wallis for TOLLIP, Games-Howell for RCN3, and the Least Significant Difference test for others. n = 6 for figure L–O, n = 3 for others.

FIGURE 6

PD + Cur inhibits the PI3K/AKT/TGF-β pathway by targeting and activating GABBR.