Icariside II modulates pulmonary fibrosis via PI3K/Akt/β-catenin pathway inhibition of M2 macrophage program

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a debilitating interstitial lung disorder characterized by its limited therapeutic interventions. Macrophages, particularly the alternatively activated macrophages (M2 subtype), have been acknowledged for their substantial involvement in the development of pulmonary fibrosis. Hence, targeting macrophages emerges as a plausible therapeutic avenue for IPF. Icariside II (ISE II) is a natural flavonoid glycoside molecule known for its excellent anti-tumor and anti-fibrotic activities. Nevertheless, the impact of ISE II on pulmonary fibrosis and the intricate mechanisms through which it operates have yet to be elucidated.

Objective: To scrutinize the impact of ISE II on the regulation of M2 macrophage polarization and its inhibitory effect on pulmonary fibrosis, as well as to delve deeper into the underlying mechanisms of its actions.

Methods: The effect of ISE II on proliferation and apoptosis in RAW264.7 cells was assessed through the use of EdU-488 labeling and the Annexin V/PI assay. Flow cytometry, western blot, and qPCR were employed to detect markers associated with the M2 polarization phenotype. The anti-fibrotic effects of ISE II in NIH-3T3 cells were investigated in a co-culture with M2 macrophages. Si-Ctnnb1 and pcDNA3.1(+)-Ctnnb1 plasmid were used to investigate the mechanism of targeted intervention. The murine model of pulmonary fibrosis was induced by intratracheal administration of bleomycin (BLM). Pulmonary function, histopathological manifestations, lung M2 macrophage infiltration, and markers associated with pulmonary fibrosis were evaluated. Furthermore, in vivo transcriptomics analysis was employed to elucidate differentially regulated genes in lung tissues. Immunofluorescence, western blot, and immunohistochemistry were conducted for corresponding validation.

Results: Our investigation demonstrated that ISE II effectively inhibited the proliferation of RAW264.7 cells and mitigated the pro-fibrotic characteristics of M2 macrophages, exemplified by the downregulation of CD206, Arg-1, and YM-1, Fizz1, through the inhibition of the PI3K/Akt/β-catenin signaling pathway. This impact led to the amelioration of myofibroblast activation and the suppression of nuclear translocation of β-catenin of NIH-3T3 cells in a co-culture. Consequently, it resulted in decreased collagen deposition, reduced infiltration of profibrotic macrophages, and a concurrent restoration of pulmonary function in mice IPF models. Furthermore, our RNA sequencing results showed that ISE II could suppress the expression of genes related to M2 polarization, primarily by inhibiting the PI3K/Akt and β-catenin signaling pathway. In essence, our findings suggest that ISE II holds potential as an anti-fibrotic agent by orchestrating macrophage polarization. This may have significant implications in clinical practice.

Conclusion: This study has provided evidence that ISE II exerts a significant anti-fibrotic effect by inhibiting macrophage M2 polarization through the suppression of the PI3K/Akt/β-catenin signaling pathway. These findings underscore the potential of ISE II as a promising candidate for the development of anti-fibrotic pharmaceuticals in the future.

Keywords: Ctnnb1; Macrophage; Myofibroblast differentiation; Pulmonary fibrosis.