Synergistic effects of diammonium glycyrrhizinate and vitamin D3 against EMT in pulmonary fibrosis through modulating the crosstalk of STAT3/HSP90AA1 and HIF-1α pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Diammonium glycyrrhizinate (DG), a triterpene saponin compound, derived from the traditional Chinese herb Licorice (Glycyrrhiza spp.). This component has been clinically applied for treating hepatitis and liver fibrosis.

AIM OF THE STUDY: This study aimed to investigate the synergistic anti-pulmonary fibrosis (PF) effect and mechanism of DG and vitamin D3 (VD3).

MATERIALS AND

METHODS

The network pharmacology, molecular docking and molecular dynamics (MD) simulations were used to rapidly screen out potential synergetic targets and pathways of anti-PF. The studies on anti-PF effect and potential mechanism including lung weight, hydroxyproline content, pathological section staining, CCK-8, cell morphology, migration and invasion experiments were comprehensively evaluated. Furthermore, the epithelial-mesenchymal transition (EMT) related markers, key targets and signaling pathways proteins were analyzed by immunohistochemical staining (IHC) and Western blot (WB).

RESULTS

Network pharmacology analysis revealed that STAT3 and HSP90AA1 were the two key synergetic targets, and HIF-1 signaling pathway was the top-ranked in KEGG analysis. Actually, DG and VD3 significantly alleviated the symptoms of the bleomycin-induced PF model mice. Moreover, the expression of N-Cadherin and α-SMA were markedly decreased and E-Cadherin were upregulated both in vivo and vitro, suggesting the reduced EMT. Importantly, the combined use of DG and VD3 dramatically decreased the levels of STAT3, HSP90AA1, and HIF-1α in vitro, which were consistent with the prediction of network pharmacology and the verification of molecular docking and MD simulations. Additionally, silencing either STAT3 or HSP90AA1 mutually suppressed each other, thus significantly affecting the HIF-1α signaling pathway and EMT.

CONCLUSION

In this study, the combination of DG and VD3 effectively ameliorated the pathological changes of PF and EMT effects by regulating the crosstalk between STAT3 and HSP90AA1 and the HIF-1α signaling pathway.