Cordyceps militaris granules reduce EMT/ECM marker expression and alleviate pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible interstitial lung disease that severely impairs patients' quality of life and overall survival, yet few therapeutic drugs are available for this disease. Cordyceps militaris (L.) Link. is an edible and medicinal fungus renowned for its lung- and kidney-nourishing properties. Decoctions containing C. militaris and other traditional Chinese medicines have been used clinically to treat elderly patients with IPF. Nevertheless, whether C. militaris or its derived products can suppress the progression of pulmonary fibrosis remains unclear.

This study aimed to evaluate the effect of a granule formulation prepared from the aqueous extract of C. militaris (CC) on pulmonary fibrosis.

An HPLC-UV method was used for the quantification of five major nucleosides in CC and its ethanol extract (CCE, with maltodextrin removed). Two pulmonary fibrosis cellular models were utilized to evaluate the bioactivity of CCE, including TGF-β1-stimulated BEAS-2B human bronchial epithelial cells and A549 human lung adenocarcinoma epithelial cells. A bleomycin-induced mouse model of pulmonary fibrosis was established to evaluate in vivo efficacy of CC. Western blot and RT-qPCR were performed to quantify the protein and mRNA expression levels of key biomarkers associated with epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in pulmonary fibrosis, including E-cadherin, N-cadherin, vimentin, α-SMA, fibronectin, collagen I and collagen III. Non-invasive whole-body plethysmography (WBP) was used to measure pulmonary function parameters in mice.

The contents of uridine, guanosine, adenosine, cordycepin, and N⁶-(2-hydroxyethyl)adenosine were 0.84, 0.56, 0.93, 1.56 and 1.15 mg/g in CC and 4.49, 1.71, 3.96, 5.57 and 4.79 mg/g in CCE, respectively, with the total nucleoside content being 5.04 and 20.52 mg/g. CCE (200-800 μg/mL) dose-dependently inhibited the mRNA and protein expression of fibronectin, collagen I, and collagen III (ECM-related markers) in TGF-β1-stimulated BEAS-2B cells, whereas CCE upregulated E-cadherin mRNA and protein expression (EMT-related marker) in TGF-β1-treated A549 cells. In bleomycin-challenged mice, intragastric administration of CC at 600 mg/kg reduced lung hydroxyproline level, mitigated elevated enhanced pause (Penh), and markedly downregulated the protein expression of fibronectin, collagen I and collagen III.

In vitro and in vivo studies indicated that CC and CCE alleviated pulmonary fibrosis, which associated with reduced EMT/ECM marker expression, in particular inhibiting overproduction of ECM components (fibronectin, collagen I, and collagen III). C. militaris granules represent a potential anti-fibrotic candidate that merits further preclinical investigation.