Endocannabinoid signalling/cannabinoid receptor 2 is involved in icariin-mediated protective effects against bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease of unknown aetiology with limited effective treatment options. It is important to explore novel therapeutic targets and develop potential drugs for IPF.The aim of the present study was to analyse nontargeted plasma metabolites in patients with IPF and investigate whether cannabinoid receptor (CB2) activation mediates the antifibrotic effect of icariin (ICA).We used an untargeted metabolomics method to detect the global metabolic profiles in the plasma of stable IPF patients and patients with stable chronic obstructive pulmonary disease (COPD), as well as healthy subjects. The untargeted liquid chromatography-mass spectrometry (LC-MS) analysis revealed that IPF showed differential metabolites and perturbed signalling pathways. ICA is pharmacologically bioactive and possesses extensive therapeutic capacities such as osteoprotective, neuroprotective, cardiovascular protective, anti-cancer, anti-inflammation and reproductive function. Therefore, ICA was administered to a pulmonary fibrosis rat model for 4 weeks and then the effect of ICA on pulmonary fibrosis was examined by dissection and histology.The metabolites in the plasma were determined by untargeted LC-MS. An unsupervised principal component analysis (PCA) was used to observe the distribution of each sample, and a supervised partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) results showed that there was significant separation between any two groups. ROC curve analyses revealed that 8 metabolites with high AUCs above 0.7 between the three groups of plasma samples. Pathway enrichment analysis revealed that 3 metabolites are involved in retrograde endocannabinoid signalling. Meanwhile, Retrograde endocannabinoid signalling was identified significantly different in IPF group from other groups by Kyoto encyclopedia of Genes and Genomes (KEGG) pathway analysis, and then we further confirmed the endocannabinoid signalling by detecting the expression of the main receptors in bleomycin-induced pulmonary fibrosis, COPD rat model and normal rats. Consistent with previous studies, we found that the elevation of CB1 and CB2 in the lung tissues could be a signature of the pulmonary fibrosis rat model. Importantly, ICA may alleviate bleomycin-induced lung injury by decreasing CB1 and CB2 expression in the bleomycin-induced rat model.Taken together, we measured the global metabolic profile of IPF patients and identified CB2 as a novel potential target. ICA treatment demonstrated outstanding therapeutic effects on bleomycin-induced pulmonary fibrosis and targeting on CB2 may be the main underlying mechanism. ICA is a promising drug candidate to cure pulmonary fibrosis and mediate antagonists of the CB2 receptor.