Screening and evaluation of quality markers from Shuangshen Pingfei formula for idiopathic pulmonary fibrosis using network pharmacology and pharmacodynamic, phytochemical, and pharmacokinetic analyses

The Shuangshen Pingfei formula (SSPF), a classic Chinese medicine derivative formula, has been shown to exert therapeutic effects on idiopathic pulmonary fibrosis (IPF). However, the quality control compounds of SSPF remain unclear.To select and confirm Q-markers of SSPF based on network pharmacology, cytobiology, animal-based pharmacodynamics, and phytochemical and pharmacokinetic analyses.A compound-target network was constructed based on previous research. In addition, high-degree compounds of SSPF were chosen as potential Q-marker candidates. Animal and cytological experiments were performed to verify key targets of IPF. Haematoxylin-eosin and Masson's trichrome staining were used to observe lung tissue pathology. Cytokine levels in the bronchoalveolar lavage fluid (BALF) were measured using ELISA kits. Gene and protein expression levels were determined using PCR and western blotting, respectively. The contents of Q-marker candidates in different batches of SSPF were then determined for traceability research, and the quality consistency of SSPF was objectively evaluated using principal component analysis (PCA). Finally, pharmacokinetic research was performed, and candidates with desirable metabolite and bioavailability parameters were confirmed as Q-markers of SSPF.The compound-target network included 56 compounds and 14 therapeutic targets. Animal experiments showed that SSPF attenuates lung fibrosis. SSPF decreased CC motif chemokine 2 (CCL2) and CC chemokine receptor type 2 (CCR2) levels in the BALF and downregulated the gene and protein expression of IPF therapy-related molecules, such as 5-hydroxytryptamine receptor 2A (HTR2A), CCL2, and CCR2, in the lungs. Cell experiments showed that nine Q-marker candidates in SSPF regulated the expression of CCL2 and CCR2, as predicted. Phytochemical analysis and PCA indicated that the qualities of SSPF in the nine batches were relatively stable. Pharmacokinetic studies demonstrated that mangiferin, salvianolic acid B, tanshinone IIA, naringin, and glycyrrhizic acid could be effectively absorbed into rat plasma, which ensured desirable bioavailability and confirmed their roles as Q-markers to represent anti-pulmonary fibrotic activity.Our study is an integrated strategy, based on network pharmacology with experimental verification and phytochemical and pharmacokinetic analyses that provides a novel approach for Q-marker selection and validation of SSPF for IPF treatment.