Antifibrotic activities of Scutellariae Radix extracts and flavonoids: Comparative proteomics reveals distinct and shared mechanisms.

Background: Scutellariae Radix (SR), the root of Scutellaria baicalensis Georgi, and SR flavonoids have antifibrotic activities. It remains obscure, however, amongst SR aqueous extract (SRA), SR methanolic extract (SRM) and five major SR flavonoids (baicalein, baicalin, wogonoside, wogonin and oroxyloside), which ones are the most promising antifibrotics and what their mechanisms are.

Purpose: To compare the antifibrotic activities of SR extracts and flavonoids, and the proteomic signatures of selected SR extract and flavonoid, versus IN1130 phosphate, an antifibrotic positive control (abbreviated as IN1130), in TGF-β1-induced in vitro model of fibrosis in NRK-49F renal fibroblasts.

Methods: Isobaric labelling-based mass spectrometry was used for proteomic studies. Differentially expressed proteins were further analyzed using Gene Ontology annotation enrichment, protein-protein interaction network analysis and pathway analysis. Selected proteins of interest were validated by enzyme-linked immunosorbent assay (ELISA).

Results: Baicalein was the SR flavonoid with the best efficacy-toxicity ratio. SRM contained 8-fold more flavonoids and was more potently antifibrotic than SRA. Proteomic analysis of cells treated by TGF-β1, with or without baicalein (40 and 80 μM), SRM (40 and 80 μg/ml) and IN1130 (1 μM) suggested that baicalein, SRM and IN1130 all repressed TGF-β1-induced ribosomal proteins in cell lysates, while baicalein and SRM, but not IN1130, regulated the intracellular lysosome pathway; secretomic analysis suggested that 40 and 80 μg/ml SRM and 80 μM baicalein, but not IN1130 and 40 μM baicalein increased ribosomal proteins in conditioned media, whereas only baicalein regulated the lysosome pathway. ELISA verified secretomic findings that baicalein, SRM and IN1130 repressed TGF-β1-induced PAI-1 (Serpine1), Plod2, Ctgf (Ccn2), Ccl2 and Ccl7; baicalein and IN1130, but not SRM, reversed TGF-β1-induced Cyr61 (Ccn1) and Tsku; only baicalein reversed TGF-β1 repression of Mmp3; only IN1130 reversed TGF-β1-repressed Nov (Ccn3). ELISA validated cell-lysate proteomic findings that baicalein, SRM and IN1130 all reversed TGF-β1-induced Enpp1; only IN1130 reversed TGF-β1-induced Impdh2 and Sqstm1 and TGF-β1-repressed Aldh3a1. Baicalein and SRM induced Ccdc80, while only baicalein induced Tfrc.

Conclusion: Baicalein, SRM and IN1130 repress TGF-β1-induced fibrogenesis in renal fibroblasts by regulating overlapping protein targets and biological pathways. Our findings offer a comprehensive view of shared, drug- and dose-specific pharmacological and toxicological mechanisms and provide a valuable resource for further research and development of more efficacious and safer antifibrotics.