TY - JOUR
T1 - Antifibrotic activities of Scutellariae Radix extracts and flavonoids
T2 - Comparative proteomics reveals distinct and shared mechanisms
AU - Zhou, Shujun
AU - Yin, Xiaoke
AU - Yuan, Jun
AU - Liang, Zhitao
AU - Song, Jingzheng
AU - Li, Yunxia
AU - Peng, Cheng
AU - Hylands, Peter J
AU - Zhao, Zhongzhen
AU - Xu, Qihe
N1 - Funding information:
This project was generously supported by a philanthropist donation of PuraPharm PhD Scholarship to King's College London (principal investigators: Qihe Xu and Peter J Hylands, a Sino-British Fellowship Trust PhD Studentship award to Shujun Zhou, a Hong Kong Baptist University Faculty Research Grant Category II (principal investigators: Zhongzhen Zhao and Qihe Xu), as well as an open fund of the Cooperation & Innovation Centre on the Genuine Medicinal Materials in Southwest China (principal investigators: Cheng Peng and Qihe Xu). The authors sincerely thank Mr Mazhar Noor (Renal Sciences Laboratory, King's College London) for his invaluable technical support and helpful discussions, and we cordially thank Dr Dae-Kee Kim (Ewha Woman’s University, Korea) for his kind gifting of IN1130. This paper is dedicated to the late Professor Peter J Hylands.
Publisher copyright:
© 2022 The Authors. Published by Elsevier GmbH.
PY - 2022/6
Y1 - 2022/6
N2 - BackgroundScutellariae 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. PurposeTo 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. MethodsIsobaric 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). ResultsBaicalein 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. ConclusionBaicalein, 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.
AB - BackgroundScutellariae 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. PurposeTo 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. MethodsIsobaric 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). ResultsBaicalein 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. ConclusionBaicalein, 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.
KW - Baicalein
KW - Fibrosis
KW - Herbal
KW - IN1130
KW - Proteomics
KW - Scutellaria baicalensis
UR - http://www.scopus.com/inward/record.url?scp=85127515928&partnerID=8YFLogxK
U2 - 10.1016/j.phymed.2022.154049
DO - 10.1016/j.phymed.2022.154049
M3 - Journal article
C2 - 35397287
AN - SCOPUS:85127515928
SN - 0944-7113
VL - 100
JO - Phytomedicine
JF - Phytomedicine
M1 - 154049
ER -