In Vitro Screening And Mechanism Study Of Traditional Chinese Medicine Against Renal Interstitial Fibrosis

Fibrosis, characterized by extracellular matrix accumulation and disruption of normal tissue architecture, is the common cause of chronic failure of multiple organs and a leading cause of morbidity and mortality worldwide. Unfortunately, there are no medicine that have convincing clinical specificity and efficacy as anti-fibrotic drugs. The aim of the present research is to apply our newly developed transforming growth factor?1 (TGF-?1)-induced,2-dimensional (2D) in vitro models into the discovery of natural anti-fibrotics.We examined the net anti-fibrotic activities of 22 herbal mixture extracts from publication review in a rat renal fibroblast-based 2D model. Net collagen accumulation was visualized by picro-Sirius red staining and quantitated by spectrophotometric analysis. Lactate dehydrogenase assay was used to monitor cytotoxicity.15 herbal mixture extracts were identified with anti-fibrotic activities.10 single herbal extracts were then picked up according to component of mixtures and previous publications and methanol extracts of 3 herbs, Scutellaria baicalensis Georgi, Rheum palmatum L. and Salvia miltiorrhiza Bunge showed in vitro anti-fibrotic activities. Finally, total 21 compounds were investigated and 3 flavonoids (quercetin, baicalein and baicalin) and 2 non-flavonoids (salvianolic acid B and emodin) demonstrated anti-fibrotic activities in both total 2D and 3D (nodule formation assays)models,The remaining 16 had little anti-fibrotic effect or were cytotoxic.Identified anti-fibrotic activities were further investigated by Western blot, RT-qPCR, SEAP report assay and high-content imaging analysis of immunofluorescence to address molecular mechanisms. These anti-fibrotic compounds suppressed collagen expression at both mRNA and protein levels and also variably suppressed fibroblasts differentiation and cell proliferation. Two flavonoids baicalein and baicalin were further found to dose-dependent inhibit cell proliferation without inducing cell apoptosis in NRK-49F cells, they significantly blocked TGF (31 induced ECM accumulation however had little effect on basal matrix production in NRK-49F and NRK-52E cells. TGF?1-Smad dependent pathway, especially endogenous TGF?1 expression and Smad3 activation were indicated to be involved.Finally, we characterized the potency and specificity of the model in detecting genuine anti-fibrotic activities from herbs. The data confirmed the beneficial activity of plant-derived compounds, single plants and mixtures in the treatment of fibrotic disease and clarified the possible mechanisms. It warrants further studies to explore the effects and mechanisms of the anti-fibrotic herbal compounds in preventing renal fibrosis and urge us to screen more plant materials to discover novel anti-fibrotic drugs.