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Title: A proteomic study of an in-vitro model of TGF-β1-induced fibrosis and mechanisms of herbal antifibrotics
Author: Zhou, Shujun
ISNI:       0000 0004 8505 260X
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2019
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Fibrosis is scarring of tissues characterised by excessive accumulation of collagen proteins, in which transforming growth factor (TGF)-β1 plays a central role. Fibrosis affects virtually all organs and is a common pathology behind chronic organ failure and mortality. Few antifibrotic drugs are available to prevent or reverse fibrosis, thus, it represents a significant unmet medical need to better understand fibrosis and to develop novel antifibrotics. Botanicals could be both cause and cure of fibrosis. Aconiti Radix (AR) and Aconiti Lateralis Radix (ALR) were suggested pro-fibrotic and potentially nephrotoxic, while Scutellariae Radix (SR) and SR flavonoids were reportedly anti-fibrotic. I hypothesised that AR/ALR and SR might affect fibrogenesis as a function of AR/ALR alkaloids and SR flavonoids, respectively. This project aimed to (i) further scrutinise effects of AR, ALR, AR/ALR alkaloids, SR and SR flavonoids in a TGF-β1-induced in-vitro model of fibrosis; and (ii) develop a proteomic view of the in-vitro model of fibrosis and the differential mechanisms of action of selected antifibrotics. In NRK-49F renal fibroblasts with and without treatment by TGF-β1 and antifibrotics, fibrogenesis was quantified by total collagen assays and examination of fibrogenic molecular markers. Cell lysates and conditioned media were harvested for proteomic analysis. Proteins of interest were validated by enzyme-linked immunosorbent assay (ELISA). Results showed that aqueous and ethanolic extracts of unprocessed and processed AR did not affect fibrogenesis. Seven of nine AR alkaloids showed anti-fibrotic effects. These disapproved my hypothesis on AR/ALR and their alkaloids. Supporting SR flavonoids as the main anti-fibrotic compounds in SR, however, methanolic extract of SR (SRM), which contained 8-fold higher flavonoids than SR aqueous (SRA) extract, is more potently antifibrotic than SRA. Five tested SR flavonoids were more or less anti-fibrotic, with baicalein being the most anti-fibrotic and least toxic. Hence, in further proteomic studies, I focused on mechanisms of TGF-β1-induced in-vitro model of fibrosis and the anti-fibrotic mechanisms of SRM and baicalein, in comparison with IN1130, an inhibitor of TGF-β type I receptor. In cell lysates of TGF-β1-stimulated NRK-49F cells, there was an apparent mismatch between increased ribosomal proteins and reduced proteins involved in multiple metabolism pathways, in between which there was increased Impdh2, a druggable target. Secretomic analysis indicated that TGF-β1-induced fibrosis was mediated by dysregulation of key regulators of matrix degradation (PAI-1and Mmp3), signalling mediators (Ccn1, Ccn2, Ccn3 and Tsku) and a collagen crosslinker (Plod2), and was coupled with increased chemokines Ccl2 and Ccl7. SRM, baicalein and IN1130 all significantly regulated the ribosome pathway; SRM and baicalein, but not IN1130, regulated the lysosome pathway, while they differentially regulated metabolism pathways. They all reversed TGF-β1-induced PAI-1, Plod2, Ccn2, Ccl2 and Ccl7. Baicalein and IN1130, but not SRM, reversed TGF-β1-induced Ccn1 and Tsku. Only baicalein reversed TGF-β1 repression of Mmp3. Only IN1130 reversed TGF-β1 induction of Impdh2 and repression of Aldh3a1 and Ccn3. Among all proteins in cell lysates, Enpp1 was most dramatically induced by TGF-β1 and it was also among the most dramatically repressed by SRM, baicalein and IN1130. In conclusion, TGF-β1-induced fibrogenesis in renal fibroblasts involves dysregulation of multiple secreted proteins involved in regulation of collagen synthesis, crosslinking and degradation, and is characterised by dysregulation of intracellular metabolism. It is inherently coupled with increased secretion of chemokines, suggestive of a proinflammatory role for TGF-β1 and myofibroblasts. Comparative proteomic analysis has uncovered overlapping mechanisms of the three antifibrotics. Besides known anti-fibrotic targets e.g. PAI-1 and Ccn2, it deserves further investigation to determine whether ribosome, lysosome, metabolism pathways and individual proteins e.g. Aldh3a1, Ccn1, Ccn3, Enpp1, Impdh2, Mmp3, Plod2 and Tsku are novel targets for developing antifibrotics.
Supervisor: Xu, Qihe ; Hylands, Peter John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available