Abstract
Plants are a validated source of novel compounds with biopharmaceutical activity, but the diversity and often low abundance of phytochemicals have hindered applications in medicine. It is paramount therefore to bridge the gap between traditional and western medicine to improve the treatment of human diseases by developing drug-like leads from man-made chemical libraries.
Liver fibrosis is an often-fatal condition that The World Health Organisation estimates affecting up to 100 million people worldwide, a number which is set to increase if diets and lifestyles continue to follow current trends.
This project takes up the challenge by using a variety of methods combining improved production of plant biomass with in vitro, high-throughput multi-readout assays designed to model the disease progression of liver fibrosis in human hepatic stellate cells. The bioassay developed uses live-cell analysis to simultaneously gather data on the cell growth and death kinetics, phenotypic analysis and detection of extracellular matrix proteins.
Plants with previously reported anti-fibrotic activity were used to generate sources of phytochemicals and in vitro liver fibrosis assays were validated with tested compounds. Astragalus membranaceus and Ocimum basilicum were genetically transformed with Agrobacterium rhizogenes to provide reproducible and rapidly growing sterile hairy root cultures as a continuous supply of plant material. Root extracts obtained with various techniques showed different anti-fibrotic activity in the in vitro bioassay. Extracellular matrix assays allowed distinction of the deposition of fibronectin and collagen following treatment. Real-time measurements provided insights on the kinetics of cell proliferation and apoptotic processes. It was possible to distinguish between Caspase-3/7 activation and maintenance of membrane integrity.
The successful development of the high-throughput bioassay provided a solid platform for future identification of novel compounds against a complex human disease. The platform requires less material than traditional methods, is less labour intensive and can be adapted to test a multitude of compounds from disparate sources.
Liver fibrosis is an often-fatal condition that The World Health Organisation estimates affecting up to 100 million people worldwide, a number which is set to increase if diets and lifestyles continue to follow current trends.
This project takes up the challenge by using a variety of methods combining improved production of plant biomass with in vitro, high-throughput multi-readout assays designed to model the disease progression of liver fibrosis in human hepatic stellate cells. The bioassay developed uses live-cell analysis to simultaneously gather data on the cell growth and death kinetics, phenotypic analysis and detection of extracellular matrix proteins.
Plants with previously reported anti-fibrotic activity were used to generate sources of phytochemicals and in vitro liver fibrosis assays were validated with tested compounds. Astragalus membranaceus and Ocimum basilicum were genetically transformed with Agrobacterium rhizogenes to provide reproducible and rapidly growing sterile hairy root cultures as a continuous supply of plant material. Root extracts obtained with various techniques showed different anti-fibrotic activity in the in vitro bioassay. Extracellular matrix assays allowed distinction of the deposition of fibronectin and collagen following treatment. Real-time measurements provided insights on the kinetics of cell proliferation and apoptotic processes. It was possible to distinguish between Caspase-3/7 activation and maintenance of membrane integrity.
The successful development of the high-throughput bioassay provided a solid platform for future identification of novel compounds against a complex human disease. The platform requires less material than traditional methods, is less labour intensive and can be adapted to test a multitude of compounds from disparate sources.
| Original language | English |
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| Qualification | Ph.D. |
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| Publication status | Unpublished - 2021 |
