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MAPK signaling pathway
Abstracts / Biochemical Pharmacology 139 (2017) 105–141
O17 Identification and characterisation of phytochemicals activating endogenous cytoprotective mechanisms Jennifer Harbottle, Andreas Kolb Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK Improved health span and lifespan extension in a wide phylogenetic range of species is associated with upregulation of the environmental stress response (ESR). The ESR, driven by the transcription factor Nrf2, provides endogenous antioxidant defence to maintain intracellular redox balance. Phytochemicals which stimulate the ESR thus upregulate cytoprotective mechanisms and ultimately contribute towards delaying the onset of age-related degenerative diseases. Two bioluminescent reporter cell lines were constructed to identify and characterise inducers of the ESR, specifically induction of Nrf2 target genes NQO1 and HMOX1. The first cell line was generated using random integration of an exogenous luciferase gene under control of an NQO1 gene promoter, and allowed rapid and sensitive high throughput screening of natural chemical libraries. The second assay system employed a more targeted strategy using an adeno-associated virus (AAV) vector to mediate the site-specific integration of a luciferase reporter into the locus of the endogenous ESR gene HMOX1, thus allowing predictive indexing of the gene’s transcription. The two cell lines were validated with potent ESR inducers sulforaphane and curcumin. A compound identified in the screening of a chemical library from the Marine Biodiscovery Centre (University of Aberdeen, UK) was further characterised in a microarray experiment and compared to known phytochemicals. In combination, these in vitro cell-based assay systems will allow a better understanding of the activity-structure relationship of ESR inducers, and assess the bioactivity of phytochemical metabolites from human serum samples following intervention trials. doi:10.1016/j.bcp.2017.06.082
O18 Revalorisation of rapeseed pomace (RSP): A study into antioxidant and DNA protective properties (in vitro) of a RSP extract and its effects on neurodegenerative disease C. elegans models Franziska Pohl a, Marie Goua a, Giovanna Bermano b, Wendy R. Russell c, Lorraine Scobbie c, Patrícia Maciel d,e, Andreia Teixeira-Castro d,e, Paul Kong Thoo Lin a a School of Pharmacy and Life Science, Robert Gordon University, Aberdeen, UK, b Centre for Obesity Research and Education (CORE), Robert Gordon University, Aberdeen, UK, c Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK, d Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal, e e ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal For a number of years there has been intense interest in phytochemicals due to their potential health benefits. One readily available and rich source of phytochemicals is Rapeseed pomace (RSP), a waste product obtained after edible oil production from Brassica napus. In this work, we analysed the ubiquitous secondary metabolites in RSP (harvest 2014, North East of Scotland) after soxhhlet ethanol/water (95:5) extraction. LC-MS/MS data of the extract revealed several secondary metabolites, with sinapic acid being the most abundant. The presence of a high level of phenolics in the extract was confirmed with the folin ciocalteu assay. Strong antioxidant activities of the extract was determined by using common antioxidant assays (FRAP, DPPH and ORAC). In addition, the extract (13.9 lg/mL) provided complete pBR322 plasmid DNA protection,
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from oxidative stress induced by AAPH (3.5 mM). Interestingly, in vitro analysis showed that the RSP extract exhibited acetylcholinesterase inhibition activity in a time and concentration dependant manner. Furthermore, cellular studies showed that incubation of neuroblastoma cells (SH Sy-5Y) with RSP extract protected cell metabolism from reactive oxygen species produced by H2O2 (1 mM). We are currently undertaking in vivo studies using the model organism C. elegans. Preliminary results showed that RSP extract is non-toxic (up to 5 mg/mL) and has neuroprotective properties in C. elegans models of neurodegenerative diseases (work in progress). The positive properties associated with the RSP extract in this study warrants further investigation for the revalorisation of rapeseed pomace in the health/food industry. doi:10.1016/j.bcp.2017.06.083
Session 7: Translating novel pathways – Diabetes and Renal O19 Ganoderma triterpenes inhibit kidney cyst development via down-regulating the Ras/MAPK signaling pathway Limin Su a, Ruoyun Chen b, Jemny Lu c, Baoxue Yang a a Peking University, Beijing, China, b Chinese Academy of Medical Sciences, Beijing, China, c Harvard Medical School, Boston, MA, USA Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenetic disease characterized by progressive development of renal cysts that leads to end stage renal disease. The aim of this study was to investigate the effect of Ganoderma triterpenes (GT), extracts from the natural product Ganoderma lucidum, on the development of kidney cysts. Our study revealed that GT significantly inhibited cyst growth in a dose-dependent manner in an in vitro cystogenesis model and an embryonic kidney cyst model. More importantly, GT was shown to attenuate the progression of cyst and reduce kidney mass in two ADPKD mouse models. To understand the underlying cyst-inhibiting mechanism of GT, a tubulogenesis assay was performed and showed that GT promoted epithelial tubule formation in MDCK cells, suggesting a possible effect on epithelial cell differentiation. The role of GT in regulating key signalling pathways that are involved in the pathogenesis of PKD was further investigated by immunoblotting. Our data revealed that GT specifically downregulated forskolin-induced activation of the Ras/MAPK signalling pathways in MDCK cells without a detectable effect on the mTOR pathway. We further screened 15 monomers that were purified from GT for their effects on inhibiting cyst formation in vitro. We demonstrated that CBLZ-7 (ethyl ganoderate C2) had a potent inhibitory effect on cyst formation in vitro. In addition, like GT, CBLZ-7 was able to downregulate FSK induced activation of the Ras/MAPK pathway. Therefore, our study demonstrated that GT and its purified monomer, CBLZ-7, are potential therapeutic regents for treating cystic kidney diseases. doi:10.1016/j.bcp.2017.06.084
O20 Low molecular weight fucoidan inhibits fibrotic processes in diabetic nephropathy Hong Zhou Peking University, Beijing, China Introduction: The aim of this study was to determine whether lowmolecular-weight fucoidan (LMWF) can reduce TGF-b-mediated
O17 Identification and characterisation of phytochemicals activating endogenous cytoprotective mechanisms Jennifer Harbottle, Andreas Kolb Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK Improved health span and lifespan extension in a wide phylogenetic range of species is associated with upregulation of the environmental stress response (ESR). The ESR, driven by the transcription factor Nrf2, provides endogenous antioxidant defence to maintain intracellular redox balance. Phytochemicals which stimulate the ESR thus upregulate cytoprotective mechanisms and ultimately contribute towards delaying the onset of age-related degenerative diseases. Two bioluminescent reporter cell lines were constructed to identify and characterise inducers of the ESR, specifically induction of Nrf2 target genes NQO1 and HMOX1. The first cell line was generated using random integration of an exogenous luciferase gene under control of an NQO1 gene promoter, and allowed rapid and sensitive high throughput screening of natural chemical libraries. The second assay system employed a more targeted strategy using an adeno-associated virus (AAV) vector to mediate the site-specific integration of a luciferase reporter into the locus of the endogenous ESR gene HMOX1, thus allowing predictive indexing of the gene’s transcription. The two cell lines were validated with potent ESR inducers sulforaphane and curcumin. A compound identified in the screening of a chemical library from the Marine Biodiscovery Centre (University of Aberdeen, UK) was further characterised in a microarray experiment and compared to known phytochemicals. In combination, these in vitro cell-based assay systems will allow a better understanding of the activity-structure relationship of ESR inducers, and assess the bioactivity of phytochemical metabolites from human serum samples following intervention trials. doi:10.1016/j.bcp.2017.06.082
O18 Revalorisation of rapeseed pomace (RSP): A study into antioxidant and DNA protective properties (in vitro) of a RSP extract and its effects on neurodegenerative disease C. elegans models Franziska Pohl a, Marie Goua a, Giovanna Bermano b, Wendy R. Russell c, Lorraine Scobbie c, Patrícia Maciel d,e, Andreia Teixeira-Castro d,e, Paul Kong Thoo Lin a a School of Pharmacy and Life Science, Robert Gordon University, Aberdeen, UK, b Centre for Obesity Research and Education (CORE), Robert Gordon University, Aberdeen, UK, c Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK, d Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal, e e ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal For a number of years there has been intense interest in phytochemicals due to their potential health benefits. One readily available and rich source of phytochemicals is Rapeseed pomace (RSP), a waste product obtained after edible oil production from Brassica napus. In this work, we analysed the ubiquitous secondary metabolites in RSP (harvest 2014, North East of Scotland) after soxhhlet ethanol/water (95:5) extraction. LC-MS/MS data of the extract revealed several secondary metabolites, with sinapic acid being the most abundant. The presence of a high level of phenolics in the extract was confirmed with the folin ciocalteu assay. Strong antioxidant activities of the extract was determined by using common antioxidant assays (FRAP, DPPH and ORAC). In addition, the extract (13.9 lg/mL) provided complete pBR322 plasmid DNA protection,
115
from oxidative stress induced by AAPH (3.5 mM). Interestingly, in vitro analysis showed that the RSP extract exhibited acetylcholinesterase inhibition activity in a time and concentration dependant manner. Furthermore, cellular studies showed that incubation of neuroblastoma cells (SH Sy-5Y) with RSP extract protected cell metabolism from reactive oxygen species produced by H2O2 (1 mM). We are currently undertaking in vivo studies using the model organism C. elegans. Preliminary results showed that RSP extract is non-toxic (up to 5 mg/mL) and has neuroprotective properties in C. elegans models of neurodegenerative diseases (work in progress). The positive properties associated with the RSP extract in this study warrants further investigation for the revalorisation of rapeseed pomace in the health/food industry. doi:10.1016/j.bcp.2017.06.083
Session 7: Translating novel pathways – Diabetes and Renal O19 Ganoderma triterpenes inhibit kidney cyst development via down-regulating the Ras/MAPK signaling pathway Limin Su a, Ruoyun Chen b, Jemny Lu c, Baoxue Yang a a Peking University, Beijing, China, b Chinese Academy of Medical Sciences, Beijing, China, c Harvard Medical School, Boston, MA, USA Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenetic disease characterized by progressive development of renal cysts that leads to end stage renal disease. The aim of this study was to investigate the effect of Ganoderma triterpenes (GT), extracts from the natural product Ganoderma lucidum, on the development of kidney cysts. Our study revealed that GT significantly inhibited cyst growth in a dose-dependent manner in an in vitro cystogenesis model and an embryonic kidney cyst model. More importantly, GT was shown to attenuate the progression of cyst and reduce kidney mass in two ADPKD mouse models. To understand the underlying cyst-inhibiting mechanism of GT, a tubulogenesis assay was performed and showed that GT promoted epithelial tubule formation in MDCK cells, suggesting a possible effect on epithelial cell differentiation. The role of GT in regulating key signalling pathways that are involved in the pathogenesis of PKD was further investigated by immunoblotting. Our data revealed that GT specifically downregulated forskolin-induced activation of the Ras/MAPK signalling pathways in MDCK cells without a detectable effect on the mTOR pathway. We further screened 15 monomers that were purified from GT for their effects on inhibiting cyst formation in vitro. We demonstrated that CBLZ-7 (ethyl ganoderate C2) had a potent inhibitory effect on cyst formation in vitro. In addition, like GT, CBLZ-7 was able to downregulate FSK induced activation of the Ras/MAPK pathway. Therefore, our study demonstrated that GT and its purified monomer, CBLZ-7, are potential therapeutic regents for treating cystic kidney diseases. doi:10.1016/j.bcp.2017.06.084
O20 Low molecular weight fucoidan inhibits fibrotic processes in diabetic nephropathy Hong Zhou Peking University, Beijing, China Introduction: The aim of this study was to determine whether lowmolecular-weight fucoidan (LMWF) can reduce TGF-b-mediated