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postgraduate thesis: Molecular mechanisms underlying the antioxidant, anti-inflammatory, and anti-obesity effects of Chinese medicine triterpene celastrol

TitleMolecular mechanisms underlying the antioxidant, anti-inflammatory, and anti-obesity effects of Chinese medicine triterpene celastrol
Authors
Issue Date2017
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Luo, D. [骆丹]. (2017). Molecular mechanisms underlying the antioxidant, anti-inflammatory, and anti-obesity effects of Chinese medicine triterpene celastrol. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractThe pathology of obesity is characterized by inflammation, oxidative stress and endoplasmic reticulum (ER) stress, leading to type-2 diabetes and related diseases. Inflammation not only disrupts blood vessel integrity but also induces macrophage infiltration into adipose tissues and livers in obesity. Proinflammatory M1 macrophages directly exacerbate inflammation and promote insulin resistance. Moreover, obesity-induced ER stress enhances macrophage M1 polarization. Natural product celastrol, a pentacyclic triterpene from herbal medicine Thunder God Vine (Tripterygium wilfordii), was recently identified as a potent anti-obesity drug. Thus, the aim of this thesis was to characterize the mechanisms underlying the anti-obesity effects of celastrol through examining blood vessel disruption, inflammation and ER stress. Tight junction proteins are well-known to regulate the integrity and functions of blood vessels. Murine endothelial bEnd3 cells were used as a model. The cell-cell tight junctions were disrupted by oxygen glucose deprivation (OGD). The endothelial permeability was assessed by measuring transepithelial electrical resistance (TEER) and the expressions of tight junction proteins were determined by RT-PCR and Western blotting. I found that celastrol profoundly induced occludin, claudin-5 and zonula occludens-1 (ZO-1) in endothelial cells and prevented TEER loss. Importantly, celastrol effectively inhibited macrophage migration through endothelial monolayers against OGD challenge. Mechanistic studies revealed that celastrol induced tight junction proteins via activating MAP kinases and PI3K/Akt/mTOR pathway. These results demonstrated the potential of celastrol in the protection of blood vessel integrity. To characterize the anti-inflammatory activities of celastrol, I induced obesity in C57BL/6N mice by high-fat diet and subsequently treated diet-induced obese mice with celastrol for three weeks. The expressions of the biomarkers associated with inflammation and macrophage polarization in adipose and hepatic tissues were determined. As result, celastrol profoundly reduced fat accumulation, ameliorated glucose tolerance and insulin sensitivity, and suppressed proinflammatory M1 macrophage polarization. The mechanisms were explored in murine macrophage RAW264.7 cells. I discovered that celastrol regulated macrophage polarization by inhibiting MAP kinases and NF-κB pathway, and activating Nrf2/HO-1 pathway. Importantly, HO-1 inhibitor SnPP diminished the effects of celastrol against M1 macrophage polarization. Thus, celastrol might attenuate diet-induced obesity via suppressing pro-inflammatory M1 macrophage polarization. To identify celastrol-protein conjugates, I synthesized a novel N-propargyl celastrol amide bearing an alkyne (-C≡CH) group. Following drug treatment, click chemistry biotinylation, affinity isolation and proteomic identification, GRP78 was identified as a major celastrol-bound protein in macrophages. GRP78 is a well-known chaperone protein controlling the unfolded protein response. Using diet-induced obese mice as model, I profiled the fatty acids with GC-MS analysis and determined the lipid metabolism genes in liver and adipose tissues by qRT-PCR. I found that celastrol reduced lipogenesis and increased lipolysis via β-oxidation and thermogenesis. Moreover, I validated that celastrol not only reduced ER stress in palmitate-challenged macrophages but also ameliorated ER stress in hepatic and adipose tissue macrophages in mice. Collectively, the results indicated that celastrol might ameliorate metabolic dysfunctions via covalent modification of GRP78. In conclusion, the results from this thesis may pave the avenue to develop celastrol into a promising drug against obesity, diabetes and insulin resistance.
DegreeDoctor of Philosophy
SubjectNatural products
Treatment - Obesity
Dept/ProgramChinese Medicine
Persistent Identifierhttp://hdl.handle.net/10722/270245

 

DC FieldValueLanguage
dc.contributor.authorLuo, Dan-
dc.contributor.author骆丹-
dc.date.accessioned2019-05-23T02:26:19Z-
dc.date.available2019-05-23T02:26:19Z-
dc.date.issued2017-
dc.identifier.citationLuo, D. [骆丹]. (2017). Molecular mechanisms underlying the antioxidant, anti-inflammatory, and anti-obesity effects of Chinese medicine triterpene celastrol. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/270245-
dc.description.abstractThe pathology of obesity is characterized by inflammation, oxidative stress and endoplasmic reticulum (ER) stress, leading to type-2 diabetes and related diseases. Inflammation not only disrupts blood vessel integrity but also induces macrophage infiltration into adipose tissues and livers in obesity. Proinflammatory M1 macrophages directly exacerbate inflammation and promote insulin resistance. Moreover, obesity-induced ER stress enhances macrophage M1 polarization. Natural product celastrol, a pentacyclic triterpene from herbal medicine Thunder God Vine (Tripterygium wilfordii), was recently identified as a potent anti-obesity drug. Thus, the aim of this thesis was to characterize the mechanisms underlying the anti-obesity effects of celastrol through examining blood vessel disruption, inflammation and ER stress. Tight junction proteins are well-known to regulate the integrity and functions of blood vessels. Murine endothelial bEnd3 cells were used as a model. The cell-cell tight junctions were disrupted by oxygen glucose deprivation (OGD). The endothelial permeability was assessed by measuring transepithelial electrical resistance (TEER) and the expressions of tight junction proteins were determined by RT-PCR and Western blotting. I found that celastrol profoundly induced occludin, claudin-5 and zonula occludens-1 (ZO-1) in endothelial cells and prevented TEER loss. Importantly, celastrol effectively inhibited macrophage migration through endothelial monolayers against OGD challenge. Mechanistic studies revealed that celastrol induced tight junction proteins via activating MAP kinases and PI3K/Akt/mTOR pathway. These results demonstrated the potential of celastrol in the protection of blood vessel integrity. To characterize the anti-inflammatory activities of celastrol, I induced obesity in C57BL/6N mice by high-fat diet and subsequently treated diet-induced obese mice with celastrol for three weeks. The expressions of the biomarkers associated with inflammation and macrophage polarization in adipose and hepatic tissues were determined. As result, celastrol profoundly reduced fat accumulation, ameliorated glucose tolerance and insulin sensitivity, and suppressed proinflammatory M1 macrophage polarization. The mechanisms were explored in murine macrophage RAW264.7 cells. I discovered that celastrol regulated macrophage polarization by inhibiting MAP kinases and NF-κB pathway, and activating Nrf2/HO-1 pathway. Importantly, HO-1 inhibitor SnPP diminished the effects of celastrol against M1 macrophage polarization. Thus, celastrol might attenuate diet-induced obesity via suppressing pro-inflammatory M1 macrophage polarization. To identify celastrol-protein conjugates, I synthesized a novel N-propargyl celastrol amide bearing an alkyne (-C≡CH) group. Following drug treatment, click chemistry biotinylation, affinity isolation and proteomic identification, GRP78 was identified as a major celastrol-bound protein in macrophages. GRP78 is a well-known chaperone protein controlling the unfolded protein response. Using diet-induced obese mice as model, I profiled the fatty acids with GC-MS analysis and determined the lipid metabolism genes in liver and adipose tissues by qRT-PCR. I found that celastrol reduced lipogenesis and increased lipolysis via β-oxidation and thermogenesis. Moreover, I validated that celastrol not only reduced ER stress in palmitate-challenged macrophages but also ameliorated ER stress in hepatic and adipose tissue macrophages in mice. Collectively, the results indicated that celastrol might ameliorate metabolic dysfunctions via covalent modification of GRP78. In conclusion, the results from this thesis may pave the avenue to develop celastrol into a promising drug against obesity, diabetes and insulin resistance. -
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshNatural products-
dc.subject.lcshTreatment - Obesity-
dc.titleMolecular mechanisms underlying the antioxidant, anti-inflammatory, and anti-obesity effects of Chinese medicine triterpene celastrol-
dc.typePG_Thesis-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineChinese Medicine-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2018-
dc.identifier.mmsid991044104149203414-

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