|Author:||Li, Hoi Lam|
|Title:||The mechanistic effects of baicalein on aqueous humor drainage and its ocular hypotensive response|
|Advisors:||Do, Chi-wai (SO)|
To, Chi-ho (SO)
Chan, Henry (SO)
Herbs -- Therapeutic use
Glaucoma -- Treatment
Hong Kong Polytechnic University -- Dissertations
|Department:||School of Optometry|
|Pages:||xix, 208 pages : color illustrations|
|Abstract:||Glaucoma is a multifactorial eye disease characterized by gradual loss of vision resulting from progressive optic neuropathy. Despite the fact that glaucoma is a leading cause of blindness worldwide, there is currently no cure yet for glaucoma. Lowering intraocular pressure (IOP) is the only clinical intervention known to slow the progression of glaucomatous blindness. Reduced treatment efficacy and adverse ocular side effects are; however, frequently reported after prolong use of existing anti-glaucoma medications. This strongly suggests an urgent need for a more potent agent with minimal side effects. Baicalein (5,6,7-trihydroxyflavone) is a natural flavonoid derived from the dried roots of Scutellaria baicalensis Georgi. It is frequently found in vegetables and commonly used in traditional Chinese medicine. It has been previously reported that baicalein inhibits swelling-activated Cl-channels in non-pigmented ciliary epithelial cells, potentially reducing the rate of aqueous humor secretion. In addition, it is anticipated that baicalein may affect cell contractility and cell volume regulation as well as extracellular matrix (ECM) remodeling. There have been no literatures to date reporting the effects of baicalein on outflow facility. The primary aim of this study was to elucidate the mechanistic effects of baicalein on conventional outflow facility. The secondary aim was to observe its potential IOP-lowering effects when administered to living rodents. First, the acute effect of baicalein on outflow facility was studied using constant pressure perfusion system in freshly enucleated C57BL/6J mouse eyes. Outflow facility was determined by measuring the flow rates at sequential pressure steps. Results demonstrated that baicalein elicited a concentration-dependent increase in outflow facility. At 10 μM, baicalein enhanced the outflow facility by approximately 90%, suggesting that baicalein may affect conventional outflow facility, in addition to its inhibitory effect on aqueous humor secretion.|
Second, the mechanisms underlying the effects of baicalein on cell volume regulation, cell contractility and cell migration was studied using human trabecular meshwork (hTM) cells. It was shown that baicalein did not affect the cell volume under isometric conditions while a significant inhibition of regulatory volume decrease (RVD) was observed when subjected to hypotonic solution in hTM cells. Likewise, it was found that baicalein triggered a concentration-dependent hTM cell relaxation and retardation of cell migration when compared to the control group. These results suggested that baicalein may enhance the outflow facility by reducing its outflow resistance through the regulation of RVD, cell contractility and migration in hTM cells. Next, we examined the patterns of differential protein expression and identified specific and novel pathways mediated by baicalein using isobaric tag for relative and absolute quantitation (iTRAQ) based quantitative proteomics. Selected candidate proteins were validated by quantitative polymerase chain reaction (qPCR). The 47 and 119 proteins were significantly regulated after a 3-hour and a 2-day baicalein treatment in hTM cells, respectively. In agreement with our foregoing results, baicalein was shown to alter the expressions of pre-B-cell leukemia transcription factor-interacting protein 1 (PBXIP1) and arylsulfatase B (ARSB), supporting the notion of baicalein-mediated inhibition of TM cell migration. Apart from that, baicalein increased the expressions of matrix metalloproteinase-14 (MMP-14) and cathepsin B that are responsible for extracellular matrix (ECM) digestion, suggesting that baicalein may decrease the outflow resistance by modulating ECM composition. In addition, novel protein changes and pathways of human TM cells related to oxidative phosphorylation, clathrin-mediated endocytosis and mitochondrial dysfunction were observed. Finally, the in vitro findings were validated by measuring IOP changes in rodents. Baicalein, when administrated intraperitoneally, topically or intravitreally, caused a significant reduction in IOP. The largest IOP-lowering effect was achieved with intravitreal injection of baicalein, in which IOP was lowered by ~9 mmHg in Sprague Dawley (SD) rats. Taken together, these findings suggest that baicalein may regulate the contractility and volume of hTM cells and the composition of the ECM in the outflow pathway; thereby, enhancing the outflow facility and potentially leading to a reduction of IOP.
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