|Title:||The effects of baicalein on chloride transport and aqueous humour formation of porcine ciliary epithelium|
|Advisors:||To, Chi-ho (SO)|
Do, Chi-wai (SO)
|Subject:||Glaucoma -- Treatment|
Chinese skullcap -- Therapeutic use.
Hong Kong Polytechnic University -- Dissertations
|Department:||School of Optometry|
|Pages:||xxv, 234 pages : illustrations ; 30 cm|
|Abstract:||Glaucoma is among the worldwide leading causes of irreversible blindness, frequently associated with elevated intraocular pressure (IOP). Currently, reducing IOP by reducing aqueous humour formation (AHF), or enhancing the outflow, is the only effective treatment in retarding the progression of vision defect. However, several current anti-glaucoma drugs have shown fading effectiveness and multiple side effects, suggesting a need in developing of novel and more potent drugs. Baicalein is a major bioactive flavone derived from a medicinal herb Scutellariae radix, with multiple biological functions, such as anti-inflammation, anti-tumor and modulation of chloride transport. AHF is also driven by chloride transport. Therefore, this present study aimed to examine if baicalein can regulate AHF and be a novel and potent medicinal for treating glaucoma. The first part of present study employed Ussing chamber techniques to investigate the effect of baicalein on short-circuit current (Isc) across porcine ciliary body/epithelium (CBE), and its underlying transport mechanisms. Addition of baicalein on aqueous side elicited significant reduction of Isc dose-dependently. Baicalein induced Isc inhibition was strongly dependent on Cl- concentration in the bath solution. Blocking Cl- channels, instead of Na+-K+-2Cl cotransporter, almost abolished the inhibition caused by baicalein, suggesting baicalein may inhibit Isc by reducing Cl- secretion via Cl- channels. Inhibition of protein kinase C (PKC), not protein kinase A or lipoxygenase, dramatically suppressed baicalein induced Isc inhibition, indicating PKC was likely involved in the signalling pathway mediating the responses. Fluid flow (FF) measurements showed that baicalein significantly reduced FF across porcine CBE, indicating the fluid secretion was markedly inhibited by baicalein.|
Due to undesirable interaction with intracellular dye, the effects of baicalein on intracellular Cl- concentration ([Cl-]i) and membrane potential of porcine ciliary epithelial cells could not be precisely quantified. Nevertheless, the electrical properties of these cells were successfully characterized by using the potential-sensitive dye bis (1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. Both pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells showed similar responses towards different interventions. Depolarization was noticed after Cl- replacement. Both NFA and baicalein significantly inhibited the depolarization induced by Cl- substitution, suggesting baicalein can also inhibit the Cl- conductance upon Cl- removal in porcine NPE cells. Whole-cell current recordings in native porcine NPE cells showed that baicalein markedly reduced swelling-activated current and the basal current with normal and increased [Cl-]i. Only the inhibitory action on basal current with normal [Cl-]i was completely blocked by PKC inhibitors (staurosporine and calphostin C), indicating under physiological condition, those active and PKC sensitive Cl- channels at NPE cells were probably the downstream cellular targets of baicalein in porcine CBE. The involvements of PKC in the baicalein induced responses were further supported by the stimulation of phospho-PKC expression in baicalein treated-porcine ciliary epithelial cells. In the final part, baicalein treated eyes showed significant lower IOP than contralateral eyes in guinea pigs from both topical administration and intravitreal injection groups. In conclusion, baicalein can inhibit the transepithelial Cl- transport and fluid secretion across porcine CBE, probably via down-regulating Cl- channel activities. This inhibition may involve PKC associated signalling pathways. Baicalein has ocular hypotensive property and it may be developed into a novel anti-glaucoma drug in the future.
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